Skin testing of guinea pigs and footpad testing of mice with a new antigen for detecting delayed hypersensitivity to Cryptococcus neoformans.

PubMed

Murphy, J W; Gregory, J A; Larsh, H W

1974-02-01

This study was undertaken to evaluate the potential of a cryptococcal culture filtrate antigen, cryptococcin C184, for detecting delayed hypersensitivity in Cryptococcus neoformans-injected animals. The antigen was tested on guinea pigs which had received saline or C. neoformans and on animals sensitized to Histoplasma capsulatum, Blastomyces dermatitidis, Candida albicans, or Sporothrix schenckii. A delayed-type hypersensitivity response was elicited by cryptococcin C184 in C. neoformans-injected guinea pigs, whereas no indurations or erythemas were seen at 48 h after skin testing of saline controls or heterologously sensitized guinea pigs. Besides being specific for Cryptococcus, the antigen showed a high degree of sensitivity and was reproducible. Footpad tests were conducted with the antigen on mice which had previously received either 10(5) viable C. neoformans cells or saline. Delayed hypersensitivity was indicated in the C. neoformans-injected mice by the increase in thickness of antigen-injected footpads when compared with the saline-injected footpads. In control mice, antigen- and saline-injected footpads were comparable in thickness 24 h after injection. Mice sensitized to B. dermatitidis were footpad tested with C184, and no cross-reactivity was demonstrated.

Evaluation of immunity to varicella zoster virus with a novel double antigen sandwich enzyme-linked immunosorbent assay.

PubMed

Liu, Jian; Chen, Chunye; Zhu, Rui; Ye, Xiangzhong; Jia, Jizong; Yang, Lianwei; Wang, Yongmei; Wang, Wei; Ye, Jianghui; Li, Yimin; Zhu, Hua; Zhao, Qinjian; Zhang, Jun; Cheng, Tong; Xia, Ningshao

2016-11-01

Varicella is a highly contagious disease caused by primary infection of Varicella zoster virus (VZV). Varicella can be severe or even lethal in susceptible adults, immunocompromised patients and neonates. Determination of the status of immunity to VZV is recommended for these high-risk populations. Furthermore, measurement of population immunity to VZV can help in developing proper varicella vaccination programmes. VZV glycoprotein E (gE) is an antigen that has been demonstrated to be a highly accurate indicator of VZV-specific immunity. In this study, recombinant gE (rgE) was used to establish a double antigen sandwich enzyme-linked immunosorbent assay (ELISA). The established sandwich ELISA showed high specificity and sensitivity in the detection of human sera, and it could detect VZV-specific antibodies at a concentration of 11.25 m IU/mL with a detection linearity interval of 11.25 to 360 m IU/mL (R 2  = 0.9985). The double gE antigen sandwich ELISA showed a sensitivity of 95.08 % and specificity of 100 % compared to the fluorescent-antibody-to-membrane-antigen (FAMA) test, and it showed a sensitivity of 100 % and a specificity of 94.74 % compared to a commercial neutralizing antibody detection kit. Thus, the established double antigen sandwich ELISA can be used as a sensitive and specific quantitative method to evaluate immunity to VZV.

Surface grafted antibodies: controlled architecture permits enhanced antigen detection.

PubMed

Sebra, Robert P; Masters, Kristyn S; Bowman, Christopher N; Anseth, Kristi S

2005-11-22

The attachment of antibodies to substrate surfaces is useful for achieving specific detection of antigens and toxins associated with clinical and field diagnostics. Here, acrylated whole antibodies were produced through conjugation chemistry, with the goal of covalently photografting these proteins from surfaces in a controlled fashion, to facilitate rapid and sensitive antigenic detection. A living radical photopolymerization chemistry was used to graft the acrylated whole antibodies on polymer surfaces at controlled densities and spatial locations by controlling the exposure time and area, respectively. Copolymer grafts containing these antibodies were synthesized to demonstrate two principles. First, PEG functionalities were introduced to prevent nonspecific protein interactions and improve the reaction kinetics by increasing solvation and mobility of the antibody-containing chains. Both of these properties lead to sensitive (pM) and rapid (<20 min) detection of antigens with this surface modification technique. Second, graft composition was tailored to include multiple antibodies on the same grafted chains, establishing a means for simultaneously detecting multiple antigens on one grafted surface area. Finally, the addition of PEG spacers between the acrylate functionality and the pendant detection antibodies was tuned to enhance the detection of a short-half-life molecule, glucagon, in a complex biological environment, plasma.

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells

PubMed Central

Cirelli, Kimberly M.; Dan, Jennifer M.; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells. PMID:29065175

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells.

PubMed

Reiss, Samantha; Baxter, Amy E; Cirelli, Kimberly M; Dan, Jennifer M; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane; Kaufmann, Daniel E

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

Development and evaluation of flow through assay for detection of antibodies against porcine cysticercosis.

PubMed

Sreedevi, C; Hafeez, Md; Subramanyam, K V; Anand Kumar, P; Chengalva Rayulu, V

2011-04-01

A flow through assay (FTA) was developed on cellulose acetate membrane for the serodiagnosis of porcine cysticercosis using cyst fluid (CFA) and whole cyst antigens (WCA) of Taenia solium metacestode. The assay consisted of antigen of T. solium metacestode coated onto membrane, mounted on a flow-through test device to provide assay capture matrix. The optimum concentration of coating antigen was 250 ng. The protein A colloidal gold conjugate served as antigen-antibody detecting reagent. A total of 225 serum samples were tested using two antigens. Results were better with CFA (96.0% sensitivity; 96.0% specificity) compared to WCA (92.0% sensitivity; 96.0% specificity). The test was also compared with enzyme-linked immunosorbent assay. The ELISA showed 96 per cent sensitivity with both the antigens whereas; the specificity was 96 and 92 per cent with CFA and WCA respectively. The sensitivity and specificity of flow through assay agrees closely with those of the ELISA. The cross-reaction was observed in one out of eight hydatidosis positive pigs (12.5%) with CFA by both the assays. The highest diagnostic accuracy (96%) was obtained with CFA-FTA and CFA-ELISA. For its high sensitivity and sporadic cross-reactions, CFA-FTA appears to be suitable for practical use at field level without instrumentation.

Impact of the rapid antigen detection test in diagnosis and treatment of acute pharyngotonsillitis in a pediatric emergency room.

PubMed

Cardoso, Débora Morais; Gilio, Alfredo Elias; Hsin, Shieh Huei; Machado, Beatriz Marcondes; de Paulis, Milena; Lotufo, João Paulo B; Martinez, Marina Baquerizo; Grisi, Sandra Josefina E

2013-01-01

To evaluate the impact of the routine use of rapid antigen detection test in the diagnosis and treatment of acute pharyngotonsillitis in children. This is a prospective and observational study, with a protocol compliance design established at the Emergency Unit of the University Hospital of Universidade de São Paulo for the care of children and adolescents diagnosed with acute pharyngitis. 650 children and adolescents were enrolled. Based on clinical findings, antibiotics would be prescribed for 389 patients (59.8%); using the rapid antigen detection test, they were prescribed for 286 patients (44.0%). Among the 261 children who would not have received antibiotics based on the clinical evaluation, 111 (42.5%) had positive rapid antigen detection test. The diagnosis based only on clinical evaluation showed 61.1% sensitivity, 47.7% specificity, 44.9% positive predictive value, and 57.5% negative predictive value. The clinical diagnosis of streptococcal pharyngotonsillitis had low sensitivity and specificity. The routine use of rapid antigen detection test led to the reduction of antibiotic use and the identification of a risk group for complications of streptococcal infection, since 42.5% positive rapid antigen detection test patients would not have received antibiotics based only on clinical diagnosis.

Diagnostic Markers of Ovarian Cancer by High-Throughput Antigen Cloning and Detection on Arrays

PubMed Central

Chatterjee, Madhumita; Mohapatra, Saroj; Ionan, Alexei; Bawa, Gagandeep; Ali-Fehmi, Rouba; Wang, Xiaoju; Nowak, James; Ye, Bin; Nahhas, Fatimah A.; Lu, Karen; Witkin, Steven S.; Fishman, David; Munkarah, Adnan; Morris, Robert; Levin, Nancy K.; Shirley, Natalie N.; Tromp, Gerard; Abrams, Judith; Draghici, Sorin; Tainsky, Michael A.

2008-01-01

A noninvasive screening test would significantly facilitate early detection of epithelial ovarian cancer. This study used a combination of high-throughput selection and array-based serologic detection of many antigens indicative of the presence of cancer, thereby using the immune system as a biosensor. This high-throughput selection involved biopanning of an ovarian cancer phage display library using serum immunoglobulins from an ovarian cancer patient as bait. Protein macroarrays containing 480 of these selected antigen clones revealed 65 clones that interacted with immunoglobulins in sera from 32 ovarian cancer patients but not with sera from 25 healthy women or 14 patients having other benign or malignant gynecologic diseases. Sequence analysis data of these 65 clones revealed 62 different antigens. Among the markers, we identified some known antigens, including RCAS1, signal recognition protein-19, AHNAK-related sequence, nuclear autoantogenic sperm protein, Nijmegen breakage syndrome 1 (Nibrin), ribosomal protein L4, Homo sapiens KIAA0419 gene product, eukaryotic initiation factor 5A, and casein kinase II, as well as many previously uncharacterized antigenic gene products. Using these 65 antigens on protein microarrays, we trained neural networks on two-color fluorescent detection of serum IgG binding and found an average sensitivity and specificity of 55% and 98%, respectively. In addition, the top 6 of the most specific clones resulted in an average sensitivity and specificity of 32% and 94%, respectively. This global approach to antigenic profiling, epitomics, has applications to cancer and autoimmune diseases for diagnostic and therapeutic studies. Further work with larger panels of antigens should provide a comprehensive set of markers with sufficient sensitivity and specificity suitable for clinical testing in high-risk populations. PMID:16424057

Use of hexyl isocyanate antigen to detect antibodies to hexamethylene diisocyanate (HDI) in sensitized guinea pigs and in a sensitized worker.

PubMed

Karol, M H; Hauth, B A

1982-01-01

Hypersensitivity to hexamethylene diisocyanate (HDI) has been reported following occupational exposure. Diagnosis of sensitivity is usually made from clinical evaluation of symptomatology. An in vitro serologic assay for HDI sensitivity was developed by immunizing guinea pigs with HDI and with hexyl isocyanate (HMI). Animals injected intradermally with HMI produced hapten-specific antibodies whereas guinea pigs injected with HDI produced antibodies specific for larger determinants which included the HDI hapten. The larger determinants were assumed to be composed of portions of "self" molecules which reacted in vivo with HDI. Serum albumin appeared to be one such molecule. No cross reactions were noted between antibodies to HDI and another widely used industrial isocyanate, toluene diisocyanate (TDI). Antigens effective in detecting antibodies to HDI or HMI were tested for ability to detect reaginic antibodies in a worker with clinical "HDI" asthma. Using a radioimmunoassay (RAST), antibodies reacted with conjugates containing either HDI or HMI as haptens. In addition, the prevalance of HDI polyisocyanates (Desmodur N) in spray paints prompted its use as a hapten. Antibodies reacted with Desmodur N antigen conjugates in RAST. RAST inhibition further indicated that Desmodur N antigen reacted more readily with the patient's antibodies than did HDI or HMI antigens. These results suggest that the patient may have been exposed to HDI polyisocyanates in spray paint application. Use of Rast inhibition for diagnosis of sensitivity may indicate the precise sensitizing agent within a mixture.

Dual Electrophoresis Detection System for Rapid and Sensitive Immunoassays with Nanoparticle Signal Amplification

NASA Astrophysics Data System (ADS)

Zhang, Fangfang; Ma, Junjie; Watanabe, Junji; Tang, Jinlong; Liu, Huiyu; Shen, Heyun

2017-02-01

An electrophoretic technique was combined with an enzyme-linked immunosorbent assay (ELISA) system to achieve a rapid and sensitive immunoassay. A cellulose acetate filter modified with polyelectrolyte multilayer (PEM) was used as a solid substrate for three-dimensional antigen-antibody reactions. A dual electrophoresis process was used to induce directional migration and local condensation of antigens and antibodies at the solid substrate, avoiding the long diffusion times associated with antigen-antibody reactions in conventional ELISAs. The electrophoretic forces drove two steps in the ELISA process, namely the adsorption of antigen, and secondary antibody-labelled polystyrene nanoparticles (NP-Ab). The total time needed for dual electrophoresis-driven detection was just 4 min, nearly 2 h faster than a conventional ELISA system. Moreover, the rapid NP-Ab electrophoresis system simultaneously achieved amplification of the specific signal and a reduction in noise, leading to a more sensitive NP-Ab immunoassay with a limit of detection (LOD) of 130 fM, and wide range of detectable concentrations from 0.13 to 130 pM. These results suggest that the combination of dual electrophoresis detection and NP-Ab signal amplification has great potential for future immunoassay systems.

Integrated Magneto-Chemical Sensor For On-Site Food Allergen Detection.

PubMed

Lin, Hsing-Ying; Huang, Chen-Han; Park, Jongmin; Pathania, Divya; Castro, Cesar M; Fasano, Alessio; Weissleder, Ralph; Lee, Hakho

2017-10-24

Adverse food reactions, including food allergies, food sensitivities, and autoimmune reaction (e.g., celiac disease) affect 5-15% of the population and remain a considerable public health problem requiring stringent food avoidance and epinephrine availability for emergency events. Avoiding problematic foods is practically difficult, given current reliance on prepared foods and out-of-home meals. In response, we developed a portable, point-of-use detection technology, termed integrated exogenous antigen testing (iEAT). The system consists of a disposable antigen extraction device coupled with an electronic keychain reader for rapid sensing and communication. We optimized the prototype iEAT system to detect five major food antigens in peanuts, hazelnuts, wheat, milk, and eggs. Antigen extraction and detection with iEAT requires <10 min and achieves high-detection sensitivities (e.g., 0.1 mg/kg for gluten, lower than regulatory limits of 20 mg/kg). When testing under restaurant conditions, we were able to detect hidden food antigens such as gluten within "gluten-free" food items. The small size and rapid, simple testing of the iEAT system should help not only consumers but also other key stakeholders such as clinicians, food industries, and regulators to enhance food safety.

Sensitive typing of reverse ABO blood groups with a waveguide-mode sensor.

PubMed

Uno, Shigeyuki; Tanaka, Torahiko; Ashiba, Hiroki; Fujimaki, Makoto; Tanaka, Mutsuo; Hatta, Yoshihiro; Takei, Masami; Awazu, Koichi; Makishima, Makoto

2018-07-01

Portable, on-site blood typing methods will help provide life-saving blood transfusions to patients during an emergency or natural calamity, such as significant earthquakes. We have previously developed waveguide-mode (WM) sensors for forward ABO and Rh(D) blood typing and detection of antibodies against hepatitis B virus and hepatitis C virus. In this study, we evaluated a WM-sensor for reverse ABO blood typing. Since reverse ABO blood typing is a method for detection of antibodies against type A and type B oligosaccharide antigens on the surface of red blood cells (RBCs), we fixed a synthetic type A or type B trisaccharide antigen on the sensor chip of the WM sensor. We obtained significant changes in the reflectance spectra from a WM sensor on type A antigen with type B plasma and type O plasma and on type B antigen with type A plasma and type O plasma, and no spectrum changes on type A antigen or type B antigen with type AB plasma. Signal enhancement with the addition of a peroxidase reaction failed to increase the sensitivity for detection on oligosaccharide chips. By utilizing hemagglutination detection using regent type A and type B RBCs, we successfully determined reverse ABO blood groups with higher sensitivity compared to a method using oligosaccharide antigens. Thus, functionality of a portable device utilizing a WM sensor can be expanded to include reverse ABO blood typing and, in combination with forward ABO typing and antivirus antibody detection, may be useful for on-site blood testing in emergency settings. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Heat treatment and false-positive heartworm antigen testing in ex vivo parasites and dogs naturally infected by Dirofilaria repens and Angiostrongylus vasorum.

PubMed

Venco, Luigi; Manzocchi, Simone; Genchi, Marco; Kramer, Laura H

2017-11-09

Heartworm antigen testing is considered sensitive and specific. Currently available tests are reported as detecting a glycoprotein found predominantly in the reproductive tract of the female worm and can reach specificity close to 100%. Main concerns regard sensitivity in the case of light infections, the presence of immature females or cases of all-male infections. Research and development have been aimed at increasing sensitivity. Recently, heat treatment of serum prior to antigen testing has been shown to result in an increase in positive antigen test results, presumably due to disruption of natural antigen-antibody complexes. Cross-reactions in dogs with both natural and experimental infections with Angiostrongylus vasorum and Spirocerca lupi have been reported, but cross-reactions with other helminths have not been extensively studied. In order to evaluate potential cross-reactivity with other canine and feline parasites, two studies were performed. Study 1: Live adults of Dirofilaria immitis, Dirofilaria repens, Toxocara canis, Toxocara cati, Dipylidium caninum, Taenia taeniaeformis and Mesocestoides spp. larvae were washed and incubated in tubes with saline solution. All worms were alive at the time of removal from the saline. Saline solutions containing excretory/secretory antigens were then tested for heartworm with six different, commercially available antigen tests. All results were evaluated blind by three of the authors. Study 2: Sera from dogs with natural infections by A. vasorum or D. repens, living in areas free of heartworm disease, were tested with the same tests before and after heat treatment (103 °C for 10 min). Results suggest that antigens detected by currently available tests are not specific for D. immitis. They may give positive results through detection of different parasites' antigens that are normally not released into the bloodstream or released in a low amount and/or bound to antibodies. Tests may even detect antigens released by male D. immitis adult worms. D. repens appears to release more detectable antigens than the other worms studied. Cross-reaction with A. vasorum and D. repens does occur in the field and could potentially occur with other helminths. Heat treatment decreases specificity by enhancing cross-reactivity.

Improved detection of acute parvovirus B19 infection by immunoglobulin M EIA in combination with a novel antigen EIA.

PubMed

Corcoran, A; Kerr, S; Elliott, G; Koppelman, M; Doyle, S

2007-10-01

Although parvovirus B19 is a significant blood product contaminant, few methods other than polymerase chain reaction (PCR) have been developed to detect the presence of the virus. A B19 antigen enzyme immunoassay (EIA) has been developed and the sensitivity of detection is ascertained using dilutions of the B19 capsid protein VP2 and 10-fold dilutions of B19 viraemic serum. Once the assay cut-off was established, a panel of viraemic donations (n = 70) was screened by the antigen EIA. The B19 immunoglobulin M (IgM) and IgG status of these specimens was also determined. During screening of blood donor units by quantitative PCR, 70 individuals were identified with levels of B19 DNA greater than 10(6) IU/ml at the time of blood donation. The sensitivity of the B19 antigen EIA was estimated to be equivalent to between 10(8) and 10(9) IU/ml B19 DNA or 1-10 pg/ml of recombinant capsid protein. B19 detection was significantly enhanced when viraemic specimens were pretreated with a low pH proprietary reagent. Unlike other virus-detection assays, detection of the B19 antigen was not affected by the presence of B19 IgM or IgG antibodies. In addition, the assay was capable of detecting all three genotypes of human erythrovirus. Combined specimen analysis by the B19 antigen assay and a B19 IgM assay facilitated the detection of 91% of acute B19 infections in the test population. In combination with B19 IgM detection, application of the B19 antigen EIA is a flexible and efficient method of detecting recent B19 infection and can be used as an alternative to PCR.

Easy-to-Fabricate and High-Sensitivity LSPR Type Specific Protein Detection Sensor Using AAO Nano-Pore Size Control

PubMed Central

Kim, Sae-Wan; Lee, Jae-Sung; Lee, Sang-Won; Kang, Byoung-Ho; Kwon, Jin-Beom; Kim, Ok-Sik; Kim, Ju-Seong; Kim, Eung-Soo; Kwon, Dae-Hyuk; Kang, Shin-Won

2017-01-01

In this study, we developed a pore size/pore area-controlled optical biosensor-based anodic aluminum oxide (AAO) nanostructure. As the pore size of AAO increases, the unit cell of AAO increases, which also increases the non-pore area to which the antibody binds. The increase in the number of antibodies immobilized on the surface of the AAO enables effective detection of trace amounts of antigen, because increased antigen-antibody bonding results in a larger surface refractive index change. High sensitivity was thus achieved through amplification of the interference wave of two vertically-incident reflected waves through the localized surface plasmon resonance phenomenon. The sensitivity of the fabricated sensor was evaluated by measuring the change in wavelength with the change in the refractive index of the device surface, and sensitivity was increased with increasing pore-size and non-pore area. The sensitivity of the fabricated sensor was improved and up to 11.8 ag/mL serum amyloid A1 antigen was detected. In addition, the selectivity of the fabricated sensor was confirmed through a reaction with a heterogeneous substance, C-reactive protein antigen. By using hard anodization during fabrication of the AAO, the fabrication time of the device was reduced and the AAO chip was fabricated quickly and easily. PMID:28406469

Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles

USDA-ARS?s Scientific Manuscript database

A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibo...

Comparison of the sensitivity of the Legionella urinary antigen EIA kits from Binax and Biotest with urine from patients with infections caused by less common serogroups and subgroups of Legionella.

PubMed

Olsen, C W; Elverdal, P; Jørgensen, C S; Uldum, S A

2009-07-01

The detection of urinary antigen is the most widely used method to diagnose Legionnaires' disease (LD), so it is important that these assays have a high sensitivity for the disease. In this study, we compare two kits for their ability to detect urinary antigen in urine samples from patients infected with Legionella species and L. pneumophila sero- and subgroups not considered as the most common causes of LD. Urine samples (n = 33) from 30 culture-proven cases of L. pneumophila serogroup (sg) 1, subgroup non-Pontiac infection, and urine samples (n = 35) from 32 cases of non-L. pneumophila species or non-sg 1 infection were examined using the Binax EIA and Biotest EIA kits. For both groups, the overall diagnostic sensitivity of the Binax kit was significantly better than the sensitivity of the Biotest kits (P < 0.0001). For the non-Pontiac group, the sensitivity was 81.8 and 42.4%, respectively, and for the non-sg1 group, it was 51.4 and 28.6%, respectively. It was concluded that the Binax kit was more suitable for the general diagnosis of LD than the Biotest kit, but we still need urinary antigen detection methods with higher sensitivity for non-sg1 LD.

Antigen detection systems

USDA-ARS?s Scientific Manuscript database

Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

Oral administration of erythrocyte membrane antigen does not suppress anti-Rh(D) antibody responses in humans.

PubMed Central

Barnes, R M; Duguid, J K; Roberts, F M; Risk, J M; Johnson, P M; Finn, R; Hardy, J; Napier, J A; Clarke, C A

1987-01-01

The effects of prior oral administration of erythrocyte membrane preparations (Oral Rh antigen) on the serum anti-Rh(D) antibody response has been evaluated in non-sensitized Rh(D)-negative male volunteers, and in female volunteers sensitized previously by Rh(D)-positive fetal blood during pregnancy. Sixty-one percent (11/18) of males who received oral Rh antigen (either D-positive or D-negative) before intravenous challenge with Rh(D)-positive cells produced detectable antibodies; of these 11, six received oral Rh(D)-negative antigen and five received oral Rh(D)-positive antigen. Seventy-two percent (13/18) of control males, who had received no prior oral Rh antigen, produced antibodies following challenge with Rh(D)-positive cells. Three out of six pre-sensitized females who received oral D-positive or D-negative Rh antigen for 4 weeks, but without intravenous challenge, increased their anti-Rh(D) antibody levels which peaked after 11-18 weeks: two had received Rh(D)-positive antigen, and one Rh(D)-negative antigen. These data indicate that administration of oral Rh antigen before parenteral immunization does not significantly suppress the anti-Rh(D) antibody response. Indeed, oral administration of either Rh(D)-positive or Rh(D)-negative antigen can boost systemic antibody in pre-sensitized females. These results do not support the rationale of treating Rh-sensitized pregnant women with oral Rh antigen. PMID:3113783

Multi-centre evaluation of the Determine HIV Combo assay when used for point of care testing in a high risk clinic-based population.

PubMed

Conway, Damian P; Holt, Martin; McNulty, Anna; Couldwell, Deborah L; Smith, Don E; Davies, Stephen C; Cunningham, Philip; Keen, Phillip; Guy, Rebecca

2014-01-01

Determine HIV Combo (DHC) is the first point of care assay designed to increase sensitivity in early infection by detecting both HIV antibody and antigen. We conducted a large multi-centre evaluation of DHC performance in Sydney sexual health clinics. We compared DHC performance (overall, by test component and in early infection) with conventional laboratory HIV serology (fourth generation screening immunoassay, supplementary HIV antibody, p24 antigen and Western blot tests) when testing gay and bisexual men attending four clinic sites. Early infection was defined as either acute or recent HIV infection acquired within the last six months. Of 3,190 evaluation specimens, 39 were confirmed as HIV-positive (12 with early infection) and 3,133 were HIV-negative by reference testing. DHC sensitivity was 87.2% overall and 94.4% and 0% for the antibody and antigen components, respectively. Sensitivity in early infection was 66.7% (all DHC antibody reactive) and the DHC antigen component detected none of nine HIV p24 antigen positive specimens. Median HIV RNA was higher in false negative than true positive cases (238,025 vs. 37,591 copies/ml; p = 0.022). Specificity overall was 99.4% with the antigen component contributing to 33% of false positives. The DHC antibody component detected two thirds of those with early infection, while the DHC antigen component did not enhance performance during point of care HIV testing in a high risk clinic-based population.

Highly Sensitive and Automated Surface Enhanced Raman Scattering-based Immunoassay for H5N1 Detection with Digital Microfluidics.

PubMed

Wang, Yang; Ruan, Qingyu; Lei, Zhi-Chao; Lin, Shui-Chao; Zhu, Zhi; Zhou, Leiji; Yang, Chaoyong

2018-04-17

Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface enhanced Raman scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated, and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4-MBA) were synthesized with a core@shell nanostructure and showed strong signals, good uniformity, and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads-antibody-antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (<1 h) and lower reagent consumption (∼30 μL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases.

Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis.

PubMed

Tiwari, Dileep; Haque, Shafiul; Tiwari, Ram P; Jawed, Arshad; Govender, Thavendran; Kruger, Hendrik G

2017-04-01

A rapid and efficient diagnostic test was developed for the detection of Mycobacterium tuberculosis antigens in serum samples of active tuberculosis (TB) and extrapulmonary TB patients via a liposomal agglutination-based method. A rapid card test has been developed to facilitate the recognition of high-affinity binding rabbit raised purified culture filtrate protein antibodies coupled on the surface of activated liposomal preparation. In the presence of TB antigens, the polyclonal antibodies bound to the liposomal particles demonstrate a visible agglutination reaction. The developed assay was simple, rapid, reliable, sensitive, and specific as a diagnostic test for the detection of antigens in serum samples of clinically confirmed cases of TB within 4-5 minutes' duration. The test was evaluated at different hospitals, medical colleges, and pathology centers, and involved 1483 participants. This investigation was conducted to detect the presence of these antigens during the period of active growth of the microorganism in serum samples for pulmonary TB and processed tissue biopsy for other extrapulmonary TB. Results obtained using this test were compared with acid-fast bacilli smear and culture results. Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application. Copyright © 2015. Published by Elsevier B.V.

Determination of ABO blood grouping and Rhesus factor from tooth material.

PubMed

Kumar, Pooja Vijay; Vanishree, M; Anila, K; Hunasgi, Santosh; Suryadevra, Sri Sujan; Kardalkar, Swetha

2016-01-01

The aim of the study was to determine blood groups and Rhesus factor from dentin and pulp using absorption-elution (AE) technique in different time periods at 0, 3, 6, 9 and 12 months, respectively. A total of 150 cases, 30 patients each at 0, 3, 6, 9 and 12 months were included in the study. The samples consisted of males and females with age ranging 13-60 years. Patient's blood group was checked and was considered as "control." The dentin and pulp of extracted teeth were tested for the presence of ABO/Rh antigen, at respective time periods by AE technique. Data were analyzed in proportion. For comparison, Chi-square test or Fisher's exact test was used for the small sample. Blood group antigens of ABO and Rh factor were detected in dentin and pulp up to 12 months. For both ABO and Rh factor, dentin and pulp showed 100% sensitivity for the samples tested at 0 month and showed a gradual decrease in the sensitivity as time period increased. The sensitivity of pulp was better than dentin for both the blood grouping systems and ABO blood group antigens were better detected than Rh antigens. In dentin and pulp, the antigens of ABO and Rh factor were detected up to 12 months but showed a progressive decrease in the antigenicity as the time period increased. When compared the results obtained of dentin and pulp in ABO and Rh factor grouping showed similar results with no statistical significance. The sensitivity of ABO blood grouping was better than Rh factor blood grouping and showed a statistically significant result.

Use of the Abbott Architect HIV antigen/antibody assay in a low incidence population.

PubMed

Dubravac, Terry; Gahan, Thomas F; Pentella, Michael A

2013-12-01

With the availability of 4th generation HIV diagnostic tests which are capable of detecting acute infection, Iowa evaluated the 3rd and 4th generation HIV test and compared the performance of these products in a low incidence population. This study was conducted to evaluate the performance of an HIV antigen/antibody combination (4th generation) assay compared to an EIA 3rd generation assay. Over a 4 month period, 2037 specimens submitted for HIV screening were tested by Bio-Rad GS HIV-1/HIV-2 Plus O EIA and the Abbott Architect i1000SR HIV Ag/Ab Combo. The performance characteristics of sensitivity, specificity, positive predictive value and negative predictive value were determined. Of the 2037 specimens tested, there were 13 (0.64%) true positives detected. None of the positive specimens were from patients in the acute phase of infection. The Abbott antigen/antibody combo assay had a sensitivity, specificity, positive-predictive value and negative predictive value of 100%, 99.85%, 81.25%, and 100% respectively. The Bio-Rad EIA assay had a sensitivity, specificity, positive-predictive value and negative predictive value of 100%, 99.80%, 76.47% and 100%, respectively. The EIA had four false positive results which tested negative by the antigen/antibody assay and western blot. In a low-incidence state where early infections are less commonly encountered, the EIA assay and the antigen/antibody assay performed with near equivalency. The antigen/antibody assay had one less false positive result. While no patients were detected in the acute stage of infection, the use of the antigen/antibody assay presents the opportunity to detect an infected patient sooner and prevent transmission to others. Copyright © 2013 Elsevier B.V. All rights reserved.

[Diagnostic advantages of the test system "DS-EIA-HBsAg-0.01" for detection of HBV surface antigen].

PubMed

Egorova, N I; Pyrenkova, I Iu; Igolkina, S N; Sharipova, I N; Puzyrev, V F; Obriadina, A P; Burkov, A N; Kornienko, N V; Fields, H A; Korovkin, A S; Shalunova, N V; Bektemirov, T A; Kuznetsov, K V; Koshcheeva, N A; Ulanova, T I

2009-01-01

The new highly sensitive test system "DS-EIA-HBsAg-0.01" (Priority Certificate No. 2006129019 of August 10, 2006) in detecting hepatitis B surface antigen (HBsAg) was assessed. The sensitivity of the test was estimated using the federal standards sample HBsAg 42-28-311-06, panels' samples Boston Biomedica Inc. (West Bridgewater, Mass, USA) and ZeptoMetrix Corp. (Buffalo, NY, USA). The findings have indicated that "DS-EIA-HBsAg-0.01" is equally effective in detecting different subtypes of HBsAg during a seroconversion period earlier than alternative assays. Along with its high analytical and diagnostic sensitivity, the system shows a high diagnostic specificity.

Serodiagnosis of bovine leptospirosis by IgG-enzyme-linked immunosorbent assay and latex agglutination test.

PubMed

Senthilkumar, T M A; Subathra, M; Ramadass, P; Ramaswamy, V

2010-02-01

The efficacy of a recombinant leptospiral outer membrane protein LipL41 as an antigen for conducting IgG-Enzyme linked immunosorbent assay (ELISA) and latex agglutination test (LAT) for serodiagnosis of bovine leptospirosis was evaluated. The recombinant LipL41 antigen developed and used for detecting the antibodies was specific in detection of the pathogenic serovars of Leptospira, as the expression of the LipL41 antigen is restricted only to pathogenic leptospires. A total of 430 bovine serum samples were subjected to IgG-ELISA and LAT, and the sensitivity and specificity were assessed in comparison with microscopic agglutination test (MAT). The sensitivity and specificity of IgG-ELISA and LAT were 86.84% and 93.16%, and 95.42% and 98.33% respectively. Both the tests are found to be sensitive, specific and concurred with the standard MAT. The study concluded that the rLipL41 protein could be used as a potential diagnostic antigen in different assay formats for bovine leptospirosis.

Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

PubMed Central

Bezek, D M; Baker, J C; Kaneene, J B

1988-01-01

A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood samples collected using ethylenediaminetetraacetic acid as an anticoagulant. Bovine viral diarrhea virus antigen was detected by immunofluorescence between 3 and 11 days postinfection in lymphocyte smears and 3 to 12 days postinfection in buffy coat smears. Isolation of virus from both lymphocytes and buffy coat preparations correlated with detection of immunofluorescence. Serum neutralizing antibody to bovine virus diarrhea virus was detected on day 10 postinfection. Buffy coat smears were as sensitive as lymphocyte smears for the detection of antigen by immunofluorescence. It appeared that immunofluorescent staining of white blood cells was an effective method of detecting bovine virus diarrhea viral antigen. PMID:2836047

Standardization of a latex agglutination test for coproantigen detection of Fasciola sp. in bovine cattle stool.

PubMed

Orejarena Ávila, Lina Paola; Inguilan Benavides, Erika Marcela; Padilla Sanabria, Leonardo; Recalde-Reyes, Delia Piedad; Rodríguez-Salazar, Carlos Andrés; Castaño-Osorio, Jhon Carlos

2018-03-01

Fasciolosis is a zoonotic parasitic disease, which affects humans and animals; diagnosed through noncommercial immunoassay tests that cannot be used on the field. Thereby, establishing the optimal conditions to develop a latex agglutination technique with IgG and IgM antibodies directed against excretion/secretion antigens of Fasciola sp. is a priority. Latex particles were sensitized with IgG and IgM antibodies directed against excretion/secretion antigens of Fasciola sp. The specificity of the antibodies was determined against antigens of different helminths and protozoa; the sensitivity and specificity of the test was evaluated against a previously standardized direct ELISA. The coupling rates of the IgG and IgM antibodies were 85.77 and 100%, respectively. The minimum detectable concentration of Fasciola sp. excretion/secretion antigens, diluted in a phosphate-buffered saline, was 1.589 mg/mL(IgG) and 0.158 mg/mL(IgM) and for the antigens incorporated in the bovine cattle stool it was 3.178 mg/mL(IgG) and 1.589 mg/mL(IgM). The test showed crossed reaction against Giardia sp., and Cryptosporidium sp. antigens. Agreement of the IgG and IgM latex test against the ELISA test was of 78.78 and 96.96%, respectively; the specificity found was of 100% for both tests and sensitivity was 78.79% (IgG) and 96.97% (IgM). This work standardized the latex agglutination technique to detect Fasciola sp. antigens in bovine cattle stool.

Evaluation of the bioNexia Legionella Test, Including Impact of Incubation Time Extension, for Detection of Legionella pneumophila Serogroup 1 Antigen in Urine.

PubMed

Badoux, Paul; Euser, Sjoerd M; Bruin, Jacob P; Mulder, Patrick P G; Yzerman, Ed P F

2017-06-01

In this study, we compared the bioNexia test (bioMérieux, Marcy-l'Étoile, France), a new immunochromatographic assay for the detection of Legionella pneumophila serogroup 1 in urine, with the BinaxNOW urinary antigen test (Alere, Waltham, Massachusetts, USA). After 15 min of incubation (in accordance with the manufacturers' instructions), the sensitivities and specificities were, respectively, 76.5% and 97.2% for the bioNexia test and 87.1% and 100% for the BinaxNOW test. After a prolonged incubation time of 60 min, the sensitivities and specificities increased to, respectively, 89.4% and 97.2% for the bioNexia test and 91.8% and 100% for the BinaxNOW test. When the tests were read after 15 min, the concentration of discrepant urine samples increased the sensitivities to 94.1% for both tests. In conclusion, we found that although the bioNexia test showed lower sensitivity for the detection of L. pneumophila antigen in nonconcentrated urine compared to the BinaxNOW test, a prolonged incubation time as well as the use of concentrated samples showed comparable sensitivities for both tests. Copyright © 2017 American Society for Microbiology.

Porous silicon photoluminescence biosensor for rapid and sensitive detection of toxins

NASA Astrophysics Data System (ADS)

Melnyk, Yulia; Pavlova, Karyna; Myndrul, Valerii; Viter, Roman; Smyntyna, Valentyn; Iatsunskyi, Igor

2017-08-01

A rapid and low cost photoluminescence (PL) immunosensor for the determination of low concentrations of Ochratoxin A(OTA) and Aflatoxine B1 (AfB1) has been developed. This biosensor was based on porous silicon (PSi) fabricated by metal-assisted chemical etching (MACE) and modified by antibodies against OTA/AfB1 (anti-OTA/anti-AfB1). Biofunctionalization method of the PSi surface by anti-OTA/ anti-AfB1 was developed. The changes of the PL intensity after interaction of the immobilized anti-OTA/anti-AfB1with OTA/AfB1 antigens were used as biosensor signal, allowing sensitive and selective detection of OTA/AfB1 antigens in BSA solution. The sensitivity of the reported optical biosensor towards OTA/AfB1 antigens is in the range from 10-3 to 102 ng/ml.

Monoclonal antibody passive hemagglutination and capture enzyme-linked immunosorbent assays for direct detection and quantitation of F41 and K99 fimbrial antigens in enterotoxigenic Escherichia coli.

PubMed Central

Raybould, T J; Crouch, C F; Acres, S D

1987-01-01

Production of diarrhea in neonatal calves by enterotoxigenic Escherichia coli depends on its ability to attach to the epithelial cells of the intestine via surface adhesins called pili or fimbriae and to secrete enterotoxins. The most important of these fimbriae are designated K99 and F41. We produced and characterized a murine monoclonal antibody specific to F41. This monoclonal antibody and a K99-specific monoclonal antibody were used to develop sensitive and specific passive hemagglutination and capture enzyme-linked immunosorbent assays (ELISAs) for detection and quantitation of F41 and K99 antigens in E. coli cultures and culture supernatants. The capture ELISA systems exhibited excellent sensitivity and specificity, whereas the passive hemagglutination systems appeared to be oversensitive. The ability of the capture ELISAs to detect K99 and F41 fimbrial antigens in fecal specimens from calves was evaluated. Fimbrial antigens were detected in six of six specimens from scouring calves but not in four of four specimens from nonscouring calves. PMID:2880866

Development of Two FhSAP2 Recombinant–Based Assays for Immunodiagnosis of Human Chronic Fascioliasis

PubMed Central

Shin, Sun Hee; Hsu, Angel; Chastain, Holly M.; Cruz, Lorna A.; Elder, Eric S.; Sapp, Sarah G. H.; McAuliffe, Isabel; Espino, Ana M.; Handali, Sukwan

2016-01-01

In the United States, infection with Fasciola hepatica has been identified as an emerging disease, primarily in immigrants, refugees, and travelers. The laboratory test of choice for diagnosis of fascioliasis is detection of disease specific antibodies, most commonly uses excretory-secretory antigens for detection of IgG antibodies. Recently, recombinant proteins such as F. hepatica antigen (FhSAP2) have been used to detect IgG antibodies. The glutathione S-transferase (GST)–FhSAP2 recombinant antigen was used to develop Western blot (WB) and fluorescent bead-based (Luminex) assays to detect F. hepatica total IgG and IgG4 antibodies. The sensitivity and specificity of GST-FhSAP2 total IgG and IgG4 WB were similar at 94% and 98%, respectively. For the IgG Luminex assay, the sensitivity and specificity were 94% and 97%, and for the IgG4, the values were 100% and 99%, respectively. In conclusion, the GST-FhSAP2 antigen performs well in several assay formats and can be used for clinical diagnosis. PMID:27549636

Development of Two FhSAP2 Recombinant-Based Assays for Immunodiagnosis of Human Chronic Fascioliasis.

PubMed

Shin, Sun Hee; Hsu, Angel; Chastain, Holly M; Cruz, Lorna A; Elder, Eric S; Sapp, Sarah G H; McAuliffe, Isabel; Espino, Ana M; Handali, Sukwan

2016-10-05

In the United States, infection with Fasciola hepatica has been identified as an emerging disease, primarily in immigrants, refugees, and travelers. The laboratory test of choice for diagnosis of fascioliasis is detection of disease specific antibodies, most commonly uses excretory-secretory antigens for detection of IgG antibodies. Recently, recombinant proteins such as F. hepatica antigen (FhSAP2) have been used to detect IgG antibodies. The glutathione S-transferase (GST)-FhSAP2 recombinant antigen was used to develop Western blot (WB) and fluorescent bead-based (Luminex) assays to detect F. hepatica total IgG and IgG 4 antibodies. The sensitivity and specificity of GST-FhSAP2 total IgG and IgG 4 WB were similar at 94% and 98%, respectively. For the IgG Luminex assay, the sensitivity and specificity were 94% and 97%, and for the IgG 4 , the values were 100% and 99%, respectively. In conclusion, the GST-FhSAP2 antigen performs well in several assay formats and can be used for clinical diagnosis. © The American Society of Tropical Medicine and Hygiene.

Femtogram Mass Biosensor Using Self-Sensing Cantilever for Allergy Check

NASA Astrophysics Data System (ADS)

Sone, Hayato; Ikeuchi, Ayumi; Izumi, Takashi; Okano, Haruki; Hosaka, Sumio

2006-03-01

A self-sensing mass biosensor with a femtogram mass sensitivity has been developed using a piezoresistive microcantilever. The mass change due to antigen and antibody adsorption on the cantilever in water was detected by the resonance frequency shift of the cantilever. We constructed a prototype harmonic vibration sensor using a commercial piezoresistive cantilever, Wheatstone bridge circuits, a positive feedback controller, an exciting piezoactuator and a phase-locked loop (PLL) demodulator. As experimental results, a mass sensitivity of about 190 fg/Hz, and a mass resolution of about 500 fg were obtained in water. The mass sensitivity is 100 times higher than that of a quartz crystal oscillation method. We demonstrated that the sensor can detect the reaction between an antibody of immunoglobulin (IgG) and an antigen of egg albumen (OVA). We confirmed that the binding ratio between the antibody and the antigen was about 1 : 2. The detection method is available for allergy check because the measured reaction ratio occurring on the cantilever concurs with the theoretical method.

Spectra analysis of coating antigen: A possible explanation for difference in anti-AFB1 polyclonal antibody sensitivity

NASA Astrophysics Data System (ADS)

Ye, Yang; Liu, Aiping; Wang, Xiaohong; Chen, Fusheng

2016-10-01

For the detection of small hapten molecules, indirect competitive enzyme-linked immunosorbent assay (icELISA) is a preferred method. However, diverse coating antigen might bring different antiserum titer and sensitivity for the identical antiserum. In the present study, four AFB1-protein (aflatoxin B1-carrier protein) conjugates were prepared by activated ester method (AFB1O-BSA/AFB1O-OVA) and mannich method (AFB1-cBSA/AFB1-cOVA), and then applied as coating antigen for titer and sensitivity detection of the identical antiserum obtained from rabbit immunized by AFB1-KLH. Afterwards, the ultraviolet-visible, fluorescence and far-ultraviolet circular dichroism (far-UV CD) spectra were recorded for understanding the difference in titer and sensitivity obtained. Results revealed that AFB1O-BSA/AFB1O-OVA showed a strong intrinsic fluorescence band centered at 450 nm that originated from the emission of AFB1, which differed from AFB1-cBSA/AFB1-cOVA, while the decrease of α-helical and increase of β-sheet in AFB1-cBSA was the most remarkable. This indicated that the better sensitivity obtained by using AFB1O-BSA as coating antigen might be caused by its extended structure, because such structure affect the binding between AFB1 and antibody. The study might offer structural information for understanding the titer and sensitivity difference caused by coating antigen.

Evaluation of a monoclonal antibody-based latex agglutination test for diagnosis of cryptococcosis: comparison with two tests using polyclonal antibodies.

PubMed Central

Temstet, A; Roux, P; Poirot, J L; Ronin, O; Dromer, F

1992-01-01

Cryptococcal antigen detection has become a routine biological test performed for patients with AIDS. The poor prognosis of cryptococcosis explains the need for reliable tests. We evaluated the performances of a newly commercialized agglutination test that uses a monoclonal antibody specific for cryptococcal capsular polysaccharide (Pastorex Cryptococcus; Sanofi-Diagnostics Pasteur, Marnes-la-Coquette, France) and compared them with those of tests that use polyclonal immune sera (Cryptococcal Antigen Latex Agglutination System, Meridian Diagnostics, Inc., Cincinnati, Ohio; and Crypto-LA, International Biological Labs Inc., Cranbury, N.J.). The sensitivities and specificities of the tests were compared by using purified polysaccharides and yeast suspensions. Clinical specimens (131 serum samples, 41 cerebrospinal fluid samples, 34 urine samples, and 19 bronchoalveolar lavage samples) from 87 human immunodeficiency virus-positive subjects with (40 patients) and without (47 patients) culture-proven cryptococcosis were retrospectively tested during a blinded study. The effect of pronase treatment of samples was assessed for Pastorex Cryptococcus and the Cryptococcal Antigen Latex Agglutination System, and the antigen titers were compared. Our results show that (i) during the screening, concordance among the three tests was 97%; (ii) the use of pronase enhanced both the sensitivities and specificities of the Pastorex Cryptococcus test; (iii) titers agreed for 67% of the cerebrospinal fluid samples and 60% of the serum samples; and (iv) cryptococcosis was detected equally well with Pastorex Cryptococcus and with the other tests, whatever the infecting serotype (A, B, or D). The meaning of in vitro sensitivity and the relationship between titers and sensitivity are discussed. The results show that Pastorex Cryptococcus is a rapid and reliable test for the detection of cryptococcal antigen in body fluids and suggest that kits cannot be used interchangeably to monitor antigen titers in patients. PMID:1400951

Determination of ABO blood grouping and Rhesus factor from tooth material

PubMed Central

Kumar, Pooja Vijay; Vanishree, M; Anila, K; Hunasgi, Santosh; Suryadevra, Sri Sujan; Kardalkar, Swetha

2016-01-01

Objective: The aim of the study was to determine blood groups and Rhesus factor from dentin and pulp using absorption-elution (AE) technique in different time periods at 0, 3, 6, 9 and 12 months, respectively. Materials and Methods: A total of 150 cases, 30 patients each at 0, 3, 6, 9 and 12 months were included in the study. The samples consisted of males and females with age ranging 13–60 years. Patient's blood group was checked and was considered as “control.” The dentin and pulp of extracted teeth were tested for the presence of ABO/Rh antigen, at respective time periods by AE technique. Statistical Analysis: Data were analyzed in proportion. For comparison, Chi-square test or Fisher's exact test was used for the small sample. Results: Blood group antigens of ABO and Rh factor were detected in dentin and pulp up to 12 months. For both ABO and Rh factor, dentin and pulp showed 100% sensitivity for the samples tested at 0 month and showed a gradual decrease in the sensitivity as time period increased. The sensitivity of pulp was better than dentin for both the blood grouping systems and ABO blood group antigens were better detected than Rh antigens. Conclusion: In dentin and pulp, the antigens of ABO and Rh factor were detected up to 12 months but showed a progressive decrease in the antigenicity as the time period increased. When compared the results obtained of dentin and pulp in ABO and Rh factor grouping showed similar results with no statistical significance. The sensitivity of ABO blood grouping was better than Rh factor blood grouping and showed a statistically significant result. PMID:27721625

Comprehensive Review of Human Sapoviruses

PubMed Central

Katayama, Kazuhiko; Saif, Linda J.

2015-01-01

SUMMARY Sapoviruses cause acute gastroenteritis in humans and animals. They belong to the genus Sapovirus within the family Caliciviridae. They infect and cause disease in humans of all ages, in both sporadic cases and outbreaks. The clinical symptoms of sapovirus gastroenteritis are indistinguishable from those caused by noroviruses, so laboratory diagnosis is essential to identify the pathogen. Sapoviruses are highly diverse genetically and antigenically. Currently, reverse transcription-PCR (RT-PCR) assays are widely used for sapovirus detection from clinical specimens due to their high sensitivity and broad reactivity as well as the lack of sensitive assays for antigen detection or cell culture systems for the detection of infectious viruses. Sapoviruses were first discovered in 1976 by electron microscopy in diarrheic samples of humans. To date, sapoviruses have also been detected from several animals: pigs, mink, dogs, sea lions, and bats. In this review, we focus on genomic and antigenic features, molecular typing/classification, detection methods, and clinical and epidemiological profiles of human sapoviruses. PMID:25567221

[Comparative research into sensitivity and specificity of immune-enzyme analysis with chemiluminescence and colorimetric detection for detecting antigens and antibodies to avian influenza viruses and newcastle disease].

PubMed

Vitkova, O N; Kapustina, T P; Mikhailova, V V; Safonov, G A; Vlasova, N N; Belousova, R V

2015-01-01

The goal of this work was to demonstrate the results of the development of the enzyme-linked immunosorbent tests with chemiluminescence detection and colorimetric detection of specific viral antigens and antibodies for identifying the avian influenza and the Newcastle disease viruses: high sensitivity and specificity of the immuno- chemiluminescence assay, which are 10-50 times higher than those of the ELISA colorimetric method. The high effectiveness of the results and the automation of the process of laboratory testing (using a luminometer) allow these methods to be recommended for including in primary screening tests for these infectious diseases.

Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

PubMed

Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

2015-05-01

Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the 30 sera of known genotypes. The antigens did not detect antibodies to genotype-3a, but detected antibodies to all genotypes and did not discriminate them genotype wise. A panel of 175 of HCV-suspected serum samples was subjected to comparative analysis with our in-house ELISA assay and with commercial HCV screening assays. After subjecting the results to the formulas for determining the quality parameters, immunoblot assay had 100% sensitivity and specificity, while the ELISA assay had 100% sensitivity and 98.8% specificity as compared to commercially available assays. This study indicates that a mixture of Core and E2 antigens are potentially valuable antigens and there is the possibility of developing serological assays for monitoring HCV infection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Serodiagnosis of parasitic diseases.

PubMed Central

Maddison, S E

1991-01-01

In this review on serodiagnosis of parasitic diseases, antibody detection, antigen detection, use of monoclonal antibodies in parasitic serodiagnosis, molecular biological technology, and skin tests are discussed. The focus at the Centers for Disease Control on developing improved antigens, a truly quantitative FAST-enzyme-linked immunosorbent assay, and the very specific immunoblot assays for antibody detection is highlighted. The last two assays are suitable for field studies. Identification of patient response in terms of immunoglobulin class or immunoglobulin G subclass isotypes or both is discussed. Immunoglobulin isotypes may asist in defining the stage of some diseases. In other instances, use of a particular anti-isotype conjugate may increase the specificity of the assay. Monoclonal antibodies have played important roles in antigen purification and identification, in competitive antibody assays with increased sensitivity and specificity, and in assays for antigen detection in serum, body fluids, or excreta. Molecular biological technology has allowed significant advances in the production of defined parasitic serodiagnostic antigens. PMID:1747862

Evaluation of ascitic soluble human leukocyte antigen-G for distinguishing malignant ascites from benign ascites.

PubMed

Sun, Juan; Chang, Yan-Xiang; Niu, Chun-Yan

2017-11-01

The overexpression of soluble human leukocyte antigen-G is associated with malignant tumours. The purpose of our study was to detect soluble human leukocyte antigen-G concentrations in ascites and to evaluate the value of ascitic soluble human leukocyte antigen-G for the diagnosis of malignant ascites. Enzyme-linked immunosorbent assay was used to detect soluble human leukocyte antigen-G levels in 64 patients with malignant ascites and 30 patients with benign ascites. Receiver operating characteristic curves were used to evaluate the diagnostic efficacy of ascitic soluble human leukocyte antigen-G for the detection of malignant ascites. Ascitic soluble human leukocyte antigen-G levels were significantly higher in the malignant ascites group than in the benign ascites group (20.718 ± 3.215 versus 12.467 ± 3.678 µg/L, t = 7.425, p < 0.001). The area under the receiver operating characteristic curve for ascitic soluble human leukocyte antigen-G was 0.957 (95% confidence interval, 0.872-0.992). At a cut-off value of 19.60 µg/L, the sensitivity and specificity of ascitic soluble human leukocyte antigen-G were 87.5% (95% confidence interval, 71.0%-96.5%) and 100% (95% confidence interval, 88.4%-100%), respectively. With respect to area under the receiver operating characteristic curve, sensitivity and specificity, ascitic carcinoembryonic antigen (0.810, 68.75% and 83.33%, respectively) and carbohydrate antigen 19-9 (0.710, 65.63% and 70%, respectively) significantly differed (all p < 0.05). In malignant ascites that were cytology-negative and biopsy-positive, the rate of positivity for ascitic soluble human leukocyte antigen-G was 75%, which was higher than the corresponding rates for ascitic carcinoembryonic antigen (31.25%) and carbohydrate antigen 19-9 (6.25%; both p < 0.05). In conclusion, The detection of ascitic soluble human leukocyte antigen-G exhibited good performance for diagnosing malignant ascites, and particularly those that were cytology-negative and biopsy-positive.

Detection of fish antigens aerosolized during fish processing using newly developed immunoassays.

PubMed

Lopata, Andreas L; Jeebhay, Mohamed F; Reese, Gerald; Fernandes, Joshua; Swoboda, Ines; Robins, Thomas G; Lehrer, Samuel B

2005-09-01

Aerosolization of fish proteins during seafood processing has been identified as a potential route for allergic sensitization and occupational asthma among workers involved in high-risk activities. The aim of this study was to develop immunological assays for the quantification of aerosolized fish antigens in a fish-processing factory. Polyclonal antibodies to the main fish species processed in the factory (anchovy and pilchard) were generated in rabbits and compared by ELISA inhibition assay and immunoblotting. These antisera were utilized to develop ELISA assays for the detection of fish antigens. The ELISA inhibition assays were evaluated by analyzing environmental air samples collected from three areas in a fish-processing factory: pilchard canning, fish meal production and lobster processing. By immunoblotting, the rabbit polyclonal antibodies demonstrated IgG antibody binding patterns comparable with IgE antibodies of fish-sensitized patients, particularly in regard to the major fish allergens parvalbumins. The sensitivity of the fish-specific ELISA assays developed was 0.5 microg/ml. The ELISA inhibition assays were able to differentiate between the two different fish species of interest but did not recognize a crustacean species. Notable differences in exposure levels to canned pilchard and anchovy antigens were demonstrated in the three different working areas of the factory, with assays having a detection limit as low as 105 ng/m(3). These ELISA-based assays are sensitive and specific to quantify differential exposure levels to fish antigens produced during fish processing, making it possible to investigate exposure-disease response relationships among workers in this industry. Copyright (c) 2005 S. Karger AG, Basel.

Comparative performance of aldolase and lactate dehydrogenase rapid diagnostic tests in Plasmodium vivax detection

PubMed Central

2014-01-01

Background Misdiagnosis of malaria by commercial rapid diagnostic tests (RDTs) is a major cause of concern in the diagnosis of malaria. This retrospective study was aimed at assessing the relative performance of four RDTs with emphasis on the detection of two Plasmodium vivax antigens: aldolase and lactate dehydrogenase (LDH). Methods Three commercially available Plasmodium LDH or aldolase antigen detection kits (One Step Malaria P.f/P.v, ParaHit Total ver. 1.0, SD Bioline Malaria) and an anti-P. vivax aldolase-specific monoclonal antibody (mAb) pair 1C3-12 F10 were evaluated with P. vivax positive as well as non-P. vivax samples and healthy samples using blood smear examination as standard. Each test was read according to the manufacturer’s instructions. Results MAb 1C3-12 F10 pair targeting P. vivax-specific aldolase exhibited very good specificity and sensitivity of 100 and 97.4%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of 100 and 99.5%, respectively, were also observed. The anti-P. vivax LDH in the One-Step Malaria P.f/P.v test showed sensitivity, specificity, PPV and NPV of 93.5, 98.0, 88.9 and 98.8%, respectively. ParaHit Total ver. 1.0 targeting the pan-aldolase antigen showed sensitivity, specificity of 97.4 and 99.6%, respectively. PPV and NPV were both 99.5%. SD Bioline had sensitivity, specificity, PPV and NPV of 93.5, 100, 100 and 98.8%, respectively. The overall sensitivity and specificity of all four RDTs were acceptable, especially for the aldolase detection tests. Five (6.5%) of the P. vivax-positive samples (n = 77) that were confirmed by microscopic examination as well as the two aldolase detection RDTs (mAb 1C3-12 F10 and ParaHit Total ver.1.0) were undetected by the two LDH detection RDTs (One Step Malaria P.f/P.v and SD Bioline). Similarly, two positive samples (2.6%) that were positively confirmed by the LDH detection RDTs were also undetected by the aldolase detection test kits. Conclusion Aldolase and LDH antigens perform differently in different P. vivax samples; hence there is a high risk of misdiagnosis when monoclonal antibodies are used against only one particular antigen in the test. A combination of both aldolase and LDH in RDTs for the rapid diagnosis of P. vivax will enhance the sensitivity of the assay and reduce misdiagnosis. PMID:25015737

Sensitivity and Specificity of Histoplasma Antigen Detection by Enzyme Immunoassay.

PubMed

Cunningham, Lauren; Cook, Audrey; Hanzlicek, Andrew; Harkin, Kenneth; Wheat, Joseph; Goad, Carla; Kirsch, Emily

2015-01-01

The objective of this study was to evaluate the sensitivity and specificity of an antigen enzyme immunoassay (EIA) on urine samples for the diagnosis of histoplasmosis in dogs. This retrospective medical records review included canine cases with urine samples submitted for Histoplasma EIA antigen assay between 2007 and 2011 from three veterinary institutions. Cases for which urine samples were submitted for Histoplasma antigen testing were reviewed and compared to the gold standard of finding Histoplasma organisms or an alternative diagnosis on cytology or histopathology. Sensitivity, specificity, negative predictive value, positive predictive value, and the kappa coefficient and associated confidence interval were calculated for the EIA-based Histoplasma antigen assay. Sixty cases met the inclusion criteria. Seventeen cases were considered true positives based on identification of the organism, and 41 cases were considered true negatives with an alternative definitive diagnosis. Two cases were considered false negatives, and there were no false positives. Sensitivity was 89.47% and the negative predictive value was 95.35%. Specificity and the positive predictive value were both 100%. The kappa coefficient was 0.9207 (95% confidence interval, 0.8131-1). The Histoplasma antigen EIA test demonstrated high specificity and sensitivity for the diagnosis of histoplasmosis in dogs.

Multiantigen Print Immunoassay for Comparison of Diagnostic Antigens for Taenia solium Cysticercosis and Taeniasis▿

PubMed Central

Handali, Sukwan; Klarman, Molly; Gaspard, Amanda N.; Noh, John; Lee, Yeuk-Mui; Rodriguez, Silvia; Gonzalez, Armando E.; Garcia, Hector H.; Gilman, Robert H.; Tsang, Victor C. W.; Wilkins, Patricia P.

2010-01-01

One of the best-characterized tests for the diagnosis of neurocysticercosis is the enzyme-linked immunoelectrotransfer blot assay, developed at the CDC, which uses lentil lectin-purified glycoproteins (LLGPs) extracted from Taenia solium cysticerci. The purification of the LLGP antigens has been difficult to standardize, and the polyacrylamide gel system used for the immunoblot assay is not easily transferable to other laboratories. In this study, we developed a multiantigen printing immunoassay (MAPIA) to compare the performance of multiple recombinant Taenia solium proteins with the potential for the detection of cysticercosis and taeniasis. We prepared MAPIA strips using six cysticercosis and two taeniasis diagnostic proteins and compared the performance of the proteins with sera collected from defined cysticercosis and taeniasis cases. Of the six cysticercosis antigens, rT24H performed well in detecting cases with two or more viable cysts in the brain (sensitivity and specificity, 97% and 99.4%, respectively); the use of a combination of cysticercosis antigens did not improve the sensitivity of the test and decreased the specificity. None of the antigens could differentiate the different clinical presentations of cysticercosis. Both of the taeniasis antigens (rES33 and rES38) had the same sensitivity of 99.4% and specificities of 93.9% and 94.5%, respectively. Some cross-reactivity against rES33 and rES38 was found, especially with sera from cases infected with Schistosoma mansoni. We conclude that MAPIA is a simple and effective tool that may be used to compare antibody responses to different cysticercosis and taeniasis antigens and, in this case, may be useful for the rapid detection of T. solium cases. PMID:19906893

Multiantigen print immunoassay for comparison of diagnostic antigens for Taenia solium cysticercosis and taeniasis.

PubMed

Handali, Sukwan; Klarman, Molly; Gaspard, Amanda N; Noh, John; Lee, Yeuk-Mui; Rodriguez, Silvia; Gonzalez, Armando E; Garcia, Hector H; Gilman, Robert H; Tsang, Victor C W; Wilkins, Patricia P

2010-01-01

One of the best-characterized tests for the diagnosis of neurocysticercosis is the enzyme-linked immunoelectrotransfer blot assay, developed at the CDC, which uses lentil lectin-purified glycoproteins (LLGPs) extracted from Taenia solium cysticerci. The purification of the LLGP antigens has been difficult to standardize, and the polyacrylamide gel system used for the immunoblot assay is not easily transferable to other laboratories. In this study, we developed a multiantigen printing immunoassay (MAPIA) to compare the performance of multiple recombinant Taenia solium proteins with the potential for the detection of cysticercosis and taeniasis. We prepared MAPIA strips using six cysticercosis and two taeniasis diagnostic proteins and compared the performance of the proteins with sera collected from defined cysticercosis and taeniasis cases. Of the six cysticercosis antigens, rT24H performed well in detecting cases with two or more viable cysts in the brain (sensitivity and specificity, 97% and 99.4%, respectively); the use of a combination of cysticercosis antigens did not improve the sensitivity of the test and decreased the specificity. None of the antigens could differentiate the different clinical presentations of cysticercosis. Both of the taeniasis antigens (rES33 and rES38) had the same sensitivity of 99.4% and specificities of 93.9% and 94.5%, respectively. Some cross-reactivity against rES33 and rES38 was found, especially with sera from cases infected with Schistosoma mansoni. We conclude that MAPIA is a simple and effective tool that may be used to compare antibody responses to different cysticercosis and taeniasis antigens and, in this case, may be useful for the rapid detection of T. solium cases.

[Clinical benefit of HCV core antigen assay in patients receiving interferon and ribavirin combination therapy].

PubMed

Higashimoto, Makiko; Takahashi, Masahiko; Jokyu, Ritsuko; Saito, Hidetsugu

2006-02-01

A highly sensitive second generation HCV core antigen assay has recently been developed. We compared viral disappearance and kinetics data between commercially available core antigen assays, Lumipulse Ortho HCV Ag, and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor Test, Version 2 to estimate the predictive benefit of sustained viral response (SVR) and non-SVR in 59 patients treated with interferon and ribavirin combination therapy. We found a good correlation between HCV core Ag and HCV RNA level regardless of genotype. Although the sensitivity of the core antigen assay was lower than PCR, the dynamic range was broader than that of the PCR assay, so that we did not need to dilute the samples in 59 patients. We detected serial decline of core Ag levels in 24 hrs, 7 days and 14 days after interferon combination therapy. The decline of core antigen levels was significant in SVR patients compared to non-SVR as well as in genotype 2a, 2b patients compared to 1b. Core antigen-negative on day 1 could predict all 10 SVR patients (PPV = 100%), whereas RNA-negative could predict 22 SVR out of 25 on day 14 (PPV = 88.0%). None of the patients who had detectable serum core antigen on day 14 became SVR(NPV = 100%), although NPV was 91.2% on RNA negativity. An easy, simple, low cost new HCV core antigen detecting system seems to be useful for assessing and monitoring IFN treatment for HCV.

Microfluidic immunosensor with integrated liquid core waveguides for sensitive Mie scattering detection of avian influenza antigens in a real biological matrix.

PubMed

Heinze, Brian C; Gamboa, Jessica R; Kim, Keesung; Song, Jae-Young; Yoon, Jeong-Yeol

2010-11-01

This work presents the use of integrated, liquid core, optical waveguides for measuring immunoagglutination-induced light scattering in a microfluidic device, towards rapid and sensitive detection of avian influenza (AI) viral antigens in a real biological matrix (chicken feces). Mie scattering simulations were performed and tested to optimize the scattering efficiency of the device through proper scatter angle waveguide geometry. The detection limit is demonstrated to be 1 pg mL(-1) in both clean buffer and real biological matrix. This low detection limit is made possible through on-chip diffusional mixing of AI target antigens and high acid content microparticle assay reagents, coupled with real-time monitoring of immunoagglutination-induced forward Mie scattering via high refractive index liquid core optical waveguides in close proximity (100 μm) to the sample chamber. The detection time for the assay is <2 min. This device could easily be modified to detect trace levels of any biological molecules that antibodies are available for, moving towards a robust platform for point-of-care disease diagnostics.

Tracking serum antibody response to viral antigens with arrayed imaging reflectometry

NASA Astrophysics Data System (ADS)

Mace, Charles R.; Rose, Robert C.; Miller, Benjamin L.

2009-02-01

Arrayed Imaging Reflectometry, or "AIR", is a new label-free technique for detecting proteins that relies on bindinginduced changes in the response of an antireflective coating on the surface of a silicon ship. Because the technique provides high sensitivity, excellent dynamic range, and readily integrates with standard silicon wafer processing technology, it is an exceptionally attractive platform on which to build systems for detecting proteins in complex solutions. In our early research, we used AIR chips bearing secreted receptor proteins from enteropathogenic E. coli to develop sensors for this pathogen. Recently, we have been exploring an alternative strategy: Rather than detecting the pathogen directly, can one immobilize antigens from a pathogen, and employ AIR to detect antibody responses to those antigens? Such a strategy would provide enhanced sensitivity for pathogen detection (as the immune system essentially amplifies the "signal" caused by the presence of an organism to which it responds), and would also potentially prove useful in the process of vaccine development. We describe herein preliminary results in the application of such a strategy to the detection of antibodies to human papillomavirus (HPV).

Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay.

PubMed

Turgeon, David K; Novicki, Thomas J; Quick, John; Carlson, LaDonna; Miller, Pat; Ulness, Bruce; Cent, Anne; Ashley, Rhoda; Larson, Ann; Coyle, Marie; Limaye, Ajit P; Cookson, Brad T; Fritsche, Thomas R

2003-02-01

Clostridium difficile is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for C. difficile toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for C. difficile detection with CBA: Prima System Clostridium difficile Tox A and VIDAS Clostridium difficile Tox A II, which detect C. difficile toxin A; Premier Cytoclone A/B and Techlab Clostridium difficile Tox A/B, which detect toxins A and B; and ImmunoCard Clostridium difficile and Triage Micro C. difficile panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.

Specific recognition of hydatid cyst antigens by serum IgG, IgE, and IgA using western blot.

PubMed

Sbihi, Y; Janssen, D; Osuna, A

1997-01-01

Diagnosis of hydatid disease in humans relies on the detection of specific antibodies against antigens of the metacestode from Echinococcus granulosus. The specificity and sensitivity of current immunological techniques based on specific serum IgG rely on the way antigens are purified. We used Western immunoblotting to detect specific IgG, IgE, and IgA antibodies in serum from patients with hydatid disease using either crude antigen preparations (total hydatid fluid), purified fractions enriched in Antigens 5 and B, and glycoproteins from hydatid fluid. Depending on whether crude HF or purified antigen fractions were used, IgG and IgE recognized specifically low-to-medium MW bands between 12 and 42 kDa. IgA recognized specifically 110 kDa band in crude hydatid fluid and in the glycoprotein fraction of hydatid fluid, and a 42 kDa band in all antigen samples used. Besides the advantage of detecting specific IgA in crude hydatid fluid, these results offer the possibility of simplifying future immunological tests if specific secretory IgA can be similarly detected.

Biconically tapered fiber optic probes for rapid label-free immunoassays.

PubMed

Miller, John; Castaneda, Angelica; Lee, Kun Ho; Sanchez, Martin; Ortiz, Adrian; Almaz, Ekrem; Almaz, Zuleyha Turkoglu; Murinda, Shelton; Lin, Wei-Jen; Salik, Ertan

2015-04-01

We report use of U-shaped biconically tapered optical fibers (BTOF) as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G) and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G)-antigen (rabbit anti-mouse IgG) reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method.

Evaluation of Gastrothylax crumenifer antigenic preparation in serodiagnosis of paramphistomiasis in sheep.

PubMed

Ahmad, Tariq; Reshi, Mohammad Latif; Cheshti, M Z; Tanveer, Syed; Shah, Zaffar Amin; Fomada, Bashir Ahmad; Raina, O K

2014-04-01

An evaluation of Gastrothylax crumenifer crude antigen preparation viz., Somatic Antigen (SAg), Excretory Secretory Antigen (ESAg) and Egg Antigen (EAg) in serodiagnosis of disease was undertaken. Test sera samples were obtained from 30 Paramphistomiasis Positive and 30 Gastrothylax free sheep slaughtered at Hazratbal Kashmir. The referral antigenic preparation were evaluated against Paramphistomiasis positive sera, via., control negative sera, using double immunodiffusion test (DID), (IEP) Immunoelectrophoretic assay and ELISA. The performance of referral antigens, as assessed from percent sensitivity and specificity, revealed an increasing trend from DID (Double immunodiffusion-An immunological technique used in the detection, identification and quantification of antibodies and antigens) to IEP (immunoelectrophoresis-A general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies), followed by ELISA, detecting higher number of sheep positive for paramphistomiasis. In ELISA the ESAg and SAg were evaluated as most reactive antigens with no significant difference and EAg was the least antigenic. In IEP, EAg had the higher sensitivity (60%) and analogous specificity of SAg and ESAg. The formation of the preceptin lines in the proximity to EAg containing wells (cathode end) in IEP was suggestive of higher molecular weight of G. crumenifer specific protein molecules with slower rate of migration. Purification and characterization of G. crumenifer and identification of specific antigenic molecules, particularly in EAg has been suggested for qualitative improvement of diagnostic value of the antigens in the tests used here in.

DELAYED HYPERSENSITIVITY

PubMed Central

Uhr, Jonathan W.; Salvin, S. B.; Pappenheimer, A. M.

1957-01-01

A general method for induction of the delayed hypersensitive state directed against single protein antigens is described. The method consists of intradermal injection of minute amounts of washed immune precipitates containing the antigen in question. Provided the specific precipitates are formed in the region of antibody excess, maximal sensitivity develops at least 2 to 3 weeks before detectable circulating antibody is formed in guinea pigs against the sensitizing antigen. Neither adjuvant nor killed acid-fast bacteria are required for induction of the delayed hypersensitive state although the degree of sensitization is considerably increased when the sensitizing material is incorporated in Freund's complete adjuvant. Characteristics of the "delayed" as opposed to the "immediate" hypersensitive states in the guinea pig are described and implications of the findings are discussed. PMID:13385403

Development and evaluation of a truncated recombinant NS3 antigen-based indirect ELISA for detection of pestivirus antibodies in sheep and goats.

PubMed

Kalaiyarasu, Semmannan; Mishra, Niranjan; Rajukumar, Katherukamem; Nema, Ram Kumar; Behera, Sthita Pragnya

2015-01-01

The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.

An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

PubMed Central

Jin, Chenggang; Roen, Diana R.; Lehmann, Paul V.; Kellermann, Gottfried H.

2013-01-01

Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-γ as a biomarker, we developed a new enzyme-linked immunospot method (iSpot LymeTM) to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease. PMID:24709800

Detecting West Nile virus in owls and raptors by an antigen-capture assay.

PubMed

Gancz, Ady Y; Campbell, Douglas G; Barker, Ian K; Lindsay, Robbin; Hunter, Bruce

2004-12-01

We evaluated a rapid antigen-capture assay (VecTest) for detection of West Nile virus in oropharyngeal and cloacal swabs, collected at necropsy from owls (N = 93) and raptors (N = 27). Sensitivity was 93.5%-95.2% for northern owl species but <42.9% for all other species. Specificity was 100% for owls and 85.7% for raptors.

Mass-tag enhanced immuno-laser desorption/ionization mass spectrometry for sensitive detection of intact protein antigens.

PubMed

Lorey, Martina; Adler, Belinda; Yan, Hong; Soliymani, Rabah; Ekström, Simon; Yli-Kauhaluoma, Jari; Laurell, Thomas; Baumann, Marc

2015-05-19

A new read-out method for antibody arrays using laser desorption/ionization-mass spectrometry (LDI-MS) is presented. Small, photocleavable reporter molecules with a defined mass called "mass-tags" are used for detection of immunocaptured proteins from human plasma. Using prostate specific antigen (PSA), a biomarker for prostate cancer, as a model antigen, a high sensitivity generic detection methodology based immunocapture with a primary antibody and with a biotin labeled secondary antibody coupled to mass-tagged avidin is demonstrated. As each secondary antibody can bind several avidin molecules, each having a large number of mass-tags, signal amplification can be achieved. The developed PSA sandwich mass-tag analysis method provided a limit of detection below 200 pg/mL (6 pM) for a 10 μL plasma sample, well below the clinically relevant cutoff value of 3-4 ng/mL. This brings the limit of detection (LOD) for detection of intact antigens with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) down to levels comparable to capture by anti-peptide antibodies selected reaction monitoring (SISCAPA SRM) and enzyme linked immunosorbent assay (ELISA), as 6 pM corresponds to a maximal amount of 60 amol PSA captured on-spot. We propose the potential use of LDI (laser desorption/ionization) with mass-tag read-out implemented in a sandwich assay format for low abundant and/or early disease biomarker detection.

Detection of anti-Yta antibodies using a sensitive and specific enzyme-linked immunosorbent assay.

PubMed

Geen, J; Hullin, D A; Hogg, S I

1999-01-01

A specific, sensitive and semi-quantitative enzyme-linked immunosorbent assay (ELISA) is described to detect anti-Yta antibodies in human serum. Recombinant acetylcholinesterase (AChE E.C.3.1.1.7) was employed as the coating antigen in the microtitre plate and horseradish peroxidase (HRP)-conjugated specific antibody (IgG) was used as the secondary antibody. The method developed showed excellent sensitivity, detecting a titre > 1 in 600,000 (3.5 ng/mL mouse IgG protein) for mouse monoclonal (mMAb) anti-AChE antibody. No cross-reaction was seen with other common blood group antibodies, confirming the specificity of the method. The recombinant antigen's AChE phenotype was confirmed as Yta, as no reaction was detected with anti-Ytb-positive sera. The ELISA method correlated closely with the established serological grading system used routinely in blood transfusion laboratories.

A self-amplified transistor immunosensor under dual gate operation: highly sensitive detection of hepatitis B surface antigen

NASA Astrophysics Data System (ADS)

Lee, I.-K.; Jeun, M.; Jang, H.-J.; Cho, W.-J.; Lee, K. H.

2015-10-01

Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor based on a self-amplified transistor under dual gate operation (immuno-DG ISFET) for the detection of hepatitis B surface antigen. To address the challenges in current ISFET-based immunosensors, we have enhanced the sensitivity of an immunosensor by precisely tailoring the nanostructure of the transistor. In the pH sensing test, the immuno-DG ISFET showed superior sensitivity (2085.53 mV per pH) to both standard ISFET under single gate operation (58.88 mV per pH) and DG ISFET with a non-tailored transistor (381.14 mV per pH). Moreover, concerning the detection of hepatitis B surface antigens (HBsAg) using the immuno-DG ISFET, we have successfully detected trace amounts of HBsAg (22.5 fg mL-1) in a non-diluted 1× PBS medium with a high sensitivity of 690 mV. Our results demonstrate that the proposed immuno-DG ISFET can be a biosensor platform for practical use in the diagnosis of various diseases.Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor based on a self-amplified transistor under dual gate operation (immuno-DG ISFET) for the detection of hepatitis B surface antigen. To address the challenges in current ISFET-based immunosensors, we have enhanced the sensitivity of an immunosensor by precisely tailoring the nanostructure of the transistor. In the pH sensing test, the immuno-DG ISFET showed superior sensitivity (2085.53 mV per pH) to both standard ISFET under single gate operation (58.88 mV per pH) and DG ISFET with a non-tailored transistor (381.14 mV per pH). Moreover, concerning the detection of hepatitis B surface antigens (HBsAg) using the immuno-DG ISFET, we have successfully detected trace amounts of HBsAg (22.5 fg mL-1) in a non-diluted 1× PBS medium with a high sensitivity of 690 mV. Our results demonstrate that the proposed immuno-DG ISFET can be a biosensor platform for practical use in the diagnosis of various diseases. Electronic supplementary information (ESI) available: Material preparation, surface functionalization and anti-HBsAg immobilization. See DOI: 10.1039/c5nr03146j

Improving dengue viral antigens detection in dengue patient serum specimens using a low pH glycine buffer treatment.

PubMed

Shen, Wen-Fan; Galula, Jedhan Ucat; Chang, Gwong-Jen J; Wu, Han-Chung; King, Chwan-Chuen; Chao, Day-Yu

2017-04-01

Early diagnosis of dengue virus (DENV) infection to monitor the potential progression to hemorrhagic fever can influence the timely management of dengue-associated severe illness. Nonstructural protein 1 (NS1) antigen detection in acute serum specimens has been widely accepted as an early diagnostic assay for dengue infection; however, lower sensitivity of the NS1 antigen-capture enzyme-linked immunosorbent assay (Ag-ELISA) in secondary dengue viral infection has been reported. In this study, we developed two forms of Ag-ELISA capable of detecting E-Ag containing virion and virus-like particles, and secreted NS1 (sNS1) antigens, respectively. The temporal kinetics of viral RNA, sNS1, and E-Ag were evaluated based on the in vitro infection experiment. Meanwhile, a panel of 62 DENV-2 infected patients' sera was tested. The sensitivity was 3.042 ng/mL and 3.840 ng/mL for sNS1 and E, respectively. The temporal kinetics of the appearance of viral RNA, E, NS1, and infectious virus in virus-infected tissue culture media suggested that viral RNAs and NS1 antigens could be detected earlier than E-Ag and infectious virus. Furthermore, a panel of 62 sera from patients infected by DENV Serotype 2 was tested. Treating clinical specimens with the dissociation buffer increased the detectable level of E from 13% to 92% and NS1 antigens from 40% to 85%. Inclusion of a low-pH glycine buffer treatment step in the commercially available Ag-ELISA is crucial for clinical diagnosis and E-containing viral particles could be a valuable target for acute DENV diagnosis, similar to NS1 detection. Copyright © 2015. Published by Elsevier B.V.

A comparative study of blood smear, QBC and antigen detection for diagnosis of malaria.

PubMed

Parija, S C; Dhodapkar, Rahul; Elangovan, Subashini; Chaya, D R

2009-01-01

Rapid diagnosis is prerequisite for effective treatment and reducing mortality and morbidity of malaria. This study was taken up to compare the efficacy of various methods available, i.e., thick and thin smear, quantitative buffy coat (QBC), plasmodium lactate dehydrogenase and aldolase in blood of patient. A total of 411 samples were collected from patients presenting with classic symptoms of malaria. For traditional microscopy; thick and thin smears were prepared and stained with Leishman's stain, taking thick smear as gold standard, thin smear had a sensitivity and specificity of 54.8% and 100%, respectively. QBC and antigen detection was done using commercially available kits; out of 411 samples, QBC and Malariagen were positive in 66 and 62 cases, with a sensitivity of 78% and 75%, respectively. Leishman's thick smear, although cost effective, is difficult to interpret for inexperienced microscopists; so if facilities are available, QBC should be used for routine diagnosis. In places where facilities are not available, rapid, simple and easy to interpret antigen detection test can be used despite low sensitivity.

Rapid, Sensitive, Enzyme-Immunodotting Assay for Detecting Cow Milk Adulteration in Sheep Milk: A Modern Laboratory Project

NASA Astrophysics Data System (ADS)

Inda, Luis A.; Razquín, Pedro; Lampreave, Fermín; Alava, María A.; Calvo, Miguel

1998-12-01

Specificity, sensitivity, and experimental simplicity make the immunoenzymatic assay suitable for a variety of laboratories dedicated to diverse activities such as research, quality control in food analysis, or clinical biochemistry. In these assays, the antibody that specifically recognizes the antigen is covalently attached to an enzyme. Once the antigen-antibody immunocomplex is formed, the enzymatic reaction gives a colored product that allows the detection of the initial antigen. The aim of this work was the design of a new laboratory project appropriate for use in courses of biochemistry, immunochemistry, or analytical chemistry. The assay described here detects the presence of cow milk in milk of other species. The main application is the detection of cow milk in sheep milk and cheese. Specific proteins, immunoglobulins (IgG) of the fraudulent bovine milk, are specifically recognized and retained by antibodies immobilized on a membrane. The binding of a second antibody covalently attached to horseradish peroxidase (HRP) allows the development of a visible signal. Thus, students can rapidly detect milk adulterations using a specific, sensitive, and safe experimental approach. The experiment allows students to apply their theoretical knowledge, resulting in a stimulating experience of solving a real problem during a 4-hour laboratory period.

RAPID DIAGNOSIS OF PNEUMOCOCCAL PNEUMONIA AMONG HIV-INFECTED ADULTS WITH URINE ANTIGEN DETECTION

PubMed Central

Boulware, David R; Daley, Charles L.; Merrifield, Cynthia; Hopewell, Philip C.; Janoff, Edward N.

2007-01-01

Objectives Streptococcus pneumoniae is the leading cause of bacterial pneumonia and associated bacteremia during HIV infection. Rapid diagnostic assays may limit inappropriate therapy. Methods Clinical signs and symptoms and sera and urine were collected prospectively from 70 adults with pneumococcal pneumonia, including 47 with HIV co-infection. Pneumococcal C-polysaccharide antigen was detected in urine using the Binax® immunochromatographic test (ICT). A systematic review of 24 published studies was conducted. Results Clinical symptoms, signs, and laboratory parameters except leukocytosis, were similar in HIV-infected and HIV-seronegative pneumonia. The performance of the urine antigen ICT was independent of HIV-status (sensitivity 81%, specificity 98%, positive (PPV) and negative predictive values (NPV) 98%, and 82%, respectively). The sensitivity of sputum Gram’s stain was 58% [34/59] with sputum unable to be provided by 16%. The CRP response was identical in HIV-infected (mean ± SD) 133 ± 88 vs. seronegative 135 ± 104 mg/L (p=0.9). In the systematic review, the ICT performance revealed 74% sensitivity (95% CI: 72% to 77%) and 94% specificity (95% CI: 93% to 95%). Urine antigen testing increases etiologic diagnosis by 23% (Range: 10% –59%) when testing adults with community acquired pneumonia of unknown etiology. Conclusions Urinary antigen detection provides a credible rapid diagnostic test for pneumococcal pneumonia regardless of HIV-status. CRP response to acute infection is similar in HIV co-infection and increases diagnostic certainty. PMID:17692384

Comparison of techniques for detecting antigens of Giardia lamblia and Cryptosporidium parvum in faeces.

PubMed Central

Tee, G H; Moody, A H; Cooke, A H; Chiodini, P L

1993-01-01

AIM--To compare the use of commercial monoclonal antibody test systems--the Giardia CEL IF test and the Crypto CEL IF test--for the detection of Giardia lamblia and Cryptosporidium parvum antigens in faeces with conventional techniques. METHODS--Sensitivity and specificity were evaluated using preparations of cysts of G lamblia and purified oocysts of C parvum. Evaluation of 59 random faecal samples passing through the Department of Clinical Parasitology, Hospital for Tropical Diseases, London, was carried out for both organisms. RESULTS--The fluorescence staining techniques proved more sensitive than other tests routinely used for diagnosis. PMID:8331181

Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1984 Annual Report.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaattari, Stephen L.

1985-06-01

The data presented here demonstrate that there is some variability to the antigenic structure of KDB. Although gel filtration of all antigenic preparations revealed a wide range of sizes for antigens, resolution on a denaturing gel revealed relatively few protein bands and immunological assays revealed the same (3) low number of antigens. It is of particular interest that there seems to be a protein of 60 kd in all preparations, but that there are not larger individual molecular species. This, in turn indicates that the larger molecular weight species detected in gel filtration are most likely aggregates or membrane fragmentsmore » composed of a lower molecular weight subunit. Use of ultrafiltration of KDM-2 medium appears to be successful in eliminating contamination of high molecular weight material found in KDM-2. There appears to be no alteration in the number of soluble antigens produced by growth in either medium, nor in the number of proteins, as detected by SDS-PAGE. However, soluble antigens isolated from UF-KDM-2 does appear to have greater heterogeneity in their isoelectric focusing (IEF) patterns than those from UF-KDM-2. Also, although there does appear to be an extended lag period in KDB growth on UF-KDM-2, there is no alteration in final O.D. or wet weight of cells. Thus, it appears that UF-KDM-2 may be an alternate medium for those wishing to isolate purified bacterial proteins or antigens. ELISA assays have been developed for the detection of soluble KDB antigens. This system is currently being developed as a sensitive measure of the presence of soluble antigen in serum and tissues of fish. Such a sensitive assay may also allow for the detection of KD+ spawners by the testing of ovarian fluid or serum. ELISA assays have also been developed to detect antibodies to soluble and cellular antigens of KDB. These systems have been proven successful in the detection of rabbit and murine monoclonal antibodies against KDB antigens. Future work will develop the use of anti-fish immunoglobulin (Ig) reagents to detect the presence of fish antibodies to KDB. This would be an extremely useful tool to be used in monitoring the immune response of salmon to the various test vaccines. The various antigens characterized in this study, along with whole KDB cells are currently being conjugated to various immunopotentiating agents. Testing of these prototype vaccines is currently under study.« less

Switching assay as a novel approach for specific antigen- antibody interaction analysis using magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Parr, M.; Illarionov, R.; Marchenko, Y.; Yakovleva, L.; Nikolaev, B.; Ischenko, A.; Shevtsov, M.

2016-08-01

Switching assay was applied for the detection of antigen-antibody interaction between 70-kDa heat shock protein (Hsp70) and anti-Hsp70 monoclonal antibodies in water solutions using conjugates with magnetic iron oxide nanoparticles (MNPs). Hsp70 is a ubiquitous intracellular protein that plays a crucial role in cancerogenesis and many other pathologies. Detection of the Hsp70 level in the biological fluids might have a prognostic and diagnostic value in clinic. The developed switch assay for the detection of Hsp70 demonstrated high sensitivity for antigen-antibody interaction analysis thus proving its potential for further preclinical and clinical studies.

Detection of Antigenic Variants of Subtype H3 Swine Influenza A Viruses from Clinical Samples

PubMed Central

Martin, Brigitte E.; Li, Lei; Nolting, Jacqueline M.; Smith, David R.; Hanson, Larry A.

2017-01-01

ABSTRACT A large population of genetically and antigenically diverse influenza A viruses (IAVs) are circulating among the swine population, playing an important role in influenza ecology. Swine IAVs not only cause outbreaks among swine but also can be transmitted to humans, causing sporadic infections and even pandemic outbreaks. Antigenic characterizations of swine IAVs are key to understanding the natural history of these viruses in swine and to selecting strains for effective vaccines. However, influenza outbreaks generally spread rapidly among swine, and the conventional methods for antigenic characterization require virus propagation, a time-consuming process that can significantly reduce the effectiveness of vaccination programs. We developed and validated a rapid, sensitive, and robust method, the polyclonal serum-based proximity ligation assay (polyPLA), to identify antigenic variants of subtype H3N2 swine IAVs. This method utilizes oligonucleotide-conjugated polyclonal antibodies and quantifies antibody-antigen binding affinities by quantitative reverse transcription-PCR (RT-PCR). Results showed the assay can rapidly detect H3N2 IAVs directly from nasal wash or nasal swab samples collected from laboratory-challenged animals or during influenza surveillance at county fairs. In addition, polyPLA can accurately separate the viruses at two contemporary swine IAV antigenic clusters (H3N2 swine IAV-α and H3N2 swine IAV-ß) with a sensitivity of 84.9% and a specificity of 100.0%. The polyPLA can be routinely used in surveillance programs to detect antigenic variants of influenza viruses and to select vaccine strains for use in controlling and preventing disease in swine. PMID:28077698

Detecting West Nile Virus in Owls and Raptors by an Antigen-capture Assay

PubMed Central

Campbell, Douglas G.; Barker, Ian K.; Lindsay, Robbin; Hunter, Bruce

2004-01-01

We evaluated a rapid antigen-capture assay (VecTest) for detection of West Nile virus in oropharyngeal and cloacal swabs, collected at necropsy from owls (N = 93) and raptors (N = 27). Sensitivity was 93.5%–95.2% for northern owl species but <42.9% for all other species. Specificity was 100% for owls and 85.7% for raptors. PMID:15663862

Comparison of Sofia Legionella FIA and BinaxNOW® Legionella urinary antigen card in two national reference centers.

PubMed

Beraud, L; Gervasoni, K; Freydiere, A M; Descours, G; Ranc, A G; Vandenesch, F; Lina, G; Gaia, V; Jarraud, S

2015-09-01

The Sofia Legionella Fluorescence Immunoassay (FIA; Quidel) is a recently introduced rapid immunochromatographic diagnostic test for Legionnaires' disease using immunofluorescence technology designed to enhance its sensitivity. The aim of this study was to evaluate its performance for the detection of urinary antigens for Legionella pneumophila serogroup 1 in two National Reference Centers for Legionella. The sensitivity and specificity of the Sofia Legionella FIA test were determined in concentrated and nonconcentrated urine samples, before and after boiling, in comparison with the BinaxNOW® Legionella Urinary Antigen Card (UAC; Alere). Compared with BinaxNOW® Legionella UAC, the sensitivity of the Sofia Legionella test was slightly higher in nonconcentrated urine samples and was identical in concentrated urine samples. The specificity of the Sofia Legionella FIA test was highly reduced by the concentration of urine samples. In nonconcentrated samples, a lack of specificity was observed in 2.3 % of samples, all of them resolved by heat treatment. The Sofia Legionella FIA is a sensitive test for detecting Legionella urinary antigens with no previous urine concentration. However, all positive samples have to be re-tested after boiling to reach a high specificity. The reading is automatized on the Sofia analyzer, which can be connected to laboratory information systems, facilitating accurate and rapid reporting of results.

Combining two serological assays optimises sensitivity and specificity for the identification of Streptococcus equi subsp. equi exposure.

PubMed

Robinson, Carl; Steward, Karen F; Potts, Nicola; Barker, Colin; Hammond, Toni-ann; Pierce, Karen; Gunnarsson, Eggert; Svansson, Vilhjálmur; Slater, Josh; Newton, J Richard; Waller, Andrew S

2013-08-01

The detection of anti-Streptococcus equi antibodies in the blood serum of horses can assist with the identification of apparently healthy persistently infected carriers and the prevention of strangles outbreaks. The aim of the current study was to use genome sequencing data to develop an indirect enzyme linked immunosorbent assay (iELISA) that targets two S. equi-specific protein fragments. The sensitivity and specificity of the antigen A and antigen C iELISAs were compared to an SeM-based iELISA marketed by IDvet - diagnostic Vétérinaire (IDvet). Individually, each assay compromised specificity in order to achieve sufficient sensitivity (SeM iELISA had a sensitivity of 89.9%, but a specificity of only 77.0%) or sensitivity to achieve high specificity. However, combining the results of the antigen A and antigen C iELISAs permitted optimisation of both sensitivity (93.3%) and specificity (99.3%), providing a robust assay for the identification of horses exposed to S. equi. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fabrication of antibody microarrays by light-induced covalent and oriented immobilization.

PubMed

Adak, Avijit K; Li, Ben-Yuan; Huang, Li-De; Lin, Ting-Wei; Chang, Tsung-Che; Hwang, Kuo Chu; Lin, Chun-Cheng

2014-07-09

Antibody microarrays have important applications for the sensitive detection of biologically important target molecules and as biosensors for clinical applications. Microarrays produced by oriented immobilization of antibodies generally have higher antigen-binding capacities than those in which antibodies are immobilized with random orientations. Here, we present a UV photo-cross-linking approach that utilizes boronic acid to achieve oriented immobilization of an antibody on a surface while retaining the antigen-binding activity of the immobilized antibody. A photoactive boronic acid probe was designed and synthesized in which boronic acid provided good affinity and specificity for the recognition of glycan chains on the Fc region of the antibody, enabling covalent tethering to the antibody upon exposure to UV light. Once irradiated with optimal UV exposure (16 mW/cm(2)), significant antibody immobilization on a boronic acid-presenting surface with maximal antigen detection sensitivity in a single step was achieved, thus obviating the necessity of prior antibody modifications. The developed approach is highly modular, as demonstrated by its implementation in sensitive sandwich immunoassays for the protein analytes Ricinus communis agglutinin 120, human prostate-specific antigen, and interleukin-6 with limits of detection of 7.4, 29, and 16 pM, respectively. Furthermore, the present system enabled the detection of multiple analytes in samples without any noticeable cross-reactivities. Antibody coupling via the use of boronic acid and UV light represents a practical, oriented immobilization method with significant implications for the construction of a large array of immunosensors for diagnostic applications.

Development of a single-antigen magnetic bead assay (SAMBA) for the sensitive detection of HPA-1a alloantibodies using tag-engineered recombinant soluble β3 integrin.

PubMed

Skaik, Younis; Battermann, Anja; Hiller, Oliver; Meyer, Oliver; Figueiredo, Constanca; Salama, Abdulgabar; Blasczyk, Rainer

2013-05-31

Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) β3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsβ3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems. Copyright © 2013 Elsevier B.V. All rights reserved.

A new technique to detect antibody-antigen reaction (biological interactions) on a localized surface plasmon resonance (LSPR) based nano ripple gold chip

NASA Astrophysics Data System (ADS)

Saleem, Iram; Widger, William; Chu, Wei-Kan

2017-07-01

We demonstrate that the gold nano-ripple localized surface plasmon resonance (LSPR) chip is a low cost and a label-free method for detecting the presence of an antigen. A uniform stable layer of an antibody was coated on the surface of a nano-ripple gold pattern chip followed by the addition of different concentrations of the antigen. A red shift was observed in the LSPR spectral peak caused by the change in the local refractive index in the vicinity of the nanostructure. The LSPR chip was fabricated using oblique gas cluster ion beam (GCIB) irradiation. The plasmon-resonance intensity of the scattered light was measured by a simple optical spectroscope. The gold nano ripple chip shows monolayer scale sensitivity and high selectivity. The LSPR substrate was used to detect antibody-antigen reaction of rabbit X-DENTT antibody and DENTT blocking peptide (antigen).

Reactive oxygen species modulator 1, a novel protein, combined with carcinoembryonic antigen in differentiating malignant from benign pleural effusion.

PubMed

Chen, Xianmeng; Zhang, Na; Dong, Jiahui; Sun, Gengyun

2017-05-01

The differential diagnosis of malignant pleural effusion and benign pleural effusion remains a clinical problem. Reactive oxygen species modulator 1 is a novel protein overexpressed in various human tumors. The objective of this study was to evaluate the diagnostic value of joint detection of reactive oxygen species modulator 1 and carcinoembryonic antigen in the differential diagnosis of malignant pleural effusion and benign pleural effusion. One hundred two consecutive patients with pleural effusion (including 52 malignant pleural effusion and 50 benign pleural effusion) were registered in this study. Levels of reactive oxygen species modulator 1 and carcinoembryonic antigen were measured by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. Results showed that the concentrations of reactive oxygen species modulator 1 both in pleural fluid and serum of patients with malignant pleural effusion were significantly higher than those of benign pleural effusion (both p < 0.05). The diagnostic sensitivity and specificity of pleural fluid reactive oxygen species modulator 1 were 61.54% and 82.00%, respectively, with the optimized cutoff value of 589.70 pg/mL. However, the diagnostic sensitivity and specificity of serum reactive oxygen species modulator 1 were only 41.38% and 86.21%, respectively, with the cutoff value of 27.22 ng/mL, indicating that serum reactive oxygen species modulator 1 may not be a good option in the differential diagnosis of malignant pleural effusion and benign pleural effusion. The sensitivity and specificity of pleural fluid carcinoembryonic antigen were 69.23% and 88.00%, respectively, at the cutoff value of 3.05 ng/mL, while serum carcinoembryonic antigen were 80.77% and 72.00% at the cutoff value of 2.60 ng/mL. The sensitivity could be raised to 88.17% in parallel detection of plural fluid reactive oxygen species modulator 1 and carcinoembryonic antigen concentration, and the specificity could be improved to 97.84% in serial detection.

Enzyme-linked immunosorbent assay for detection of antibodies to Epstein-Barr virus antigens.

PubMed

Voevodin, A F; Pácsa, A S

1983-01-01

Enzyme-Linked Immunosorbent Assay (ELISA) was standardized for measurement of antibody activity of reference human and baboon (Papio hamadryas) sera to soluble Epstein-Barr virus (EBV) antigens. A comparison with the immunofluorescent (IF) method showed that ELISA detects antibody specifically and sensitivity. In ELISA, Herpesvirus Papio (HVP) nuclear antigen (HUPNA) positive baboon serum reacted with EBV nuclear antigen (EBNA), as a further indication of the antigenic similarity between HVP and EBV. Forty-two baboon sera were tested with EBV antigens in both ELISA and IF test. The results showed an agreement between the two methods and also that by the use of EBV antigens, ELISA measures anti-HVP activity of baboon sera. ELISA did not reveal significant difference in antibody activity of 23 baboons with lymphoma and that of 24 healthy baboons. Results provide further data that ELISA can be used effectively in the field of EBV serology.

Generation and partial characterization of an eosinophil chemotactic cytokine produced by sensitized equine mononuclear cells stimulated with Strongylus vulgaris antigen.

PubMed

Dennis, V A; Klei, T R; Chapman, M R

1993-07-01

Supernatants generated by stimulation of peripheral blood mononuclear cells (PBMC) from Strongylus vulgaris sensitized or immunized ponies were assayed in vitro for eosinophil chemotactic activity (ECA) using the filter system in blind well chambers. The supernatants from these cultures were chemotactic for eosinophils, but not for neutrophils. Supernates from cultures of unsensitized PBMC stimulated with S. vulgaris antigen were not chemotactic for eosinophils. ECA was first detected in culture supernatants after 1.5 h of incubation and was dependent on both antigen and PBMC concentrations, but independent of serum concentrations. Both female and male S. vulgaris worm antigens stimulated ECA production from sensitized PBMC. ECA was not induced by in vitro stimulation of sensitized S. vulgaris PBMC by female Strongylus edentatus worm antigen. Partial characterization of the eosinophil chemotactic cytokine showed it to be nondialyzable, greater than 8000 molecular weight (MW), and sensitive to heating (56 and 95 degrees C), trypsin, and sodium metaperiodate treatments, suggesting that the cytokine is a protein containing some essential carbohydrate moieties. The cytokine described in this paper could partially contribute to the in vivo blood and tissue eosinophilia in experimental S. vulgaris infection.

Early Diagnosis of Scrub Typhus with a Rapid Flow Assay Using Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi

PubMed Central

Ching, W.-M.; Rowland, D.; Zhang, Z.; Bourgeois, A. L.; Kelly, D.; Dasch, G. A.; Devine, P. L.

2001-01-01

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to O. tsutsugamushi. The RFA employs a truncated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of the RFA was evaluated with a panel of 321 sera (serial bleedings of 85 individuals suspected of scrub typhus) which were collected in the Pescadore Islands, Taiwan, from 1976 to 1977. Among these 85 individuals, IgM tests were negative for 7 cases by both RFA and indirect fluorescence assay (IFA) using Karp whole-cell antigen. In 29 cases specific responses were detected by the RFA earlier than by IFA, 44 cases had the same detection time, and 5 cases were detected earlier by IFA than by RFA. For IgG responses, 4 individuals were negative with both methods, 37 cases exhibited earlier detection by RFA than IFA, 42 cases were detected at the same time, and 2 cases were detected earlier by IFA than by RFA. The sensitivities of RFA detection of antibody in sera from confirmed cases were 74 and 86% for IgM and IgG, respectively. When IgM and IgG results were combined, the sensitivity was 89%. A panel of 78 individual sera collected from patients with no evidence of scrub typhus was used to evaluate the specificity of the RFA. The specificities of the RFA were 99% for IgM and 97% for IgG. The sensitivities of IFA were 53 and 73% for IgM and IgG, respectively, and were 78% when the results of IgM and IgG were combined. The RFA test was significantly better than the IFA test for the early detection of antibody to scrub typhus in primary infections, while both tests were equally sensitive with reinfected individuals. PMID:11238230

Development of animal models and sandwich-ELISA tests to detect the allergenicity and antigenicity of fining agent residues in wines.

PubMed

Lifrani, Awatif; Dos Santos, Jacinthe; Dubarry, Michel; Rautureau, Michelle; Blachier, Francois; Tome, Daniel

2009-01-28

Food allergy can cause food-related anaphylaxis. Food allergen labeling is the principal means of protecting sensitized individuals. This motivated European Directive 2003/89 on the labeling of ingredients or additives that could trigger adverse reactions, which has been in effect since 2005. During this study, we developed animal models with allergy to ovalbumin, caseinate, and isinglass in order to be able to detect fining agent residues that could induce anaphylactic reactions in sensitized mice. The second aim of the study was to design sandwich ELISA tests specific to each fining agent in order to detect their residue antigenicity, both during wine processing and in commercially available bottled wines. Sensitized mice and sandwich ELISA methods were established to test a vast panel of wines. The results showed that although they were positive to our highly sensitive sandwich-ELISA tests, some commercially available wines are not allergenic in sensitized mice. Commercially available bottled wines made using standardized processes, fining, maturation, and filtration, do not therefore represent any risk of anaphylactic reactions in sensitized mice.

Diagnostic Performance of Tuberculosis-Specific IgG Antibody Profiles in Patients with Presumptive Tuberculosis from Two Continents.

PubMed

Broger, Tobias; Basu Roy, Robindra; Filomena, Angela; Greef, Charles H; Rimmele, Stefanie; Havumaki, Joshua; Danks, David; Schneiderhan-Marra, Nicole; Gray, Christen M; Singh, Mahavir; Rosenkrands, Ida; Andersen, Peter; Husar, Gregory M; Joos, Thomas O; Gennaro, Maria L; Lochhead, Michael J; Denkinger, Claudia M; Perkins, Mark D

2017-04-01

Development of rapid diagnostic tests for tuberculosis is a global priority. A whole proteome screen identified Mycobacterium tuberculosis antigens associated with serological responses in tuberculosis patients. We used World Health Organization (WHO) target product profile (TPP) criteria for a detection test and triage test to evaluate these antigens. Consecutive patients presenting to microscopy centers and district hospitals in Peru and to outpatient clinics at a tuberculosis reference center in Vietnam were recruited. We tested blood samples from 755 HIV-uninfected adults with presumptive pulmonary tuberculosis to measure IgG antibody responses to 57 M. tuberculosis antigens using a field-based multiplexed serological assay and a 132-antigen bead-based reference assay. We evaluated single antigen performance and models of all possible 3-antigen combinations and multiantigen combinations. Three-antigen and multiantigen models performed similarly and were superior to single antigens. With specificity set at 90% for a detection test, the best sensitivity of a 3-antigen model was 35% (95% confidence interval [CI], 31-40). With sensitivity set at 85% for a triage test, the specificity of the best 3-antigen model was 34% (95% CI, 29-40). The reference assay also did not meet study targets. Antigen performance differed significantly between the study sites for 7/22 of the best-performing antigens. Although M. tuberculosis antigens were recognized by the IgG response during tuberculosis, no single antigen or multiantigen set performance approached WHO TPP criteria for clinical utility among HIV-uninfected adults with presumed tuberculosis in high-volume, urban settings in tuberculosis-endemic countries. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

PubMed

Varghese, Anju; Raina, Opinder K; Chandra, Dinesh; Mirdha, Bijay R; Kelawala, Naresh H; Solanki, Jayesh B; Kumar, Niranjan; Ravindran, Reghu; Arun, Anandanarayanan; Rialch, Ajayta; Lalrinkima, Hniang; Kelawala, Rohan N; Samanta, Subhamoy

2017-12-20

Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

Highly sensitive and selective lateral flow immunoassay based on magnetic nanoparticles for quantitative detection of carcinoembryonic antigen.

PubMed

Liu, Fangming; Zhang, Honglian; Wu, Zhenhua; Dong, Haidao; Zhou, Lin; Yang, Dawei; Ge, Yuqing; Jia, Chunping; Liu, Huiying; Jin, Qinghui; Zhao, Jianlong; Zhang, Qiqing; Mao, Hongju

2016-12-01

Carcinoembryonic antigen (CEA) is an important biomarker in cancer diagnosis. Here, we present an efficient, selective lateral-flow immunoassay (LFIA) based on magnetic nanoparticles (MNPs) for in situ sensitive and accurate point-of-care detection of CEA. Signal amplification mechanism involved linking of detection MNPs with signal MNPs through biotin-modified single-stranded DNA (ssDNA) and streptavidin. To verify the effectiveness of this modified LFIA system, the sensitivity and specificity were evaluated. Sensitivity evaluation showed a broad detection range of 0.25-1000ng/ml for CEA protein by the modified LFIA, and the limit of detection (LOD) of the modified LFIA was 0.25ng/ml, thus producing significant increase in detection threshold compared with the traditional LFIA. The modified LFIA could selectively recognize CEA in presence of several interfering proteins. In addition, this newly developed assay was applied for quantitative detection of CEA in human serum specimens collected from 10 randomly selected patients. The modified LFIA system detected minimum 0.27ng/ml of CEA concentration in serum samples. The results were consistent with the clinical data obtained using commercial electrochemiluminescence immunoassay (ECLIA) (p<0.01). In conclusion, the MNPs based LFIA system not only demonstrated enhanced signal to noise ratio, it also detected CEA with higher sensitivity and selectivity, and thus has great potential to be commercially applied as a sensitive tumor marker filtration system. Copyright © 2016 Elsevier B.V. All rights reserved.

Isotope labeled immunoassay for environmental chemical detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Velez, M.M.

1994-05-06

Altrazine, one of the most heavily used agricultural pesticides in North America, has been identified as a major groundwater contaminant in the U.S. Research provides evidence that under certain conditions atrazine and some of its derivatives may prove to be carcinogenic and mutagenic. Immunossays are one of the most powerful of all analytical immunochemical techniques. They employ a wide range of methods to detect and quantitate antigens or antibodies, and to study the structure of antigens. With the appropriate assay, they can be remarkably quick and easy, to yield information that would be difficult to determine by other techniques. Themore » development of the appropriate assay; however, requires clean and precise separation of antigens bound to antibodies from those that remain free. Sensitive assays depend on quantification of these bound antigens at very low levels. We are making direct and competitive immunoassays with atrazine and its antibodies using accelerator mass spectrometry (AMS) in order to obtain a sensitive immunoassay for atrazine in environmental samples.« less

A Nano-Au/C-MWCNT based label free amperometric immunosensor for the detection of capsicum chlorosis virus in bell pepper.

PubMed

Sharma, Anshul; Kaushal, Ankur; Kulshrestha, Saurabh

2017-07-01

Accurate and on time diagnosis of plant viruses is an essential prerequisite for efficient control in field conditions. A number of diagnostic methods have been reported with the required level of sensitivity. Here, we propose a label free immunosensor for efficient and sensitive detection of capsicum chlorosis virus (CaCV) in bell pepper. Antigen was immobilized over the surface of gold nanoparticle/multi-walled carbon nanotube (Nano-Au/C-MWCNT) screen printed electrodes using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) cross linking chemistry followed by interaction with groundnut bud necrosis virus (GBNV)/CaCV specific polyclonal antibody. The electrochemical response was measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV) using the redox indicator. Electrode surface characterization was done by performing scanning electron microscopy (SEM). Electrochemical studies showed positive results at different antigenic dilutions ranging from 10 -2 - 8x10 -5 . The sensitivity of the immunosensor developed has been compared with direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) and the results showed that the immunosensor developed was 800-1000 times more sensitive, when compared to DAC-ELISA for CaCV detection. The immunosensor we have developed is economical and sensitive and could be used for immediate determination of the presence of virus in extracts from bell pepper leaves.

Development and Evaluation of a Sensitive and Specific Assay for Diagnosis of Human Toxocariasis by Use of Three Recombinant Antigens (TES-26, TES-30USM, and TES-120)▿

PubMed Central

Mohamad, Suharni; Azmi, Norhaida Che; Noordin, Rahmah

2009-01-01

Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis. PMID:19369434

Development and evaluation of a sensitive and specific assay for diagnosis of human toxocariasis by use of three recombinant antigens (TES-26, TES-30USM, and TES-120).

PubMed

Mohamad, Suharni; Azmi, Norhaida Che; Noordin, Rahmah

2009-06-01

Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.

Histoplasma Urinary Antigen Testing Obviates the Need for Coincident Serum Antigen Testing.

PubMed

Libert, Diane; Procop, Gary W; Ansari, Mohammad Q

2018-03-07

Serum and urine antigen (SAg, UAg) detection are common tests for Histoplasma capsulatum. UAg detection is more widely used and reportedly has a higher sensitivity. We investigated whether SAg detection contributes meaningfully to the initial evaluation of patients with suspected histoplasmosis. We reviewed 20,285 UAg and 1,426 SAg tests ordered from 1997 to 2016 and analyzed paired UAg and SAg tests completed on the same patient within 1 week. We determined the positivity rate for each test. Of 601 paired specimens, 542 were concurrent negatives and 48 were concurrent positives (98% agreement). Medical records were available for eight of 11 pairs with discrepant results. UAg was falsely positive in six instances, truly positive once, and falsely negative once. These findings support using a single antigen detection test, rather than both UAg and SAg, as an initial screen for suspected histoplasmosis. This aligns with the current practice of most physicians.

Comparison of nonstructural protein-1 antigen detection by rapid and enzyme-linked immunosorbent assay test and its correlation with polymerase chain reaction for early diagnosis of dengue

PubMed Central

Gaikwad, Seema; Sawant, Sandhya S.; Shastri, Jayanthi S.

2017-01-01

INTRODUCTION: Early diagnosis of dengue is important for appropriate clinical management and vector control. Different serological tests based on the principle of immunochromatography and enzyme-linked immunosorbent assay (ELISA) are commonly used for detection of antigen and antibodies of dengue virus. The performance of these tests depends on the sensitivity and specificity. Hence, the study was undertaken to compare nonstructural protein-1 (NS1) antigen detection by rapid and ELISA with real-time polymerase chain reaction (RT-PCR) for diagnosis of dengue. MATERIALS AND METHODS: Prospective laboratory study was carried out on sera samples (n = 200) from clinically suspected cases of dengue. The sera samples were subjected for NS1 antigen detection test by rapid test, NS1 ELISA, and RT-PCR. The results of rapid and ELISA tests were compared with real Time PCR. RESULTS: The sensitivity, specificity, positive, and negative predictive value of rapid dengue NS1 antigen test were 81.5%, 66.7%, 78.2%, and 71.1%, respectively whereas that of NS1 ELISA were 89.9%, 100%, 100%, and 94%, respectively. Concordance of Rapid NS1 and NS1 ELISA with PCR was 75.5% and 94%. DISCUSSION AND CONCLUSION: NS1 antigen ELISA can be implemented in diagnostic laboratories for diagnosis of dengue in the acute phase of illness. The test also has great potential value for use in epidemic situations, as it could facilitate the early screening of patients and limit disease expansion. PMID:28706387

A Multiplex Cancer/Testis Antigen-Based Biomarker Panel to Predict the Aggressive Phenotype of Prostate Cancer

DTIC Science & Technology

2016-09-01

US (Siegel et al., 2014). The introduction of the prostate specific antigen ( PSA ) test has greatly aided to the early detection of PCa. Detectable...levels of PSA are the earliest sign of recurrent disease after radical prostatectomy (RP) (Pound et al., 1999). Besides its sensitiveness, it is...estimated that 23-44% of patients submitted to RP will progress with detectable PSA levels and will never present recurrence (Draisma et al., 2009). Thus

Comparison of a newly developed automated and quantitative hepatitis C virus (HCV) core antigen test with the HCV RNA assay for clinical usefulness in confirming anti-HCV results.

PubMed

Kesli, Recep; Polat, Hakki; Terzi, Yuksel; Kurtoglu, Muhammet Guzel; Uyar, Yavuz

2011-12-01

Hepatitis C virus (HCV) is a global health care problem. Diagnosis of HCV infection is mainly based on the detection of anti-HCV antibodies as a screening test with serum samples. Recombinant immunoblot assays are used as supplemental tests and for the final detection and quantification of HCV RNA in confirmatory tests. In this study, we aimed to compare the HCV core antigen test with the HCV RNA assay for confirming anti-HCV results to determine whether the HCV core antigen test may be used as an alternative confirmatory test to the HCV RNA test and to assess the diagnostic values of the total HCV core antigen test by determining the diagnostic specificity and sensitivity rates compared with the HCV RNA test. Sera from a total of 212 treatment-naive patients were analyzed for anti-HCV and HCV core antigen both with the Abbott Architect test and with the molecular HCV RNA assay consisting of a reverse transcription-PCR method as a confirmatory test. The diagnostic sensitivity, specificity, and positive and negative predictive values of the HCV core antigen assay compared to the HCV RNA test were 96.3%, 100%, 100%, and 89.7%, respectively. The levels of HCV core antigen showed a good correlation with those from the HCV RNA quantification (r = 0.907). In conclusion, the Architect HCV antigen assay is highly specific, sensitive, reliable, easy to perform, reproducible, cost-effective, and applicable as a screening, supplemental, and preconfirmatory test for anti-HCV assays used in laboratory procedures for the diagnosis of hepatitis C virus infection.

ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts.

PubMed

Dalton, K P; Podadera, A; Granda, V; Nicieza, I; Del Llano, D; González, R; de Los Toyos, J R; García Ocaña, M; Vázquez, F; Martín Alonso, J M; Prieto, J M; Parra, F; Casais, R

2018-01-01

The emergence and rapid spread of variant of the rabbit hemorrhagic disease virus (RHDV2) require new diagnostic tools to ensure that efficient control measures are adopted. In the present study, a specific sandwich enzyme-linked immunosorbent assay (ELISA) for detection of RHDV2 antigens in rabbit liver homogenates, based on the use of an RHDV2-specific monoclonal antibody (Mab) 2D9 for antigen capture and an anti-RHDV2 goat polyclonal antibody (Pab), was developed. This ELISA was able to successfully detect RHDV2 and RHDV2 recombinant virions with high sensitivity (100%) and specificity (97.22%). No cross-reactions were detected with RHDV G1 viruses while low cross-reactivity was detected with one of the RHDVa samples analyzed. The ELISA afforded good repeatability and had high analytical sensitivity as it was able to detect a dilution 1:163,640 (6.10ng/mL) of purified RHDV-N11 VLPs, which contained approximately 3.4×10 8 molecules/mL particles. The reliable discrimination between closely related viruses is crucial to understand the epidemiology and the interaction of co-existing pathogens. In the work described here we design and validate an ELISA for laboratory based, specific, sensitive and reliable detection of RHDVb/RHDV2. This ELISA is a valuable, specific virological tool for monitoring virus circulation, which will permit a better control of this disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Chicken egg yolk antibodies (IgY) for detecting circulating antigens of Schistosoma japonicum.

PubMed

Cai, Yu-Chun; Guo, Jian; Chen, Shao-Hong; Tian, Li-Guang; Steinmann, Peter; Chen, Mu-Xin; Li, Hao; Ai, Lin; Chen, Jia-Xu

2012-09-01

IgY isolated from egg yolk has been widely used in immunodiagnostic tests, including tests to detect circulating antigen (soluble egg antigen or SEA) of Schistosoma japonicum. A sandwich ELISA was established using a combination of anti-S. japonicum SEA-IgY polyclonal antibodies and IgM monoclonal antibodies. To explore sensitivity and specificity of the sandwich ELISA, serum samples from 43 patients infected with S. japonicum were tested. All acute cases and 91.3% of the chronic cases showed a positive reaction. Only 5% of the control sera from healthy persons gave a positive response. Cross-reactions with antibodies to nine other parasites were rare. The developed immunoassay is reasonably sensitive and specific. It could be used for field research and treatment efficacy assessments. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

An interlaboratory study on efficient detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O121, O145, and O157 in food using real-time PCR assay and chromogenic agar.

PubMed

Hara-Kudo, Yukiko; Konishi, Noriko; Ohtsuka, Kayoko; Iwabuchi, Kaori; Kikuchi, Rie; Isobe, Junko; Yamazaki, Takumiko; Suzuki, Fumie; Nagai, Yuhki; Yamada, Hiroko; Tanouchi, Atsuko; Mori, Tetsuya; Nakagawa, Hiroshi; Ueda, Yasufumi; Terajima, Jun

2016-08-02

To establish an efficient detection method for Shiga toxin (Stx)-producing Escherichia coli (STEC) O26, O103, O111, O121, O145, and O157 in food, an interlaboratory study using all the serogroups of detection targets was firstly conducted. We employed a series of tests including enrichment, real-time PCR assays, and concentration by immunomagnetic separation, followed by plating onto selective agar media (IMS-plating methods). This study was particularly focused on the efficiencies of real-time PCR assays in detecting stx and O-antigen genes of the six serogroups and of IMS-plating methods onto selective agar media including chromogenic agar. Ground beef and radish sprouts samples were inoculated with the six STEC serogroups either at 4-6CFU/25g (low levels) or at 22-29CFU/25g (high levels). The sensitivity of stx detection in ground beef at both levels of inoculation with all six STEC serogroups was 100%. The sensitivity of stx detection was also 100% in radish sprouts at high levels of inoculation with all six STEC serogroups, and 66.7%-91.7% at low levels of inoculation. The sensitivity of detection of O-antigen genes was 100% in both ground beef and radish sprouts at high inoculation levels, while at low inoculation levels, it was 95.8%-100% in ground beef and 66.7%-91.7% in radish sprouts. The sensitivity of detection with IMS-plating was either the same or lower than those of the real-time PCR assays targeting stx and O-antigen genes. The relationship between the results of IMS-plating methods and Ct values of real-time PCR assays were firstly analyzed in detail. Ct values in most samples that tested negative in the IMS-plating method were higher than the maximum Ct values in samples that tested positive in the IMS-plating method. This study indicates that all six STEC serogroups in food contaminated with more than 29CFU/25g were detected by real-time PCR assays targeting stx and O-antigen genes and IMS-plating onto selective agar media. Therefore, screening of stx and O-antigen genes followed by isolation of STECs by IMS-plating methods may be an efficient method to detect the six STEC serogroups. Copyright © 2016 Elsevier B.V. All rights reserved.

Two-dimensional Layered MoS2 Biosensors Enable Highly Sensitive Detection of Biomolecules

NASA Astrophysics Data System (ADS)

Lee, Joonhyung; Dak, Piyush; Lee, Yeonsung; Park, Heekyeong; Choi, Woong; Alam, Muhammad A.; Kim, Sunkook

2014-12-01

We present a MoS2 biosensor to electrically detect prostate specific antigen (PSA) in a highly sensitive and label-free manner. Unlike previous MoS2-FET-based biosensors, the device configuration of our biosensors does not require a dielectric layer such as HfO2 due to the hydrophobicity of MoS2. Such an oxide-free operation improves sensitivity and simplifies sensor design. For a quantitative and selective detection of PSA antigen, anti-PSA antibody was immobilized on the sensor surface. Then, introduction of PSA antigen, into the anti-PSA immobilized sensor surface resulted in a lable-free immunoassary format. Measured off-state current of the device showed a significant decrease as the applied PSA concentration was increased. The minimum detectable concentration of PSA is 1 pg/mL, which is several orders of magnitude below the clinical cut-off level of ~4 ng/mL. In addition, we also provide a systematic theoretical analysis of the sensor platform - including the charge state of protein at the specific pH level, and self-consistent channel transport. Taken together, the experimental demonstration and the theoretical framework provide a comprehensive description of the performance potential of dielectric-free MoS2-based biosensor technology.

Rapid diagnosis of equine influenza by highly sensitive silver amplification immunochromatography system.

PubMed

Yamanaka, Takashi; Nemoto, Manabu; Bannai, Hiroshi; Tsujimura, Koji; Kondo, Takashi; Matsumura, Tomio; Fu, Tao Qi Huang; Fernandez, Charlene Judith; Gildea, Sarah; Cullinane, Ann

2017-06-16

Equine influenza (EI) is a respiratory disease caused by equine influenza A virus (EIV, H3N8) infection. Rapid diagnosis is essential to limit the disease spread. We previously reported that some rapid antigen detection (RAD) tests are fit for diagnosing EI although their sensitivity is not optimal. Here, we evaluated the performance of the newly developed RAD test using silver amplification immunochromatography (Quick Chaser Auto Flu A, B: QCA) to diagnose EI. The detection limits of QCA for EIVs were five-fold lower than the conventional RAD tests. The duration of virus antigen detection in the infected horses was longer than the conventional RAD tests. We conclude that QCA could be a valuable diagnostic method for EI.

Magneto immuno-PCR: a novel immunoassay based on biogenic magnetosome nanoparticles.

PubMed

Wacker, Ron; Ceyhan, Buelent; Alhorn, Petra; Schueler, Dirk; Lang, Claus; Niemeyer, Christof M

2007-06-01

We describe an innovative modification of the Immuno-PCR technology for automatable high sensitive antigen detection. The Magneto Immuno-PCR (M-IPCR) is based on antibody-functionalized biogenic magnetosome nanoparticles revealing major advantages over synthetic magnetic particles. The general principle of the M-IPCR is similar to that of a two-sided (sandwich) immunoassay. However, antibody-functionalized magnetosome conjugates were employed for the immobilization and magnetic enrichment of the signal generating detection complex enabling the establishment of a surface independent immunoassay. To this end, the M-IPCR was carried out by simultaneously tagging the antigen with the reagent for read-out, i.e., a conjugate comprising the specific antibody and DNA fragments, in the presence of the antibody-functionalized magnetosomes. To demonstrate the general functionality of the M-IPCR, the detection of recombinant Hepatitis B surface Antigen (HBsAg) in human serum was established. We observed a detection limit of 320pg/ml of HBsAg using the M-IPCR, which was about 100-fold more sensitive than the analogous Magneto-ELISA, established in parallel for comparison purposes.

Sensitive and specific enzyme-linked immunosorbent assay for detecting serum antibodies against Mycobacterium avium subsp. paratuberculosis in fallow deer.

PubMed

Prieto, José M; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

2014-08-01

The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting Serum Antibodies against Mycobacterium avium subsp. paratuberculosis in Fallow Deer

PubMed Central

Prieto, José M.; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E.; Garrido, Joseba M.; Juste, Ramon A.

2014-01-01

The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations. PMID:24872517

IP-FCM measures physiologic protein-protein interactions modulated by signal transduction and small-molecule drug inhibition.

PubMed

Smith, Stephen E P; Bida, Anya T; Davis, Tessa R; Sicotte, Hugues; Patterson, Steven E; Gil, Diana; Schrum, Adam G

2012-01-01

Protein-protein interactions (PPI) mediate the formation of intermolecular networks that control biological signaling. For this reason, PPIs are of outstanding interest in pharmacology, as they display high specificity and may represent a vast pool of potentially druggable targets. However, the study of physiologic PPIs can be limited by conventional assays that often have large sample requirements and relatively low sensitivity. Here, we build on a novel method, immunoprecipitation detected by flow cytometry (IP-FCM), to assess PPI modulation during either signal transduction or pharmacologic inhibition by two different classes of small-molecule compounds. First, we showed that IP-FCM can detect statistically significant differences in samples possessing a defined PPI change as low as 10%. This sensitivity allowed IP-FCM to detect a PPI that increases transiently during T cell signaling, the antigen-inducible interaction between ZAP70 and the T cell antigen receptor (TCR)/CD3 complex. In contrast, IP-FCM detected no ZAP70 recruitment when T cells were stimulated with antigen in the presence of the src-family kinase inhibitor, PP2. Further, we tested whether IP-FCM possessed sufficient sensitivity to detect the effect of a second, rare class of compounds called SMIPPI (small-molecule inhibitor of PPI). We found that the first-generation non-optimized SMIPPI, Ro-26-4550, inhibited the IL-2:CD25 interaction detected by IP-FCM. This inhibition was detectable using either a recombinant CD25-Fc chimera or physiologic full-length CD25 captured from T cell lysates. Thus, we demonstrate that IP-FCM is a sensitive tool for measuring physiologic PPIs that are modulated by signal transduction and pharmacologic inhibition.

[Pernicious anaemia--diagnostic benefit of the detection of autoantibodies against intrinsic factor and gastric parietal cells antigen H+/K+ ATPase].

PubMed

Sedláková, L; Dubská, L; Průcha, M

2010-08-01

Pernicious anaemia is an autoimmune disease that causes acquired vitamin B12 deficiency. The diagnostic process includes the detection of typical changes in the blood count, low serum levels of vitamin B12, endoscopic and histological signs of gastritis and autoantibodies against the gastric parietal cells antigen H+/K+ ATPase and intrinsic factor. Our aims were to establish immunological tests for the detection of autoantibodies against intrinsic factor and target gastric parietal cell antigen H+/K+ ATPase and to evaluate their diagnostic benefits in patients with pernicious anaemia. Sera from 95 patients were tested for autoantibodies against H+/K+ ATPase and intrinsic factor by multiplex Luminex assay. The results were compared with those of the immunofluorescence assay for the detection of autoantibodies against gastric parietal cells and with the diagnostic criteria. The autoantibodies against gastric parietal cell H+/K+ ATPase had a sensitivity of 68.2% with a specificity of 91.7% for the diagnosis of pernicious anaemia. The respective rates for the autoantibodies against intrinsic factor were 40.9% and 98.6%. The combined sensitivity and specificity rates for both autoantibodies were 86.36% and 90.28%, respectively, the combined positive predictive value was 73.08% and the combined negative predictive value was 95.59%. The detection of both autoantibodies is helpful in diagnosing pernicious anaemia and the combination of the two assays increases diagnostic sensitivity.

Highly sensitive determination of diclofenac based on resin beads and a novel polyclonal antibody by using flow injection chemiluminescence competitive immunoassay

NASA Astrophysics Data System (ADS)

Shi, Jing; Xu, Mingxia; Tang, Qinghui; Zhao, Kang; Deng, Anping; Li, Jianguo

2018-02-01

A novel flow injection chemiluminescence immunoassay for simple, sensitive and low-cost detection of diclofenac was established based on specific binding of antigen and antibody. Carboxylic resin beads used as solid phase carrier materials provided good biocompatibility and large surface-to-volume ratio for modifying more coating antigen. There was a competitive process between the diclofenac in solution and the immobilized coating antigen to react with the limited binding sites of the polyclonal antibody to form the immunocomplex. The second antibody labelled with horseradish peroxidase was introduced into the immunosensor and trapped by captured polyclonal antibody against diclofenac, which could effectively amplify chemiluminescence signals of luminol-PIP-H2O2. Under optimal conditions, the diclofenac could be detected quantitatively. The chemiluminescence intensity decreased linearly with the logarithm of the diclofenac concentration in the range of 0.1-100 ng mL- 1 with a detection limit of 0.05 ng mL- 1 at a signal-to-noise ratio of 3. The immunosensor exhibited high sensitivity, specificity and acceptable stability. This easy-operated and cost-effective analytical method could be valuable for the diclofenac determination in real water samples.

A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.

PubMed

Suin, Vanessa; Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael; Van Gucht, Steven

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test

PubMed Central

Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible. PMID:24822188

Detection of Antigenic Variants of Subtype H3 Swine Influenza A Viruses from Clinical Samples.

PubMed

Martin, Brigitte E; Bowman, Andrew S; Li, Lei; Nolting, Jacqueline M; Smith, David R; Hanson, Larry A; Wan, Xiu-Feng

2017-04-01

A large population of genetically and antigenically diverse influenza A viruses (IAVs) are circulating among the swine population, playing an important role in influenza ecology. Swine IAVs not only cause outbreaks among swine but also can be transmitted to humans, causing sporadic infections and even pandemic outbreaks. Antigenic characterizations of swine IAVs are key to understanding the natural history of these viruses in swine and to selecting strains for effective vaccines. However, influenza outbreaks generally spread rapidly among swine, and the conventional methods for antigenic characterization require virus propagation, a time-consuming process that can significantly reduce the effectiveness of vaccination programs. We developed and validated a rapid, sensitive, and robust method, the polyclonal serum-based proximity ligation assay (polyPLA), to identify antigenic variants of subtype H3N2 swine IAVs. This method utilizes oligonucleotide-conjugated polyclonal antibodies and quantifies antibody-antigen binding affinities by quantitative reverse transcription-PCR (RT-PCR). Results showed the assay can rapidly detect H3N2 IAVs directly from nasal wash or nasal swab samples collected from laboratory-challenged animals or during influenza surveillance at county fairs. In addition, polyPLA can accurately separate the viruses at two contemporary swine IAV antigenic clusters (H3N2 swine IAV-α and H3N2 swine IAV-ß) with a sensitivity of 84.9% and a specificity of 100.0%. The polyPLA can be routinely used in surveillance programs to detect antigenic variants of influenza viruses and to select vaccine strains for use in controlling and preventing disease in swine. Copyright © 2017 American Society for Microbiology.

Comparison of Detection Limits of 4th Generation Combination HIV Antigen/Antibody, p24 Antigen and Viral Load Assays on Diverse HIV Isolates.

PubMed

Stone, Mars; Bainbridge, John; Sanchez, Ana M; Keating, Sheila M; Pappas, Andrea; Rountree, Wes; Todd, Chris; Bakkour, Sonia; Manak, Mark; Peel, Sheila A; Coombs, Robert W; Ramos, Eric M; Shriver, M Kathleen; Contestable, Paul; Nair, Sangeetha Vijaysri; Wilson, David H; Stengelin, Martin; Murphy, Gary; Hewlett, Indira; Denny, Thomas N; Busch, Michael P

2018-05-23

Detection of acute HIV infection is critical for HIV public health and diagnostics. Clinical 4 th generation antigen-antibody (Ag/Ab) combination (combo) and p24 Ag immunoassays have enhanced detection of acute infection compared to Ab alone assays, but require ongoing evaluation with currently circulating diverse subtypes. Genetically and geographically diverse HIV clinical isolates were used to assess clinical HIV diagnostic, blood screening and next generation assays. Blinded 300 member panels of 20 serially diluted well-characterized antibody negative HIV isolates were distributed to manufacturers and end-user labs to assess relative analytic sensitivity of currently approved and pre-approved clinical HIV 4 th generation Ag/Ab combo or p24 Ag alone immunoassays across diverse subtypes. The limits of virus detection (LODs) were estimated for different subtypes relative to confirmed viral loads. Analysis of immunoassay sensitivity was benchmarked against confirmed viral load measurements on the blinded panel. Based on the proportion of positive results on 300 observations all Ag/Ab combo and standard sensitivity p24 Ag assays performed similarly and within half log LODs, illustrating similar breadth of reactivity and diagnostic utility. Ultrasensitive p24 Ag assays achieved dramatically increased sensitivities, while the rapid combo-assays performed poorly. Similar performance of the different commercially available 4 th gen assays on diverse subtypes supports their use in broad geographic settings with locally circulating HIV clades and recombinant strains. Next generation pre-clinical ultrasensitive p24 Ag assays achieved dramatically improved sensitivity, while p24 Ag detection by rapid 4 th gen assays performed poorly. Copyright © 2018 American Society for Microbiology.

Comparative efficacy of antigen and antibody detection tests for human trichinellosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ivanoska, D.; Cuperlovic, K.; Gamble, H.R.

1989-02-01

Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory productsmore » of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation.« less

SPR platform based on image acquisition for HER2 antigen detection

NASA Astrophysics Data System (ADS)

Monteiro, Johny P.; Predabon, Sheila M.; Bonafé, Elton G.; Martins, Alessandro F.; Brolo, Alexandre G.; Radovanovic, Eduardo; Girotto, Emerson M.

2017-01-01

HER2 antigen is a marker used for breast cancer diagnosis and prevention. Its determination has great importance since breast cancer is one of the most insidious types of cancer in women. HER2 antigen assessment in human serum is traditionally achieved by enzyme-linked immunosorbent assay (ELISA method), but it has some disadvantages, such as suppressing the thermodynamic-kinetic studies regarding the antibody-antigen interaction, and the use of labeled molecules that can promote false positive responses. Biosensors based on surface plasmon resonance (SPR) are sensitive optical techniques widely applied on bioassays. The plasmonic devices do not operate with labeled molecules, overcoming conventional immunoassay limitations, and enabling a direct detection of target analytes. In this way, a new SPR biosensor to assess HER2 antigen has been proposed, using nanohole arrays on a gold thin film by signal transduction of transmitted light measurements from array image acquisitions. These metallic nanostructures may couple the light directly on surface plasmons using a simple collinear arrangement. The proposed device reached an average sensitivity for refractive index (RI) variation on a metal surface of 4146 intensity units/RIU (RIU = RI units). The device feasibility on biomolecular assessment was evaluated. For this, 3 ng ml-1 known HER2 antigen concentration was efficiently flowed (using a microfluidic system) and detected from aqueous solutions. This outcome shows that the device may be a powerful apparatus for bioassays, particularly toward breast cancer diagnosis and prognosis.

Dye sensitized photoelectrochemical immunosensor for the tumor marker CEA by using a flower-like 3D architecture prepared from graphene oxide and MoS2.

PubMed

Song, Kaijing; Ding, Chuanmin; Zhang, Bing; Chang, Honghong; Zhao, Zhihuan; Wei, Wenlong; Wang, Junwen

2018-06-01

The authors describe a dye-sensitized photoelectrochemical immunoassay for the tumor marker carcinoembryonic antigen (CEA). The method employs the rhodamine dye Rh123 with red color and absorption maximum at 500 nm for spectral sensitization, and a 3D nanocomposite prepared from graphene oxide and MoS 2 acting as the photoelectric conversion layer. The nanocomposite with flower-like 3D architectures was characterized by transmission electron microscopy, scanning electron microscopy, X-ray powder diffraction, and UV-vis diffuse reflectometry. A photoelectrochemical sandwich immunoassay was developed that is based on the use of the nanocomposite and based on the specific binding of antibody and antigen, and by using a secondary antibody labeled with Rh123 and CdS (Ab 2 -Rh123@CdS). Under optimal conditions and at a typical working voltage of 0 V (vs. Hg/HgCl 2 ), the photocurrent increases linearly 10 pg mL -1 to 80 ng mL -1 CEA concentration range, with a 3.2 pg mL -1 detection limit. Graphical abstract Flower-like GO-MoS 2 complex with high efficiency of electron transport was synthesized to construct photoelectrochemical platform. The sandwich-type immunoassay was built on this platform based on specific binding of antigen and antibody. Carcinoembryonic antigen in sample was detected sensitively by using sensitization of rhodamine dye Rh123 as signal amplification strategy.

A prospective field evaluation of an enzyme immunoassay: Detection of eastern equine encephalomyelitis virus antigen in pools of Culiseta melanura

USGS Publications Warehouse

Scott, T.W.; Olson, J.G.; Lewis, T.E.; Carpenter, J.W.; Lorenz, L.H.; Lembeck, L.A.; Joseph, S.R.; Pagac, B.B.

1987-01-01

A prospective field study was conducted to determine the sensitivity and specificity of an enzyme immunoassay (EIA) compared to virus isolation in cell culture for the detection of eastern equine encephalomyelitis (EEE) virus in naturally infected mosquitoes. A total of 10,811 adult female Culiseta melanura were collected in light traps during 1985 from four locations in Maryland. Eastern equine encephalomyelitis virus was isolated from 5 of 495 mosquito pools in African green monkey kidney and baby hamster kidney cell cultures. All five virus-infected pools were detected by the EIA, and all 490 uninfected pools were correctly scored as not containing virus. The EIA did not produce false positive or false negative results. Results support the assertion of previous researchers that the antigen detection EIA is a rapid, sensitive, specific, and simple alternative to traditional bioassays for the detection of EEE virus in mosquitoes.

Engineered biomarkers for leprosy diagnosis using labeled and label-free analysis.

PubMed

de Santana, Juliana F; da Silva, Mariângela R B; Picheth, Guilherme F; Yamanaka, Isabel B; Fogaça, Rafaela L; Thomaz-Soccol, Vanete; Machado-de-Avila, Ricardo A; Chávez-Olórtegui, Carlos; Sierakowski, Maria Rita; de Freitas, Rilton Alves; Alvarenga, Larissa M; de Moura, Juliana

2018-09-01

The biotechnological evolution towards the development of antigens to detect leprosy has been progressing. However, the identification of leprosy in paucibacillary patients, based solely on the antigen-antibody interaction still remains a challenge. The complexity of clinical manifestations requires innovative approaches to improve the sensitivity of assays to detect leprosy before the onset of symptoms, thus avoiding disabilities and contributing, indirectly, to reduce transmission. In this study, the strategies employed for early leprosy diagnosis were: i. using a phage-displayed mimotope (APDDPAWQNIFNLRR) which mimics an immunodominant sequence (PPNDPAWQRNDPILQ) of an antigen of Mycobacterium leprae known as Ag85B; ii. engineering the mimotope by adding a C-terminal flexible spacer (SGSG-C); iii. conjugating the mimotope to a carrier protein to provide better exposure to antibodies; iv. amplifying the signal using biotin-streptavidin detection system in an ELISA; and v. coating the optimized mimotope on a quartz crystal microbalance (QCM) sensor for label-free biosensing. The ELISA sensitivity increased up to 91.7% irrespective of the immunological profile of the 132 patients assayed. By using comparative modeling, the M. tuberculosis Ag85B was employed as a template to ascertain which features make the mimotope a good antigen in terms of its specificity. For the first time, a sensitive QCM-based immunosensor to detect anti M. leprae antibodies in human serum was used. M. leprae antibodies could also be detected in the sera of paucibacillary patients; thus, the use of a mimotope-derived synthetic peptide as bait for antibodies in a novel analytical label-free immunoassay for leprosy diagnosis exhibits great potential. Copyright © 2018 Elsevier B.V. All rights reserved.

Enhancement of sensitivity using hybrid stimulus for the diagnosis of prostate cancer based on polydiacetylene (PDA) supramolecules.

PubMed

Kwon, Il Kyoung; Kim, Jun Pyo; Sim, Sang Jun

2010-12-15

In this study, hybrid stimulus was initially introduced to improve the sensitivity of PDA vesicle chip for detection of prostate-specific antigen-α1-antichymotrypsin (PSA-ACT) complex. The strategy of hybrid stimulus on PDA vesicle chip offers the amplification method of fluorescent signal which combines a primary response by the immune reaction of antigen-antibody and a secondary response by the mechanical pressure of pAb-conjugated magnetic beads. As the primary response result on PDA vesicle chip, the PSA-ACT complex in PBS buffer was detected at 10 ng/mL. However, this detection sensitivity was insufficient for diagnosis of prostate cancer because the normal human PSA concentration is less than 4.0 ng/mL. To solve this problem, polyclonal PSA antibody-conjugated magnetic beads were used as an amplifying agent after primary immunoresponse. As a result, the PSA-ACT complex concentrations (as low as 0.1 ng/mL) could be detected in the PBS buffer sample. Therefore, this result can be applied to various fields, such as the detection of cells, proteins, and DNA for sensitive and specific biosensing based on PDA supramolecules. Copyright © 2010 Elsevier B.V. All rights reserved.

Development of a Multiantigen Panel for Improved Detection of Borrelia burgdorferi Infection in Early Lyme Disease

PubMed Central

Panas, Michael W.; Mao, Rong; Delanoy, Michelle; Flanagan, John J.; Binder, Steven R.; Rebman, Alison W.; Montoya, Jose G.; Soloski, Mark J.; Steere, Allen C.; Dattwyler, Raymond J.; Arnaboldi, Paul M.; Aucott, John N.

2015-01-01

The current standard for laboratory diagnosis of Lyme disease in the United States is serologic detection of antibodies against Borrelia burgdorferi. The Centers for Disease Control and Prevention recommends a two-tiered testing algorithm; however, this scheme has limited sensitivity for detecting early Lyme disease. Thus, there is a need to improve diagnostics for Lyme disease at the early stage, when antibiotic treatment is highly efficacious. We examined novel and established antigen markers to develop a multiplex panel that identifies early infection using the combined sensitivity of multiple markers while simultaneously maintaining high specificity by requiring positive results for two markers to designate a positive test. Ten markers were selected from our initial analysis of 62 B. burgdorferi surface proteins and synthetic peptides by assessing binding of IgG and IgM to each in a training set of Lyme disease patient samples and controls. In a validation set, this 10-antigen panel identified a higher proportion of early-Lyme-disease patients as positive at the baseline or posttreatment visit than two-tiered testing (87.5% and 67.5%, respectively; P < 0.05). Equivalent specificities of 100% were observed in 26 healthy controls. Upon further analysis, positivity on the novel 10-antigen panel was associated with longer illness duration and multiple erythema migrans. The improved sensitivity and comparable specificity of our 10-antigen panel compared to two-tiered testing in detecting early B. burgdorferi infection indicates that multiplex analysis, featuring the next generation of markers, could advance diagnostic technology to better aid clinicians in diagnosing and treating early Lyme disease. PMID:26447113

Comparison of (1->3)-β-D-glucan, mannan/anti-mannan antibodies, and Cand-Tec Candida antigen as serum biomarkers for candidemia.

PubMed

Held, Jürgen; Kohlberger, Isabelle; Rappold, Elfriede; Busse Grawitz, Andrea; Häcker, Georg

2013-04-01

We conducted a case-control study using the Fungitell assay, the novel Platelia Candida Antigen (Ag) Plus and Candida Antibody (Ab) Plus assays, and the Cand-Tec latex agglutination test to evaluate the usefulness of (1→3)-β-D-glucan (BDG), mannan antigen with/without anti-mannan antibody, and Cand-Tec Candida antigen measurement for the diagnosis of candidemia. A total of 56 patients fulfilled the inclusion criteria and were enrolled. One hundred patients with bacteremia and 100 patients with sterile blood cultures served as negative controls. In the candidemia group, median (1→3)-β-D-glucan, mannan antigen, and anti-mannan antibody levels were 427 pg/ml, 190 pg/ml, and 18.6 antibody units (AU)/ml, respectively. All three parameters were significantly elevated in patients with candidemia. The sensitivity and specificity were, respectively, 87.5% and 85.5% for (1→3)-β-D-glucan, 58.9% and 97.5% for mannan antigen, 62.5% and 65.0% for anti-mannan antibody, 89.3% and 63.0% for mannan antigen plus anti-mannan antibody, 89.3% and 85.0% for BDG plus mannan antigen, and 13.0% and 93.9% for Cand-Tec Candida antigen. The low mannan antigen sensitivity was in part caused by Candida parapsilosis and Candida guilliermondii fungemias, which were not detected by the Platelia Candida Ag Plus assay. When the cutoff was lowered from 125 pg/ml to 50 pg/ml, mannan antigen sensitivity increased to 69.6% without severely affecting the specificity (93.5%). Contrary to recently published data, superficial candidiasis was not associated with elevated mannan antigen levels, not even after the cutoff was lowered. Combining procalcitonin (PCT) with (1→3)-β-D-glucan to increase specificity provided a limited advantage because the benefit of the combination did not outweigh the loss of sensitivity. Our results demonstrate that the Cand-Tec Candida antigen and the mannan antigen plus anti-mannan antibody measurements have unacceptably low sensitivity or specificity. Of the four tests compared, (1→3)-β-D-glucan and mannan antigen are the superior biomarkers, depending on whether a sensitivity-driven or specificity-driven approach is used.

Comparison of (1→3)-β-d-Glucan, Mannan/Anti-Mannan Antibodies, and Cand-Tec Candida Antigen as Serum Biomarkers for Candidemia

PubMed Central

Kohlberger, Isabelle; Rappold, Elfriede; Busse Grawitz, Andrea; Häcker, Georg

2013-01-01

We conducted a case-control study using the Fungitell assay, the novel Platelia Candida Antigen (Ag) Plus and Candida Antibody (Ab) Plus assays, and the Cand-Tec latex agglutination test to evaluate the usefulness of (1→3)-β-d-glucan (BDG), mannan antigen with/without anti-mannan antibody, and Cand-Tec Candida antigen measurement for the diagnosis of candidemia. A total of 56 patients fulfilled the inclusion criteria and were enrolled. One hundred patients with bacteremia and 100 patients with sterile blood cultures served as negative controls. In the candidemia group, median (1→3)-β-d-glucan, mannan antigen, and anti-mannan antibody levels were 427 pg/ml, 190 pg/ml, and 18.6 antibody units (AU)/ml, respectively. All three parameters were significantly elevated in patients with candidemia. The sensitivity and specificity were, respectively, 87.5% and 85.5% for (1→3)-β-d-glucan, 58.9% and 97.5% for mannan antigen, 62.5% and 65.0% for anti-mannan antibody, 89.3% and 63.0% for mannan antigen plus anti-mannan antibody, 89.3% and 85.0% for BDG plus mannan antigen, and 13.0% and 93.9% for Cand-Tec Candida antigen. The low mannan antigen sensitivity was in part caused by Candida parapsilosis and Candida guilliermondii fungemias, which were not detected by the Platelia Candida Ag Plus assay. When the cutoff was lowered from 125 pg/ml to 50 pg/ml, mannan antigen sensitivity increased to 69.6% without severely affecting the specificity (93.5%). Contrary to recently published data, superficial candidiasis was not associated with elevated mannan antigen levels, not even after the cutoff was lowered. Combining procalcitonin (PCT) with (1→3)-β-d-glucan to increase specificity provided a limited advantage because the benefit of the combination did not outweigh the loss of sensitivity. Our results demonstrate that the Cand-Tec Candida antigen and the mannan antigen plus anti-mannan antibody measurements have unacceptably low sensitivity or specificity. Of the four tests compared, (1→3)-β-d-glucan and mannan antigen are the superior biomarkers, depending on whether a sensitivity-driven or specificity-driven approach is used. PMID:23363830

Monoclonal antibody DS6 detects a tumor-associated sialoglycotope expressed on human serous ovarian carcinomas.

PubMed

Kearse, K P; Smith, N L; Semer, D A; Eagles, L; Finley, J L; Kazmierczak, S; Kovacs, C J; Rodriguez, A A; Kellogg-Wennerberg, A E

2000-12-15

A newly developed murine monoclonal antibody, DS6, immunohistochemically reacts with an antigen, CA6, that is expressed by human serous ovarian carcinomas but not by normal ovarian surface epithelium or mesothelium. CA6 has a limited distribution in normal adult tissues and is most characteristically detected in fallopian tube epithelium, inner urothelium and type 2 pneumocytes. Pre-treatment of tissue sections with either periodic acid or neuraminidase from Vibrio cholerae abolishes immunoreactivity with DS6, indicating that CA6 is a neuraminidase-sensitive and periodic acid-sensitive sialic acid glycoconjugate ("sialoglycotope"). SDS-PAGE of OVCAR5 cell lysates has revealed that the CA6 epitope is expressed on an 80 kDa non-disulfide-linked glycoprotein containing N-linked oligosaccharides. Two-dimensional non-equilibrium pH gradient gel electrophoresis indicates an isoelectric point of approximately 6.2 to 6.5. Comparison of the immunohistochemical distribution of CA6 in human serous ovarian adenocarcinomas has revealed similarities to that of CA125; however, distinct differences and some complementarity of antigen expression were revealed by double-label, 2-color immunohistochemical studies. The DS6-detected CA6 antigen appears to be distinct from other well-characterized tumor-associated antigens, including MUC1, CA125 and the histo-blood group-related antigens sLea, sLex and sTn. Copyright 2000 Wiley-Liss, Inc.

Immune Response to Giardia duodenalis

PubMed Central

Faubert, Gaétan

2000-01-01

The intestinal protozoan Giardia duodenalis is a widespread opportunistic parasite of humans and animals. This parasite inhabits the upper part of the small intestine and has a direct life cycle. After ingestion of cysts, which are the infective stage, the trophozoites emerge from the cysts in the duodenum and attach to the small intestinal mucosa of the host. Since the migration of trophozoites from the lumen of the intestine into surrounding tissues is an unusual occurrence, the immune response to Giardia remains localized. The identification of antigens that play a role in acquired immunity has been difficult because of the occurrence of antigenic variation and because, Giardia being an ubiquituous organism, it is possible that the antigenic profiles of isolates from different geographic areas will vary. Innate-immunity mechanisms play a role in the control and/or severity of the infection. Both humoral and cell-mediated immune responses play a role in acquired immunity, but the mechanisms involved are unknown. A variety of serological assays have been used to detect circulating antibodies in serum. Because of the biological characteristics of the parasite and the lack of suitable antigens, the sensitivity of serological assays remains poor. On the other hand, detection of antigens in feces of infected patients has met with success. Commercial kits are available, and they are reported to be more sensitive than microscopic examination for the detection of giardiasis on a single specimen. PMID:10627490

Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

PubMed Central

Chen, Mu-Xin; Chen, Jia-Xu; Chen, Shao-Hong; Huang, Da-Na; Ai, Lin; Zhang, Ren-Li

2016-01-01

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis. PMID:27417097

Novel graphene-based biosensor for early detection of Zika virus infection.

PubMed

Afsahi, Savannah; Lerner, Mitchell B; Goldstein, Jason M; Lee, Joo; Tang, Xiaoling; Bagarozzi, Dennis A; Pan, Deng; Locascio, Lauren; Walker, Amy; Barron, Francie; Goldsmith, Brett R

2018-02-15

We have developed a cost-effective and portable graphene-enabled biosensor to detect Zika virus with a highly specific immobilized monoclonal antibody. Field Effect Biosensing (FEB) with monoclonal antibodies covalently linked to graphene enables real-time, quantitative detection of native Zika viral (ZIKV) antigens. The percent change in capacitance in response to doses of antigen (ZIKV NS1) coincides with levels of clinical significance with detection of antigen in buffer at concentrations as low as 450pM. Potential diagnostic applications were demonstrated by measuring Zika antigen in a simulated human serum. Selectivity was validated using Japanese Encephalitis NS1, a homologous and potentially cross-reactive viral antigen. Further, the graphene platform can simultaneously provide the advanced quantitative data of nonclinical biophysical kinetics tools, making it adaptable to both clinical research and possible diagnostic applications. The speed, sensitivity, and selectivity of this first-of-its-kind graphene-enabled Zika biosensor make it an ideal candidate for development as a medical diagnostic test. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

The detection of African horse sickness virus antigens and antibodies in young Equidae.

PubMed Central

Hamblin, C.; Anderson, E. C.; Mellor, P. S.; Graham, S. D.; Mertens, P. P.; Burroughs, J. N.

1992-01-01

Four ponies were each inoculated with a different serotype of African horse sickness virus (AHSV) which had been passaged through cell culture in order to achieve attenuation. Three of the ponies died suddenly after showing mild clinical signs, the fourth pony remained clinically normal and was killed at day 38. Infectious AHSV was isolated from blood samples collected at intervals from all four ponies. Positive antigen ELISA reactions were only observed with blood samples from two of the ponies on the two days preceding death. Specific AHSV antibodies were detected by ELISA in serum samples from the other two ponies although one eventually died. African horse sickness viral antigens were detected by ELISA in post-mortem tissue samples collected from all four ponies. No infectious virus could be detected in tissue samples taken post-mortem from the pony which survived African horse sickness (AHS) infection. In the event of a suspected outbreak of AHS it is recommended that sera and heparinized blood should be tested for specific antibodies and AHSV antigen respectively. When available, post-mortem tissues, including spleen, heart, lung and liver, should also be tested for AHSV antigen. Although the ELISA used for the detection of AHSV antigen is highly sensitive and specific, negative ELISA results should be confirmed by virus isolation attempts. PMID:1547837

Development of enzyme linked immunosorbent assay (ELISA) for the detection of root-knot nematode Meloidogyne incognita.

PubMed

Kapur-Ghai, J; Kaur, M; Goel, P

2014-09-01

Root-knot nematodes (Meloidogyne incognita) are obligate, sedentary plant endoparasites that are extremely polyphagous in nature and cause severe economic losses in agriculture. Hence, it is essential to control the parasite at an early stage. For any control strategy to be effective, an early and accurate diagnosis is of paramount importance. Immunoassays have the inherent advantages of sensitivity and specificity; have the potential to identify and quantify these plant-parasitic nematodes. Hence, in the present studies, enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of M.incognita antigens. First an indirect ELISA was developed for detection and titration of anti-M.incognita antibodies. Results indicated as high as 320 K titre of the antisera. Finally competitive inhibition ELISA was developed employing these anti-M.incognita antibodies for detection of M.incognita antigens. Sensitivity of ELISA was 10 fg. Competitive inhibition ELISA developed in the present studies has the potential of being used as an easy, rapid, specific and sensitive diagnostic tool for the detection of M.incognita infection.

Novel rolling circle amplification and DNA origami-based DNA belt-involved signal amplification assay for highly sensitive detection of prostate-specific antigen (PSA).

PubMed

Yan, Juan; Hu, Chongya; Wang, Ping; Liu, Rui; Zuo, Xiaolei; Liu, Xunwei; Song, Shiping; Fan, Chunhai; He, Dannong; Sun, Gang

2014-11-26

Prostate-specific antigen (PSA) is one of the most important biomarkers for the early diagnosis and prognosis of prostate cancer. Although many efforts have been made to achieve significant progress for the detection of PSA, challenges including relative low sensitivity, complicated operation, sophisticated instruments, and high cost remain unsolved. Here, we have developed a strategy combining rolling circle amplification (RCA)-based DNA belts and magnetic bead-based enzyme-linked immunosorbent assay (ELISA) for the highly sensitive and specific detection of PSA. At first, a 96-base circular DNA template was designed and prepared for the following RCA. Single stranded DNA (ssDNA) products from RCA were used as scaffold strand for DNA origami, which was hybridized with three staple strands of DNA. The resulting DNA belts were conjugated with multiple enzymes for signal amplification and then employed to magnetic bead based ELISA for PSA detection. Through our strategy, as low as 50 aM of PSA can be detected with excellent specificity.

Electron spin resonance spectroscopy for immunoassay using iron oxide nanoparticles as probe.

PubMed

Jiang, Jia; Tian, Sizhu; Wang, Kun; Wang, Yang; Zang, Shuang; Yu, Aimin; Zhang, Ziwei

2018-02-01

With the help of iron oxide nanoparticles, electron spin resonance spectroscopy (ESR) was applied to immunoassay. Iron oxide nanoparticles were used as the ESR probe in order to achieve an amplification of the signal resulting from the large amount of Fe 3+ ion enclosed in each nanoparticle. Rabbit IgG was used as antigen to test this method. Polyclonal antibody of rabbit IgG was used as antibody to detect the antigen. Iron oxide nanoparticle with a diameter of either 10 or 30 nm was labeled to the antibody, and Fe 3+ in the nanoparticle was probed for ESR signal. The sepharose beads were used as solid phase to which rabbit IgG was conjugated. The nanoparticle-labeled antibody was first added in the sample containing antigen, and the antigen-conjugated sepharose beads were then added into the sample. The nanoparticle-labeled antibody bound to the antigen on sepharose beads was separated from the sample by centrifugation and measured. We found that the detection ranges of the antigen obtained with nanoparticles of different sizes were different because the amount of antibody on nanoparticles of 10 nm was about one order of magnitude higher than that on nanoparticles of 30 nm. When 10 nm nanoparticle was used as probe, the upper limit of detection was 40.00 μg mL -1 , and the analytical sensitivity was 1.81 μg mL -1 . When 30 nm nanoparticle was used, the upper limit of detection was 3.00 μg mL -1 , and the sensitivity was 0.014 and 0.13 μg mL -1 depending on the ratio of nanoparticle to antibody. Graphical abstract Schematic diagram of procedure and ESR spectra.

Circulating antigen tests and urine reagent strips for diagnosis of active schistosomiasis in endemic areas

PubMed Central

Ochodo, Eleanor A; Gopalakrishna, Gowri; Spek, Bea; Reitsma, Johannes B; van Lieshout, Lisette; Polman, Katja; Lamberton, Poppy; Bossuyt, Patrick Mm; Leeflang, Mariska Mg

2015-01-01

Background Point-of-care (POC) tests for diagnosing schistosomiasis include tests based on circulating antigen detection and urine reagent strip tests. If they had sufficient diagnostic accuracy they could replace conventional microscopy as they provide a quicker answer and are easier to use. Objectives To summarise the diagnostic accuracy of: a) urine reagent strip tests in detecting active Schistosoma haematobium infection, with microscopy as the reference standard; and b) circulating antigen tests for detecting active Schistosoma infection in geographical regions endemic for Schistosoma mansoni or S. haematobium or both, with microscopy as the reference standard. Search methods We searched the electronic databases MEDLINE, EMBASE, BIOSIS, MEDION, and Health Technology Assessment (HTA) without language restriction up to 30 June 2014. Selection criteria We included studies that used microscopy as the reference standard: for S. haematobium, microscopy of urine prepared by filtration, centrifugation, or sedimentation methods; and for S. mansoni, microscopy of stool by Kato-Katz thick smear. We included studies on participants residing in endemic areas only. Data collection and analysis Two review authors independently extracted data, assessed quality of the data using QUADAS-2, and performed meta-analysis where appropriate. Using the variability of test thresholds, we used the hierarchical summary receiver operating characteristic (HSROC) model for all eligible tests (except the circulating cathodic antigen (CCA) POC for S. mansoni, where the bivariate random-effects model was more appropriate). We investigated heterogeneity, and carried out indirect comparisons where data were sufficient. Results for sensitivity and specificity are presented as percentages with 95% confidence intervals (CI). Main results We included 90 studies; 88 from field settings in Africa. The median S. haematobium infection prevalence was 41% (range 1% to 89%) and 36% for S. mansoni (range 8% to 95%). Study design and conduct were poorly reported against current standards. Tests for S. haematobium Urine reagent test strips versus microscopy Compared to microscopy, the detection of microhaematuria on test strips had the highest sensitivity and specificity (sensitivity 75%, 95% CI 71% to 79%; specificity 87%, 95% CI 84% to 90%; 74 studies, 102,447 participants). For proteinuria, sensitivity was 61% and specificity was 82% (82,113 participants); and for leukocyturia, sensitivity was 58% and specificity 61% (1532 participants). However, the difference in overall test accuracy between the urine reagent strips for microhaematuria and proteinuria was not found to be different when we compared separate populations (P = 0.25), or when direct comparisons within the same individuals were performed (paired studies; P = 0.21). When tests were evaluated against the higher quality reference standard (when multiple samples were analysed), sensitivity was marginally lower for microhaematuria (71% vs 75%) and for proteinuria (49% vs 61%). The specificity of these tests was comparable. Antigen assay Compared to microscopy, the CCA test showed considerable heterogeneity; meta-analytic sensitivity estimate was 39%, 95% CI 6% to 73%; specificity 78%, 95% CI 55% to 100% (four studies, 901 participants). Tests for S. mansoni Compared to microscopy, the CCA test meta-analytic estimates for detecting S. mansoni at a single threshold of trace positive were: sensitivity 89% (95% CI 86% to 92%); and specificity 55% (95% CI 46% to 65%; 15 studies, 6091 participants) Against a higher quality reference standard, the sensitivity results were comparable (89% vs 88%) but specificity was higher (66% vs 55%). For the CAA test, sensitivity ranged from 47% to 94%, and specificity from 8% to 100% (4 studies, 1583 participants). Authors' conclusions Among the evaluated tests for S. haematobium infection, microhaematuria correctly detected the largest proportions of infections and non-infections identified by microscopy. The CCA POC test for S. mansoni detects a very large proportion of infections identified by microscopy, but it misclassifies a large proportion of microscopy negatives as positives in endemic areas with a moderate to high prevalence of infection, possibly because the test is potentially more sensitive than microscopy. Plain Language Summary How well do point-of-care tests detect Schistosoma infections in people living inendemic areas? Schistosomiasis, also known as bilharzia, is a parasitic disease common in the tropical and subtropics. Point-of-care tests and urine reagent strip tests are quicker and easier to use than microscopy. We estimate how well these point-of-care tests are able to detect schistosomiasis infections compared with microscopy. We searched for studies published in any language up to 30 June 2014, and we considered the study’s risk of providing biased results. What do the results say? We included 90 studies involving almost 200,000 people, with 88 of these studies carried out in Africa in field settings. Study design and conduct were poorly reported against current expectations. Based on our statistical model, we found: • Among the urine strips for detecting urinary schistosomiasis, the strips for detecting blood were better than those detecting protein or white cells (sensitivity and specificity for blood 75% and 87%; for protein 61% and 82%; and for white cells 58% and 61%, respectively). • For urinary schistosomiasis, the parasite antigen test performance was worse (sensitivity, 39% and specificity, 78%) than urine strips for detecting blood. • For intestinal schistosomiasis, the parasite antigen urine test, detected many infections identified by microscopy but wrongly labelled many uninfected people as sick (sensitivity, 89% and specificity, 55%). What are the consequences of using these tests? If we take 1000 people, of which 410 have urinary schistosomiasis on microscopy testing, then using the strip detecting blood in the urine would misclassify 77 uninfected people as infected, and thus may receive unnecessary treatment; and it would wrongly classify 102 infected people as uninfected, who thus may not receive treatment. If we take 1000 people, of which 360 have intestinal schistosomiasis on microscopy testing, then the antigen test would misclassify 288 uninfected people as infected. These people may be given unnecessary treatment. This test also would wrongly classify 40 infected people as uninfected who thus may not receive treatment. Conclusion of review For urinary schistosomiasis, the urine strip for detecting blood leads to some infected people being missed and some non-infected people being diagnosed with the condition, but is better than the protein or white cell tests. The parasite antigen test is not accurate. For intestinal schistosomiasis, the parasite antigen urine test can wrongly classify many uninfected people as infected. PMID:25758180

Serologic reactivity to purified recombinant and native 29-kilodalton peripheral membrane protein of pathogenic Entamoeba histolytica.

PubMed Central

Flores, B M; Reed, S L; Ravdin, J I; Torian, B E

1993-01-01

The 29-kDa peripheral membrane protein of Entamoeba histolytica has recently been demonstrated to have epitopes on pathogenic clinical isolates which were not detected by monoclonal antibodies on nonpathogenic isolates. To analyze the serological response to this protein, we tested 93 serum specimens (from 33 patients with amebic liver abscess, 7 patients with colitis, 2 patients with ameboma, 18 individuals harboring a nonpathogenic zymodeme strain, 10 healthy Mexican migrant workers, and 23 healthy controls) by enzyme-linked immunosorbent assay (ELISA) using immunoaffinity-purified native or recombinant protein. When tested by ELISA with the native antigen, 79% (26 of 33) of the serum specimens from patients with amebic liver abscess, 4 of 9 serum specimens from symptomatic patients with colitis or ameboma, and serum from one migrant worker were positive. None of the 18 subjects harboring a nonpathogenic strain or 23 control individuals were seropositive to the native antigen (sensitivity, 71%; specificity, 98%). Of 30 serum specimens from patients with amebic liver abscess tested with recombinant antigen, 27 were seropositive (90%). In addition, six patients with colitis or ameboma and two individuals who harbored a nonpathogenic strain were seropositive to the recombinant antigen. One healthy Mexican migrant worker tested positive by both ELISAs (sensitivity, 87%; specificity, 94%). Immunoblotting of 51 serum specimens to sodium dodecyl sulfate-denatured native 29-kDa protein was less sensitive (65%) than ELISA in detecting serum antibodies to the antigen. These results suggest a similar antibody response to native and recombinant antigens (r = 0.86) and support the potential utility of a quantitative assay with defined recombinant antigen for the serodiagnosis of invasive amebiasis in nonendemic areas in conjunction with other diagnostic tools. Images PMID:8314979

Highly sensitive nano-porous lattice biosensor based on localized surface plasmon resonance and interference.

PubMed

Yeom, Se-Hyuk; Kim, Ok-Geun; Kang, Byoung-Ho; Kim, Kyu-Jin; Yuan, Heng; Kwon, Dae-Hyuk; Kim, Hak-Rin; Kang, Shin-Won

2011-11-07

We propose a design for a highly sensitive biosensor based on nanostructured anodized aluminum oxide (AAO) substrates. A gold-deposited AAO substrate exhibits both optical interference and localized surface plasmon resonance (LSPR). In our sensor, application of these disparate optical properties overcomes problems of limited sensitivity, selectivity, and dynamic range seen in similar biosensors. We fabricated uniform periodic nanopore lattice AAO templates by two-step anodizing and assessed their suitability for application in biosensors by characterizing the change in optical response on addition of biomolecules to the AAO template. To determine the suitability of such structures for biosensing applications, we immobilized a layer of C-reactive protein (CRP) antibody on a gold coating atop an AAO template. We then applied a CRP antigen (Ag) atop the immobilized antibody (Ab) layer. The shift in reflectance is interpreted as being caused by the change in refractive index with membrane thickness. Our results confirm that our proposed AAO-based biosensor is highly selective toward detection of CRP antigen, and can measure a change in CRP antigen concentration of 1 fg/ml. This method can provide a simple, fast, and sensitive analysis for protein detection in real-time.

Comparison of antigen-capture ELISA to stool-culture methods for the detection of asymptomatic Entamoeba species infection in Kafer Daoud, Egypt.

PubMed

Abd-Alla, M D; Wahib, A A; Ravdin, J I

2000-05-01

We performed a prospective field study in the village of Kafer Daoud in Menofia, Egypt to compare the fecal culture method with enzyme linked immuno assay (ELISA) for detection of 170 kDa lectin antigen in feces for diagnosis of asymptomatic Entamoeba histolytica and Entamoeba dispar infection. All subjects with E. histolytica or E. dispar infection detected by culture also had positive ELISA for amebic antigen in their feces and an additional 57 Entameoba infections missed by culture were detected by ELISA (P < 0.001 compared to culture). The presence of fecal anti-lectin IgA antibodies and serum anti-LC3 (recombinant cysteine-rich lectin protein) IgG antibodies were positive predictors for E. histolytica infection (P < 0.03). Of interest, infection with Trichomonas hominis but not Blastocystis hominis was positively associated with E. histolytica infection (P < 0.05). In conclusion, ELISA for detection of fecal lectin antigen is a more sensitive method than fecal culture for detecting asymptomatic E. histolytica infection.

Improvement of antigen detection efficiency with the use of two-dimensional photonic crystal as a substrate

NASA Astrophysics Data System (ADS)

Dovzhenko, Dmitriy; Terekhin, Vladimir; Vokhmincev, Kirill; Sukhanova, Alyona; Nabiev, Igor

2017-01-01

Multiplex detection of different antigens in human serum in order to reveal diseases at the early stage is of interest nowadays. There are a lot of biosensors, which use the fluorescent labels for specific detection of analytes. For instance, common method for detection of antigens in human serum samples is enzyme-linked immunosorbent assay (ELISA). One of the most effective ways to improve the sensitivity of this detection method is the use of a substrate that could enhance the fluorescent signal and make it easier to collect. Two-dimensional (2D) photonic crystals are very suitable structures for these purposes because of the ability to enhance the luminescent signal, control the light propagation and perform the analysis directly on its surface. In our study we have calculated optimal parameters for 2D-dimensional photonic crystal consisting of the array of silicon nano-rods, fabricated such photonic crystal on a silicon substrate using reactive ion etching and showed the possibility of its efficient application as a substrate for ELISA detection of human cancer antigens.

Performance comparison of the 4th generation Bio-Rad Laboratories GS HIV Combo Ag/Ab EIA on the EVOLIS™ automated system versus Abbott ARCHITECT HIV Ag/Ab Combo, Ortho Anti-HIV 1+2 EIA on Vitros ECi and Siemens HIV-1/O/2 enhanced on Advia Centaur.

PubMed

Mitchell, Elizabeth O; Stewart, Greg; Bajzik, Olivier; Ferret, Mathieu; Bentsen, Christopher; Shriver, M Kathleen

2013-12-01

A multisite study was conducted to evaluate the performance of the Bio-Rad 4th generation GS HIV Combo Ag/Ab EIA versus Abbott 4th generation ARCHITECT HIV Ag/Ab Combo. The performance of two 3rd generation EIAs, Ortho Diagnostics Anti-HIV 1+2 EIA and Siemens HIV 1/O/2 was also evaluated. Study objective was comparison of analytical HIV-1 p24 antigen detection, sensitivity in HIV-1 seroconversion panels, specificity in blood donors and two HIV false reactive panels. Analytical sensitivity was evaluated with International HIV-1 p24 antigen standards, the AFFSAPS (pg/mL) and WHO 90/636 (IU/mL) standards; sensitivity in acute infection was compared on 55 seroconversion samples, and specificity was evaluated on 1000 negative blood donors and two false reactive panels. GS HIV Combo Ag/Ab demonstrated better analytical HIV antigen sensitivity compared to ARCHITECT HIV Ag/Ab Combo: 0.41 IU/mL versus 1.2 IU/mL (WHO) and 12.7 pg/mL versus 20.1 pg/mL (AFSSAPS); GS HIV Combo Ag/Ab EIA also demonstrated slightly better specificity compared to ARCHITECT HIV Ag/Ab Combo (100% versus 99.7%). The 4th generation HIV Combo tests detected seroconversion 7-11 days earlier than the 3rd generation HIV antibody only EIAs. Both 4th generation immunoassays demonstrated excellent performance in sensitivity, with the reduction of the serological window period (7-11 days earlier detection than the 3rd generation HIV tests). However, GS HIV Combo Ag/Ab demonstrated improved HIV antigen analytical sensitivity and slightly better specificity when compared to ARCHITECT HIV Ag/Ab Combo assay, with higher positive predictive values (PPV) for low prevalence populations. Copyright © 2013 Elsevier B.V. All rights reserved.

Detection of tumor markers in prostate cancer and comparison of sensitivity between real time and nested PCR.

PubMed

Matsuoka, Takayuki; Shigemura, Katsumi; Yamamichi, Fukashi; Fujisawa, Masato; Kawabata, Masato; Shirakawa, Toshiro

2012-06-27

The objective of this study is to investigate and compare the sensitivity in conventional PCR, quantitative real time PCR, nested PCR and western blots for detection of prostate cancer tumor markers using prostate cancer (PCa) cells. We performed conventional PCR, quantitative real time PCR, nested PCR, and western blots using 5 kinds of PCa cells. Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and androgen receptor (AR) were compared for their detection sensitivity by real time PCR and nested PCR. In real time PCR, there was a significant correlation between cell number and the RNA concentration obtained (R(2)=0.9944) for PSA, PSMA, and AR. We found it possible to detect these markers from a single LNCaP cell in both real time and nested PCR. By comparison, nested PCR reached a linear curve in fewer PCR cycles than real time PCR, suggesting that nested PCR may offer PCR results more quickly than real time PCR. In conclusion, nested PCR may offer tumor maker detection in PCa cells more quickly (with fewer PCR cycles) with the same high sensitivity as real time PCR. Further study is necessary to establish and evaluate the best tool for PCa tumor marker detection.

Duplex microfluidic SERS detection of pathogen antigens with nanoyeast single-chain variable fragments.

PubMed

Wang, Yuling; Rauf, Sakandar; Grewal, Yadveer S; Spadafora, Lauren J; Shiddiky, Muhammad J A; Cangelosi, Gerard A; Schlücker, Sebastian; Trau, Matt

2014-10-07

Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications.

Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

PubMed

Pritt, Bobbi S; Patel, Robin; Kirn, Thomas J; Thomson, Richard B

2016-10-01

Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis

PubMed Central

Nascimento, Marilia B.; Santos, Wagner J. T.; Medeiros, Zulma M.; Lima Neto, Adelino S.; Costa, Dorcas L.; Costa, Carlos H. N.; dos Santos, Washington L. C.; Pontes de Carvalho, Lain C.; Oliveira, Geraldo G. S.

2017-01-01

Current strategies for the control of zoonotic visceral leishmaniasis (VL) rely on its efficient diagnosis in both human and canine hosts. The most promising and cost effective approach is based on serologic assays with recombinant proteins. However, no single antigen has been found so far which can be effectively used to detect the disease in both dogs and humans. In previous works, we identified Leishmania infantum antigens with potential for the serodiagnosis of VL. Here, we aimed to expand the panel of the available antigens for VL diagnosis through another screening of a genomic expression library. Seven different protein-coding gene fragments were identified, five of which encoding proteins which have not been previously studied in Leishmania and rich in repetitive motifs. Poly-histidine tagged polypeptides were generated from six genes and evaluated for their potential for diagnosis of VL by ELISA (Enzyme Linked ImmunoSorbent Assay) with sera from infected humans and dogs. None of those was valid for the detection of human VL (26–52% sensitivity) although their performance was increased in the canine sera (48–91% sensitivity), with one polypeptide useful for the diagnosis of canine leishmaniasis. Next, we assayed a mixture of three antigens, found to be best for human or canine VL, among 13 identified through different screenings. This “Mix” resulted in similar levels of sensitivity for both human (84%) and canine (88%) sera. With improvements, this validates the use of multiple proteins, including antigens identified here, as components of a single system for the diagnosis of both forms of leishmaniasis. PMID:28957332

Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis.

PubMed

Magalhães, Franklin B; Castro Neto, Artur L; Nascimento, Marilia B; Santos, Wagner J T; Medeiros, Zulma M; Lima Neto, Adelino S; Costa, Dorcas L; Costa, Carlos H N; Dos Santos, Washington L C; Pontes de Carvalho, Lain C; Oliveira, Geraldo G S; de Melo Neto, Osvaldo P

2017-01-01

Current strategies for the control of zoonotic visceral leishmaniasis (VL) rely on its efficient diagnosis in both human and canine hosts. The most promising and cost effective approach is based on serologic assays with recombinant proteins. However, no single antigen has been found so far which can be effectively used to detect the disease in both dogs and humans. In previous works, we identified Leishmania infantum antigens with potential for the serodiagnosis of VL. Here, we aimed to expand the panel of the available antigens for VL diagnosis through another screening of a genomic expression library. Seven different protein-coding gene fragments were identified, five of which encoding proteins which have not been previously studied in Leishmania and rich in repetitive motifs. Poly-histidine tagged polypeptides were generated from six genes and evaluated for their potential for diagnosis of VL by ELISA (Enzyme Linked ImmunoSorbent Assay) with sera from infected humans and dogs. None of those was valid for the detection of human VL (26-52% sensitivity) although their performance was increased in the canine sera (48-91% sensitivity), with one polypeptide useful for the diagnosis of canine leishmaniasis. Next, we assayed a mixture of three antigens, found to be best for human or canine VL, among 13 identified through different screenings. This "Mix" resulted in similar levels of sensitivity for both human (84%) and canine (88%) sera. With improvements, this validates the use of multiple proteins, including antigens identified here, as components of a single system for the diagnosis of both forms of leishmaniasis.

Construction and Potential Applications of Biosensors for Proteins in Clinical Laboratory Diagnosis

PubMed Central

Liu, Xuan

2017-01-01

Biosensors for proteins have shown attractive advantages compared to traditional techniques in clinical laboratory diagnosis. In virtue of modern fabrication modes and detection techniques, various immunosensing platforms have been reported on basis of the specific recognition between antigen-antibody pairs. In addition to profit from the development of nanotechnology and molecular biology, diverse fabrication and signal amplification strategies have been designed for detection of protein antigens, which has led to great achievements in fast quantitative and simultaneous testing with extremely high sensitivity and specificity. Besides antigens, determination of antibodies also possesses great significance for clinical laboratory diagnosis. In this review, we will categorize recent immunosensors for proteins by different detection techniques. The basic conception of detection techniques, sensing mechanisms, and the relevant signal amplification strategies are introduced. Since antibodies and antigens have an equal position to each other in immunosensing, all biosensing strategies for antigens can be extended to antibodies under appropriate optimizations. Biosensors for antibodies are summarized, focusing on potential applications in clinical laboratory diagnosis, such as a series of biomarkers for infectious diseases and autoimmune diseases, and an evaluation of vaccine immunity. The excellent performances of these biosensors provide a prospective space for future antibody-detection-based disease serodiagnosis. PMID:29207528

Construction and Potential Applications of Biosensors for Proteins in Clinical Laboratory Diagnosis.

PubMed

Liu, Xuan; Jiang, Hui

2017-12-04

Biosensors for proteins have shown attractive advantages compared to traditional techniques in clinical laboratory diagnosis. In virtue of modern fabrication modes and detection techniques, various immunosensing platforms have been reported on basis of the specific recognition between antigen-antibody pairs. In addition to profit from the development of nanotechnology and molecular biology, diverse fabrication and signal amplification strategies have been designed for detection of protein antigens, which has led to great achievements in fast quantitative and simultaneous testing with extremely high sensitivity and specificity. Besides antigens, determination of antibodies also possesses great significance for clinical laboratory diagnosis. In this review, we will categorize recent immunosensors for proteins by different detection techniques. The basic conception of detection techniques, sensing mechanisms, and the relevant signal amplification strategies are introduced. Since antibodies and antigens have an equal position to each other in immunosensing, all biosensing strategies for antigens can be extended to antibodies under appropriate optimizations. Biosensors for antibodies are summarized, focusing on potential applications in clinical laboratory diagnosis, such as a series of biomarkers for infectious diseases and autoimmune diseases, and an evaluation of vaccine immunity. The excellent performances of these biosensors provide a prospective space for future antibody-detection-based disease serodiagnosis.

Use of long term dermal sensitization followed by intratracheal challenge method to identify low-dose chemical-induced respiratory allergic responses in mice.

PubMed

Fukuyama, Tomoki; Ueda, Hideo; Hayashi, Koichi; Tajima, Yukari; Shuto, Yasufumi; Saito, Toru R; Harada, Takanori; Kosaka, Tadashi

2008-10-01

The inhalation of many types of chemicals, including pesticides, perfumes, and other low-molecular weight chemicals, is a leading cause of allergic respiratory diseases. We attempted to develop a new test protocol to detect environmental chemical-related respiratory hypersensitivity at low and weakly immunogenic doses. We used long-term dermal sensitization followed by a low-dose intratracheal challenge to evaluate sensitization by the well-known respiratory sensitizers trimellitic anhydride (TMA) and toluene diisocyanate (TDI) and the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). After topically sensitizing BALB/c mice (9 times in 3 weeks) and challenging them intratracheally with TMA, TDI, or DNCB, we assayed differential cell counts and chemokine levels in bronchoalveolar lavage fluid (BALF); lymphocyte counts, surface antigen expression of B cells, and local cytokine production in lung-associated lymph nodes (LNs); and antigen-specific IgE levels in serum and BALF. TMA induced marked increases in antigen-specific IgE levels in both serum and BALF, proliferation of eosinophils and chemokines (MCP-1, eotaxin, and MIP-1beta) in BALF, and proliferation of Th2 cytokines (interleukin (IL)-4, IL-10, and IL-13) in restimulated LN cells. TDI induced marked increases in levels of cytokines (IL-4, IL-10, IL-13, and IFN-gamma) produced by restimulated LN cells. In contrast, DNCB treatment yielded, at most, small, nonsignificant increases in all parameters. Our protocol thus detected respiratory allergic responses to low-molecular weight chemicals and may be useful for detecting environmental chemical-related respiratory allergy.

Detection of Chagas infections using Trypanosoma evansi crude antigen demonstrates high cross-reactions with Trypanosoma cruzi.

PubMed

Desquesnes, Marc; Bosseno, Marie-France; Brenière, Simone Frédérique

2007-07-01

Antigenic similarities between salivarian trypanosomes are known for a long time, but similarities between salivarian and stercorarian trypanosomes have been very little investigated. Phylogenetically, these genus and species appear to be far. However, in a preliminary work we had shown strong reactions of chagasic human sera using T. evansi antigens in Western-blotting and ELISA. In the current work an ELISA test using T. evansi crude antigens was probed with one hundred and two sera of chagasic Bolivian patients previously diagnosed which presented different pathologies. The sensitivity of the ELISA T. evansi was 92.6% similar to that of ELISA T. cruzi. The specificity evaluated using 20 sera of patients infected by Leishmania sp. reaches a comparable value of that obtained with the T. cruzi immunofluorescent assay. Finally, the sensitivity and the specificity of the ELISA T. evansi were not really different from conventional serology of Chagas. In spite of their taxonomic position in various sections and their old divergence, these observations prove a strong antigenic community between T. cruzi and T. evansi. Consequently, the common antigens which remain to be characterized, could be an alternative source of antigen for the detection of antibodies against T. cruzi. Given that T. evansi seems to have strong antigenic communities with the majority of the pathogenic current trypanosomoses of mammals, it is very attractive to identify and characterize these highly conserved antigens which could be suitable targets to develop tools for diagnosis, prophylaxy and chemotherapy against several human and animal trypanosomoses.

Stool antigen immunodetection for diagnosis of Giardia duodenalis infection in human subjects with HIV and cancer.

PubMed

Nooshadokht, Maryam; Kalantari-Khandani, Behjat; Sharifi, Iraj; Kamyabi, Hossein; Liyanage, Namal P M; Lagenaur, Laurel A; Kagnoff, Martin F; Singer, Steven M; Babaei, Zahra; Solaymani-Mohammadi, Shahram

2017-10-01

Human infection with the protozoan parasite Giardia duodenalis is one the most common parasitic diseases worldwide. Higher incidence rates of giardiasis have been reported from human subjects with multiple debilitating chronic conditions, including hypogammaglobulinemia and common variable immunodeficiency (CVID). In the current study, stool specimens were collected from 199 individuals diagnosed with HIV or cancer and immunocompetent subjects. The sensitivity of microscopy-based detection on fresh stool preparations, trichrome staining and stool antigen immunodetection for the diagnosis of G. duodenalis were 36%, 45.5% and 100%, respectively when compared with a highly sensitive stool-based PCR method as the gold standard. Further multilocus molecular analyses using glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) loci demonstrated that the AI genotype of G. duodenalis was the most prevalent, followed by the AII genotype and mixed (AI+B) infections. We concluded that stool antigen immunodetection-based immunoassays and stool-based PCR amplification had comparable sensitivity and specificity for the diagnosis of G. duodenalis infections in these populations. Stool antigen detection-based diagnostic modalities are rapid and accurate and may offer alternatives to conventional microscopy and PCR-based diagnostic methods for the diagnosis of G. duodenalis in human subjects living with HIV or cancer. Copyright © 2017. Published by Elsevier B.V.

Detection of prostate-specific antigen with biomolecule-gated AlGaN/GaN high electron mobility transistors

NASA Astrophysics Data System (ADS)

Li, Jia-dong; Cheng, Jun-jie; Miao, Bin; Wei, Xiao-wei; Xie, Jie; Zhang, Jin-cheng; Zhang, Zhi-qiang; Wu, Dong-min

2014-07-01

In order to improve the sensitivity of AlGaN/GaN high electron mobility transistor (HEMT) biosensors, a simple biomolecule-gated AlGaN/GaN HEMT structure was designed and successfully fabricated for prostate specific antigen (PSA) detection. UV/ozone was used to oxidize the GaN surface and then a 3-aminopropyl trimethoxysilane (APTES) self-assembled monolayer was bound to the sensing region. This monolayer serves as a binding layer for attachment of the prostate specific antibody (anti-PSA). The biomolecule-gated AlGaN/GaN HEMT sensor shows a rapid and sensitive response when the target prostate-specific antigen in buffer solution was added to the antibody-immobilized sensing area. The current change showed a logarithm relationship against the PSA concentration from 0.1 pg/ml to 0.993 ng/ml. The sensitivity of 0.215% is determined for 0.1 pg/ml PSA solution. The above experimental result of the biomolecule-gated AlGaN/GaN HEMT biosensor suggested that this biosensor might be a useful tool for prostate cancer screening.

Detection of antibody-antigen reaction by silicon nitride slot-ring biosensors using protein G

NASA Astrophysics Data System (ADS)

Taniguchi, Tomoya; Hirowatari, Anna; Ikeda, Takeshi; Fukuyama, Masataka; Amemiya, Yoshiteru; Kuroda, Akio; Yokoyama, Shin

2016-04-01

Biosensors using ring resonators with silicon nitride (SiN) slot waveguides have been fabricated. The temperature coefficient of the resonance wavelength of the SiN resonator is 0.006 nm/°C, which is one order of magnitude smaller than that of Si. The sensitivity of the biosensor has been improved by using slot waveguide together with Si-binding protein (designated as Si-tag), which bonds to SiN or SiO2 surface, as an anchoring molecule to immobilize bioreceptors on the SiN rings in an oriented manner. Furthermore, the protein G, which strongly bonds to many kinds of mammalian antibodies only by mixing the antibody solution, is used to efficiently immobilize the antigen on the sensor surface. By means of these devises the sensitivity of the biosensor has been improved by factor of 10-100 compared with that of normal Si ring resonator sensors without slot. Then the detection of prostate specific antigen (PSA) with the sensitivity of ~1×10-8 g/ml, which is the concentration of strongly suspicious for the prostate cancer, has been achieved.

Evaluation of whole blood IFNgamma test using PPD and recombinant antigen challenge for diagnosis of pulmonary and extra-pulmonary tuberculosis.

PubMed

Kalantri, Yatiraj; Hemvani, Nanda; Chitnis, D S

2009-06-01

Quantiferon TB gold (QFT-G) with recombinant antigen cocktail is well evaluated for diagnosis of pulmonary tuberculosis (PTB). However, diagnosis of extra-pulmonary tuberculosis (EPTB) is more difficult due to limitations of conventional techniques. This study compares recombinant antigens based QFT-G and low cost PPD based interferon test for the diagnosis of PTB and EPTB. IFNgamma release, with recombinant antigens and PPD, was assayed by ELISA from 140 cases of EPTB, 100 cases of PTB along with acid fast bacillus (AFB) detection, AFB culture on LJ and MGIT BACTEC. Sensitivity and specificity for QFT-G recombinant antigens was 84.29% and 96%, while for PPD based interferon was 70% and 84% for EPTB group. The sensitivity was far superior to AFB smear and culture for both the antigens. Nine samples were identified as non-tubercular mycobacteria (NTM) in the EPTB group and all were negative for QFT-G, but six of them were positive for PPD based test. Results of the study show that QFT-G using recombinant antigen is sensitive and specific for both PTB and EPTB diagnosis. The PPD based test is economic and offers comparable performance for PTB and EPTB diagnosis and also useful for diagnosis of NTM.

Preliminary multiplex microarray IgG immunoassay for the diagnosis of toxoplasmosis and rubella.

PubMed

Baschirotto, Priscila T; Krieger, Marco A; Foti, Leonardo

2017-06-01

During pregnancy, toxoplasmosis and rubella can cause serious damage to the mother and the foetus through vertical transmission. Early diagnosis enables implementation of health measures aimed at preventing vertical transmission and minimising damage caused by these diseases. Here, we report the development of a multiplex assay for simultaneous detection of IgG antibodies produced during toxoplasmosis and rubella infection. This assay is based on xMap technology. Initially, by singleplex assays, we evaluated the following antigens: one Toxoplasma gondii lysate; two antigenic extracts of T. gondii (TOX8131 and TOX8122); fragments of T. gondii antigens [SAG-1 (amino acids 45-198), GRA-7 (24-100), GRA-1 (57-149), ROP-4, and MIC-3 (234-306)]; two chimeric antigens composed of fragments of SAG-1, GRA-7, and P35 (CTOX and CTOXH); and fragments of Rubella virus antigens [E-1 (157-176, 213-239, 374-390), E-2 (31-105), and C (1-123)]. A multiplex assay to simultaneously diagnose toxoplasmosis and rubella was designed with the best-performing antigens in singleplex and multiplex assays, which included CTOXH, T. gondii lysate, TOX8131, E-1, and E-2. The multiplex assay showed 100% sensitivity and specificity for anti-T. gondii IgG detection and 95.6% sensitivity and 100% specificity for anti-R. virus IgG detection. We found that, despite the difficulties related to developing multiplex systems, different types of antigens (extracts and recombinant proteins) can be used to develop high-performance diagnostic tests. The assay developed is suitable to screen for prior T. gondii and R. virus infections, because it is a rapid, high-throughput, low-cost alternative to the current standard diagnostic tools, which require multiple individual tests.

Anti-human neutrophil antigen-1a, -1b, and -2 antibodies in neonates and children with immune neutropenias analyzed by extracted granulocyte antigen immunofluorescence assay.

PubMed

Onodera, Rie; Kurita, Emi; Taniguchi, Kikuyo; Karakawa, Shuhei; Okada, Satoshi; Kihara, Hirotaka; Fujii, Teruhisa; Kobayashi, Masao

2017-11-01

Anti-human neutrophil antigen (HNA) antibodies have been implicated in the development of neonatal alloimmune neutropenia (NAN) and autoimmune neutropenia (AIN). There are many conventional assay methods that detect anti-HNA antibodies. However, a method to measure multiple samples and detect several anti-HNA antibodies simultaneously is needed. We developed a new method, the extracted granulocyte antigen immunofluorescence assay (EGIFA), to analyze anti-HNA-1a, -1b, and -2 antibodies in sera. The results obtained by EGIFA were evaluated in comparison with those from several standard assay methods. Anti-HNA antibodies in serum samples from nine familial cases with suspected NAN (n = 19) and children with suspected AIN (n = 88) were also measured by EGIFA. The evaluation of nine serum samples with anti-HNA antibodies suggested that EGIFA demonstrated equivalent specificity and superior sensitivity to monoclonal antibody-specific immobilization of granulocyte antigens and had comparable sensitivity to the granulocyte indirect immunofluorescence test. EGIFA successfully detected anti-HNA-1a or -1b antibodies in seven of nine familial cases with suspected NAN. EGIFA detected anti-HNA antibodies in 40.9% of children with suspected AIN. Among them, isolated anti-HNA-1a or -1b antibody was detected in 4.5 or 12.5% of children, respectively, and anti-HNA-2 antibody was identified in 3.4% of children. The 30.8% (16 of 52) of children negative for anti-HNA antibody by EGIFA were positive for anti-HLA antibody. EGIFA facilitated the measurement of anti-HNA-1a, -1b, and/or -2 antibodies in sera. The prompt measurement of anti-HNA antibodies will improve the diagnosis and clinical management of patients with suspected NAN or AIN. © 2017 AABB.

Assignment of C1q-binding HLA antibodies as unacceptable HLA antigens avoids positive CDC-crossmatches prior to transplantation of deceased donor organs.

PubMed

Juhl, David; Marget, Matthias; Hallensleben, Michael; Görg, Siegfried; Ziemann, Malte

2017-03-01

Soon, a virtual crossmatch shall replace the complement-dependent cytotoxicity (CDC) allocation crossmatch in the Eurotransplant region. To prevent positive CDC-crossmatches in the recipient centre, careful definition of unacceptable antigens is necessary. For highly sensitized patients, this is difficult by CDC alone. Assignment of all antibodies detected by sensitive assays, however, could prevent organ allocation. To assess the usefulness of the Luminex C1q-assay to prevent positive CDC-crossmatches, all CDC-crossmatches performed prior to deceased kidney transplantation in a 16-month-period were reviewed. Sera causing positive crossmatches were investigated by the C1q-assay. 31 out of 1432 crossmatches (2.2%) were positive. Sera involved in 26 positive crossmatches were available. C1q-binding donor-specific antibodies were detected in 19 sera (73.1%). The other sera were from recipients without any HLA antibodies detectable by CDC or common solid phase assays. Three patients had known Non-HLA antibodies causing positive CDC-results. Four crossmatches were only weak positive. Therefore, avoidance of donors with HLA antigens against whom C1q-binding antibodies were detected would have prevented all positive crossmatches due to HLA antibodies. Provided that all HLA specificities against which antibodies are detected by the Luminex C1q-assay are considered as unacceptable antigens, CDC-crossmatches prior to transplantation might safely be omitted in many patients. They should be maintained in highly immunized patients, however, for whom assignment of all C1q-positive antibodies as unacceptable antigens could lead to a significant delay or even prevention of transplantation. Copyright © 2017 Elsevier B.V. All rights reserved.

Organic electrochemical transistor based immunosensor for prostate specific antigen (PSA) detection using gold nanoparticles for signal amplification.

PubMed

Kim, Duck-Jin; Lee, Nae-Eung; Park, Joon-Shik; Park, In-Jun; Kim, Jung-Gu; Cho, Hyoung J

2010-07-15

We demonstrated a highly sensitive organic electrochemical transistor (OECT) based immunosensor with a low detection limit for prostate specific antigen/alpha1-antichymotrypsin (PSA-ACT) complex. The poly(styrenesulfonate) doped poly(3,4-ethylenedioxythiophene) (PEDOT:PSS) based OECT with secondary antibody conjugated gold nanoparticles (AuNPs) provided a detection limit of the PSA-ACT complex as low as 1pg/ml, as well as improved sensitivity and a dynamic range, due to the role of AuNPs in the signal amplification. The sensor performances were particularly improved in the lower concentration range where the detection is clinically important for the preoperative diagnosis and screening of prostate cancer. This result shows that the OECT-based immunosensor can be used as a transducer platform acceptable to the point-of-care (POC) diagnostic systems and demonstrates adaptability of organic electronics to clinical applications. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Performance of ELISA antigens prepared from 8 isolates of porcine reproductive and respiratory syndrome virus with homologous and heterologous antisera.

PubMed Central

Cho, H J; Entz, S C; Magar, R; Joo, H S

1997-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) ELISA antigens of high quality were produced using 8 different isolates of PRRSV: the European Lelystad virus (LV), the U.S. MN-1b, 89-46448, 93-44927, and 93-24025B, and the Canadian LHVA-93-3, PA-8 and GH-6 virus isolates. The performance of each of these 8 antigens and a commercial PRRSV antibody test kit (Idexx's HerdChek) were measured against antisera raised in 5 groups of 6 piglets inoculated with either LV, MN-1b, 89-46448, 93-44927, or 93-24025B. Among the 8 isolates, the 89-46448 isolate produced the broadest spectrum of antigen and resulted in earlier detection of antibodies to various North American PRRSV isolates, followed by MN-1b as the 2nd best ELISA antigen for the detection of North American PRRSV antibodies. The GH-6 and PA-8 viral antigens exhibited restricted detection of PRRSV antibodies. The LV and 89-46448 combined antigens produced the best performance for the detection of antibodies against both European and North American antigenic types of PRRSV. Using 173 panel samples collected at 11 to 60 d after intranasal inoculation with 1 of the 5 PRRSV isolates, the sensitivities of the indirect ELISA used were 73.4%, 98.3%, 90.8%, 98.3%, 83.2%, 93.1%, 77.1%, 64.2%, 98.8% and 95.9% for LV, MN-1b, LHVA-93-3, 89-46448, 93-44927, 93-24025B, PA-8, GH-6 antigens, 89-46448-LV combined antigens and Idexx's PRRSV antibody test kit, respectively. All 8 antigens gave negative results with preinfection porcine sera (n = 30); high background or nonspecific reactions were not observed with the antigens. PMID:9342455

Imaging of blood antigen distribution on blood cells by thermal lens microscopy

NASA Astrophysics Data System (ADS)

Kimura, Hiroko; Sekiguchi, Kazuya; Nagao, Fumiko; Mukaida, Masahiro; Kitamori, Takehiko; Sawada, Tsuguo

2000-05-01

Blood group antigens on a cell were measured by a new microscopic method, i.e. thermal lens microscopy which involves spectrometry using a laser-induced thermal-lens effect. The blood group antigen was immunologically stained using antibody labeled with colloidal gold. Human leukocyte antigens (HLA) on lymphocytes and mononuclear leukocytes were observed by the thermal lens microscope, and Lewis blood group antigens on erythrocytes and polymorphonuclear leukocytes were also observed. The antigen distribution on each cell-surface was imaged using this technique. In spite of convex surface of living cells, colloidal gold was correctly quantified by adjusting the deviation of the focal point of the probe laser by the phase of the signal. In the measurement of leukocyte antigens, antigens of HLA-A, -B, -C loci on the lymphocytes were identified and quantitated by using a single cell. The image of HLA-A, -B, -C antigen distribution on a mononuclear leukocyte was obtained. In the measurement of erythrocyte antigens, a small quantity of Lewis antigens was detected on the cord erythrocytes. Localized small quantities of membrane antigens are better quantitated without extraction or cytolysis. Our thermal lens microscope is a powerful and highly sensitive analytical tool for detecting and quantitating localized antigens in single cells and/or cell-surface-associated molecules.

A Highly Selective and Sensitive Fluorescence Detection Method of Glyphosate Based on an Immune Reaction Strategy of Carbon Dot Labeled Antibody and Antigen Magnetic Beads.

PubMed

Wang, Duo; Lin, Bixia; Cao, Yujuan; Guo, Manli; Yu, Ying

2016-08-03

A sensitive fluorescence detection method for glyphosate (GLY) was established based on immune reaction. First, carbon dot labeled antibodies (lgG-CDs) which were able to specifically identify glyphosate were prepared with the environmentally friendly carbon dots (CDs) and glyphosate antibody (lgG). lgG-CDs could be used to in situ visualize the distribution of glyphosate in plant tissues. In order to eliminate the effects of excess lgG-CDs on the determination of GLY, antigen magnetic beads Fe3O4-GLY based on magnetic nanoparticles Fe3O4 and glyphosate were constructed and utilized to couple with the excess lgG-CDs. After magnetic separation to remove antigen magnetic beads, there was a linear relationship between the fluorescence intensity of lgG-CDs and the logarithmic concentration of glyphosate in the range of 0.01-80 μg/mL with a detection limit of 8 ng/mL. The method was used for the detection of glyphosate in Pearl River water, tea, and soil samples with satisfactory recovery ratio between 87.4% and 103.7%.

Surface stress sensor based on MEMS Fabry-Perot interferometer with high wavelength selectivity for label-free biosensing

NASA Astrophysics Data System (ADS)

Takahashi, Toshiaki; Hizawa, Takeshi; Misawa, Nobuo; Taki, Miki; Sawada, Kazuaki; Takahashi, Kazuhiro

2018-05-01

We have developed a surface stress sensor based on a microelectromechanical Fabry-Perot interferometer with high wavelength selectivity by using Au half-mirrors, for highly sensitive label-free biosensing. When the target molecule is adsorbed by the antigen-antibody reaction onto a movable membrane with a thin Au film, which acts as an upper mirror of the optical interferometer, the amount of deflection of the movable membrane deflected by the change in surface stress can be detected with high sensitivity. To improve the signal at the small membrane deflection region of this biosensor resulting in detection of low concentration molecules, by integrating 50 nm-thick Au half-mirrors, the wavelength selectivity of the optical interferometer has been successfully improved 6.6 times. Furthermore, the peak shift in the reflection spectrum due to the adsorption of bovine serum albumin (BSA) antigen with a concentration of 10 ng ml-l by the antigen-antibody reaction was spectroscopically measured on the fabricated optical interferometer, and the deflection amount of the movable membrane after 10 min treatment was 2.4 times larger than that of nonspecific adsorption with the avidin molecules. This result indicated that the proposed sensor can be used for selective detection of low-concentration target antigen molecules.

Sonicated Diagnostic Immunoblot for Bartonellosis

PubMed Central

Mallqui, Vania; Speelmon, Emily C.; Verástegui, Manuela; Maguiña-Vargas, Ciro; Pinell-Salles, Paula; Lavarello, Rosa; Delgado, Jose; Kosek, Margaret; Romero, Sofia; Arana, Yanina; Gilman, Robert H.

2000-01-01

Two simple Bartonella bacilliformis immunoblot preparation methods were developed. Antigen was prepared by two different methods: sonication of whole organisms or glycine extraction. Both methods were then tested for sensitivity and specificity. Well-defined control sera were utilized in the development of these diagnostic immunoblots, and possible cross-reactions were thoroughly examined. Sera investigated for cross-reaction with these diagnostic antigens were drawn from patients with brucellosis, chlamydiosis, Q fever, and cat scratch disease, all of whom were from regions where bartonellosis is not endemic. While both immunoblots yielded reasonable sensitivity and high specificity, we recommend the use of the sonicated immunoblot, which has a higher sensitivity when used to detect acute disease and produces fewer cross-reactions. The sonicated immunoblot reported here is 94% sensitive to chronic bartonellosis and 70% sensitive to acute bartonellosis. In a healthy group, it is 100% specific. This immunoblot preparation requires a simple sonication protocol for the harvesting of B. bacilliformis antigens and is well suited for use in regions of endemicity. PMID:10618267

The Role of TPS and TPA in the Diagnostics of Distant Metastases.

PubMed

Kucera, Radek; Topolcan, Ondrej; Fiala, Ondrej; Kinkorova, Judita; Treska, Vladislav; Zedníková, Ilona; Slouka, David; Simanek, Vaclav; Safanda, Martin; Babuska, Vaclav

2016-02-01

The aim of the study was to assess the degree to which tissue polypeptide antigen (TPA) and tissue polypeptide-specific antigen (TPS), as well as carcinoembryonic antigen (CEA), can assist in the detection of distant metastases. We assessed 157 patients with colorectal and breast cancer divided into two groups. The first was a group of patients with cancer at stages 1, 2 and 3; the second was a group of patients with cancer at stage 4 with metastasis. We found significantly higher levels of all biomarkers in the metastatic group compared to the group with cancer at stages 1-3 (p<0.0001). The calculated area under the receiver operating characteristic (ROC) curve was 0.9929 for TPS, 0.9337 for TPA and 0.7234 for CEA. The cut-off was calculated for each biomarker at 95% specificity, TPS cut-off=255 IU/l (sensitivity 95%), TPA cut-off=200 IU/l (sensitivity 70%) and CEA cut-off=18 μg/l (sensitivity 37%). We suggest combining CEA with TPS or TPA in the detection of distant metastases or using only cytokeratins. This approach can significantly increase the quality of detection of the metastatic process. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

A new real-time method for investigation of affinity properties and binding kinetics of magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Orlov, Alexey V.; Nikitin, Maxim P.; Bragina, Vera A.; Znoyko, Sergey L.; Zaikina, Marina N.; Ksenevich, Tatiana I.; Gorshkov, Boris G.; Nikitin, Petr I.

2015-04-01

A method for quantitative investigation of affinity constants of receptors immobilized on magnetic nanoparticles (MP) is developed based on spectral correlation interferometry (SCI). The SCI records with a picometer resolution the thickness changes of a layer of molecules or nanoparticles due to a biochemical reaction on a cover slip, averaged over the sensing area. The method is compatible with other types of sensing surfaces employed in biosensing. The measured values of kinetic association constants of magnetic nanoparticles are 4 orders of magnitude higher than those of molecular antibody association with antigen. The developed method also suggests highly sensitive detection of antigens in a wide dynamic range. The limit of detection of 92 pg/ml has been demonstrated for prostate-specific antigen (PSA) with 50-nm MP employed as labels, which produce 3-order amplification of the SCI signals. The calibration curve features high sensitivity (slope) of 3-fold signal raise per 10-fold increase of PSA concentration within 4-order dynamic range, which is an attractive compromise for precise quantitative and highly sensitive immunoassay. The proposed biosensing technique offers inexpensive disposable sensor chips of cover slips and represents an economically sound alternative to traditional immunoassays for disease diagnostics, detection of pathogens in food and environmental monitoring.

Diagnostic potential of Fasciola gigantica-derived 14.5 kDa fatty acid binding protein in the immunodiagnosis of bubaline fascioliasis.

PubMed

Allam, G; Bauomy, I R; Hemyeda, Z M; Diab, T M; Sakran, T F

2013-06-01

The 14.5 kDa fatty acid binding protein (FABP) was isolated from the crude extract of adult Fasciola gigantica worms. Polyclonal anti-FABP IgG was generated in rabbits immunized with prepared FABP antigen. Sandwich enzyme-linked immunosorbent assay (ELISA) was applied to detect coproantigen in stools and circulating Fasciola antigen (CA) in sera of 126 water buffaloes by using purified and horseradish peroxidase (HRP)-conjugated anti-FABP IgG. Sandwich ELISA sensitivity was 96.97% and 94.95%; while specificity was 94.12% and 82.35% for coproantigen and CA detection, respectively. However, sensitivity and specificity of the Kato-Katz technique was 73.74% and 100%, respectively. The diagnostic efficacy of sandwich ELISA was 96.55% and 93.1% for coproantigen and CA detection, respectively. In contrast, the diagnostic efficacy of the Kato-Katz technique was 77.59%. In conclusion, these results demonstrate that the purified 14.5 kDa FABP provides a more suitable antigen for immunodiagnosis of early and current bubaline fascioliasis by using sandwich ELISA.

Quantifying serum antibody in bird fanciers' hypersensitivity pneumonitis.

PubMed

McSharry, Charles; Dye, George M; Ismail, Tengku; Anderson, Kenneth; Spiers, Elizabeth M; Boyd, Gavin

2006-06-26

Detecting serum antibody against inhaled antigens is an important diagnostic adjunct for hypersensitivity pneumonitis (HP). We sought to validate a quantitative fluorimetric assay testing serum from bird fanciers. Antibody activity was assessed in bird fanciers and control subjects using various avian antigens and serological methods, and the titer was compared with symptoms of HP. IgG antibody against pigeon serum antigens, quantified by fluorimetry, provided a good discriminator of disease. Levels below 10 mg/L were insignificant, and increasing titers were associated with disease. The assay was unaffected by total IgG, autoantibodies and antibody to dietary hen's egg antigens. Antigens from pigeon serum seem sufficient to recognize immune sensitivity to most common pet avian species. Decreasing antibody titers confirmed antigen avoidance. Increasing antibody titer reflected the likelihood of HP, and decreasing titers confirmed antigen avoidance. Quantifying antibody was rapid and the increased sensitivity will improve the rate of false-negative reporting and obviate the need for invasive diagnostic procedures. Automated fluorimetry provides a method for the international standardization of HP serology thereby improving quality control and improving its suitability as a diagnostic adjunct.

Ultra-Sensitive Detection of Prion Protein in Blood Using Isothermal Amplification Technology

DTIC Science & Technology

2005-12-01

cattle products are admitted into the food chain, the Western Blot or ELISA in an antigen detection format are used to test the bovine brain stems...the most sensitive fluorescent dye (SYBR Green I, SYBR Green II, or SYBR Gold) detection of transcription products using real-time technology...RNA products from ug quantities of DNA template. Figure 3 shows that SYBR Green II exhibited higher relative fluorescence units (RFU) than SYBR

Development of a Fluorescent Based Immunosensor for the Serodiagnosis of Canine Leishmaniasis Combining Immunomagnetic Separation and Flow Cytometry

PubMed Central

Sousa, Susana; Cardoso, Luís; Reed, Steven G.; Reis, Alexandre B.; Martins-Filho, Olindo A.; Silvestre, Ricardo; Cordeiro da Silva, Anabela

2013-01-01

Background An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce. Methodology Ni-NTA magnetic microspheres with 1,7 µm and 8,07 µm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry. Principal Findings A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%). Conclusions Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis. PMID:23991232

Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis

PubMed Central

Wu, Xiaoxin; Wang, Yeping; Weng, Tianhao; Hu, Chenyu; Wang, Frederick X.C.; Wu, Zhigang; Yu, Dongshan; Lu, Huoquan; Yao, Hangping

2017-01-01

Abstract The early secreted protein early secretory antigenic target 6(ESAT-6) and the culture filtrate protein 10 (CFP-10) are 2 antigens that are specific to Mycobacterium tuberculosis. These 2 antigens are good targets for tuberculosis (TB) detection. To rapidly diagnose TB across a variety of samples, we developed colloidal gold immunochromatographic strips (ICSs) based on ESAT-6 and CFP-10. The strips were evaluated using 233 samples, including sputum, plasma, and pleural effusion samples. The positive detection rates for ICSs for ESAT-6 and CFP-10 in sputum (culture-positive for M tuberculosis) were 100% and 91.2%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in plasma were 34.1% and 29.4%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in pleural effusion were 64.7% and 55.9%, respectively. Experimental analysis of culture supernatant showing that the ICS developed for ESAT-6 had a sensitivity of 100% and a specificity of 91.2%. While the ICS developed for CFP-10 had a sensitivity of 91.2% and a specificity of 88.2%. The validity of the test is limited by source of sample. The technique is sensitive and specific for samples in sputum and culture media but not for plasma or pleural effusion samples. Detection of M tuberculosis using ICSs is rapid, simple, and relatively effective; thus, ICSs are a potential screening tool for TB. PMID:29390519

Development and Validation of Monoclonal Antibody-Based Antigen Capture ELISA for Detection of Group A Porcine Rotavirus.

PubMed

Memon, Atta Muhammad; Bhuyan, Anjuman Ara; Chen, Fangzhou; Guo, Xiaozhen; Menghwar, Harish; Zhu, Yinxing; Ku, Xugang; Chen, Shuhua; Li, Zhonghua; He, Qigai

2017-05-01

Porcine rotavirus-A (PoRVA) is one of the common causes of mild to severe dehydrating diarrhea, leading to losses in weaning and postweaning piglets. A rapid, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of PoRVA, by using VP6 (a highly conserved and antigenic protein of group-A rotavirus)-directed rabbit polyclonal antibodies (capture antibody) and murine monoclonal antibodies (detector antibody). The detection limit of AC-ELISA was found to be equal to that of conventional reverse transcription-polymerase chain reaction (RT-PCR; about 10 2.5 TCID 50 /mL). For validation of the in-house AC-ELISA, 295 porcine fecal/diarrhea samples, collected from different provinces of China, were evaluated and compared with conventional RT-PCR and TaqMan RT-quantitative PCR (qPCR). The sensitivity and specificity of this in-house AC-ELISA relative to RT-qPCR were found to be 91.67% and 100%, respectively, with the strong agreement (kappa = 0.972) between these two techniques. Total detection rate with AC-ELISA, conventional RT-PCR, and RT-qPCR were found to be 11.2%, 11.5%, and 12.2%, respectively, without any statistical significant difference. Moreover, AC-ELISA failed to detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastroenteritis virus, pseudorabies virus, and porcine circovirus-2. These results suggested that our developed method was rapid, highly specific, and sensitive, which may help in large-scale surveillance, timely detection, and preventive control of rotavirus infection in porcine farms.

Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis.

PubMed

Wu, Xiaoxin; Wang, Yeping; Weng, Tianhao; Hu, Chenyu; Wang, Frederick X C; Wu, Zhigang; Yu, Dongshan; Lu, Huoquan; Yao, Hangping

2017-12-01

The early secreted protein early secretory antigenic target 6(ESAT-6) and the culture filtrate protein 10 (CFP-10) are 2 antigens that are specific to Mycobacterium tuberculosis. These 2 antigens are good targets for tuberculosis (TB) detection.To rapidly diagnose TB across a variety of samples, we developed colloidal gold immunochromatographic strips (ICSs) based on ESAT-6 and CFP-10.The strips were evaluated using 233 samples, including sputum, plasma, and pleural effusion samples.The positive detection rates for ICSs for ESAT-6 and CFP-10 in sputum (culture-positive for M tuberculosis) were 100% and 91.2%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in plasma were 34.1% and 29.4%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in pleural effusion were 64.7% and 55.9%, respectively. Experimental analysis of culture supernatant showing that the ICS developed for ESAT-6 had a sensitivity of 100% and a specificity of 91.2%. While the ICS developed for CFP-10 had a sensitivity of 91.2% and a specificity of 88.2%.The validity of the test is limited by source of sample. The technique is sensitive and specific for samples in sputum and culture media but not for plasma or pleural effusion samples. Detection of M tuberculosis using ICSs is rapid, simple, and relatively effective; thus, ICSs are a potential screening tool for TB. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

Indirect enzyme-linked immunosorbent assay method based on Streptococcus agalactiae rSip-Pgk-FbsA fusion protein for detection of bovine mastitis.

PubMed

Bu, Ri-E; Wang, Jin-Liang; Wu, Jin-Hua; Xilin, Gao-Wa; Chen, Jin-Long; Wang, Hua

2017-03-01

The aim of this study was to establish a rapid and accurate method for the detection of the Streptococcus agalactiae antibody (SA-Ab) to determine the presence of the bovine mastitis (BM)-causative pathogen. The multi-subunit fusion protein rSip-Pgk-FbsA was prokaryotically expressed and purified. The triple activities of the membrane surface-associated proteins Sip, phosphoglycerate kinase (Pgk), and fibronectin (FbsA) were used as the diagnostic antigens to establish an indirect enzyme-linked immunosorbent assay (ELISA) method for the detection of SA-Ab in BM. The optimal antigen coating concentration was 2 μg/mL, the optimal serum dilution was 1:160, and the optimal dilution of the enzyme-labeled secondary antibody was 1:6000. The sensitivity, specificity, and repeatability tests showed that the method established in this study had no cross-reaction with antibodies to Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis in the sera. The results of the sensitivity test showed that a positive result could be obtained even if the serum dilution reached 1:12,800, indicating the high sensitivity and good repeatability of the method. The positive coincidence rate of this method was 98.6%, which is higher than that of previous tests established with the Sip or Pgk mono-antigen fusion protein, respectively, demonstrating the relatively higher sensitivity of this newly established method. The detection rate for 389 clinical samples was 46.53%. The indirect ELISA method established in this study could provide a more accurate and reliable serological method for the rapid detection of S. agalactiae in cases of BM.

Immunofluorescence Technique Using HeLa Cells Expressing Recombinant Nucleoprotein for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus

PubMed Central

Saijo, Masayuki; Qing, Tang; Niikura, Masahiro; Maeda, Akihiko; Ikegami, Tetsuro; Sakai, Koji; Prehaud, Christophe; Kurane, Ichiro; Morikawa, Shigeru

2002-01-01

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections. PMID:11825944

Highly sensitive detection of multiple tumor markers for lung cancer using gold nanoparticle probes and microarrays.

PubMed

Gao, Wanlei; Wang, Wentao; Yao, Shihua; Wu, Shan; Zhang, Honglian; Zhang, Jishen; Jing, Fengxiang; Mao, Hongju; Jin, Qinghui; Cong, Hui; Jia, Chunping; Zhang, Guojun; Zhao, Jianlong

2017-03-15

Assay of multiple serum tumor markers such as carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen (CYFRA21-1), and neuron specific enolase (NSE), is important for the early diagnosis of lung cancer. Dickkopf-1 (DKK1), a novel serological and histochemical biomarker, was recently reported to be preferentially expressed in lung cancer. Four target proteins were sandwiched by capture antibodies attached to microarrays and detection antibodies carried on modified gold nanoparticles. Optical signals generated by the sandwich structures were amplified by gold deposition with HAuCl 4 and H 2 O 2 , and were observable by microscopy or the naked eye. The four tumor markers were subsequently measured in 106 lung cancer patients and 42 healthy persons. The assay was capable of detecting multiple biomarkers in serum sample at concentration of <1 ng mL -1 in 1 h. Combined detection of the four tumor markers highly improved the sensitivity (to 87.74%) for diagnosis of lung cancer compared with sensitivity of single markers. A rapid, highly sensitive co-detection method for multiple biomarkers based on gold nanoparticles and microarrays was developed. In clinical use, it would be expected to improve the early diagnosis of lung cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

Asymmetric split-ring resonator-based biosensor for detection of label-free stress biomarkers

NASA Astrophysics Data System (ADS)

Lee, Hee-Jo; Lee, Jung-Hyun; Choi, Suji; Jang, Ik-Soon; Choi, Jong-Soon; Jung, Hyo-Il

2013-07-01

In this paper, an asymmetric split-ring resonator, metamaterial element, is presented as a biosensing transducer for detection of highly sensitive and label-free stress biomarkers. In particular, the two biomarkers, cortisol and α-amylase, are used for evaluating the sensitivity of the proposed biosensor. In case of cortisol detection, the competitive reaction between cortisol-bovine serum albumin and free cortisol is employed, while alpha-amylase is directly detected by its antigen-antibody reaction. From the experimental results, we find that the limit of detection and sensitivity of the proposed sensing device are about 1 ng/ml and 1.155 MHz/ng ml-1, respectively.

Biomolecule detection based on Si single-electron transistors for practical use

NASA Astrophysics Data System (ADS)

Nakajima, Anri; Kudo, Takashi; Furuse, Sadaharu

2013-07-01

Experimental and theoretical analyses demonstrated that ultra-sensitive biomolecule detection can be achieved using a Si single-electron transistor (SET). A multi-island channel structure was used to enable room-temperature operation. Coulomb oscillation increases transconductance without increasing channel width, which increases detection sensitivity to a charged target. A biotin-modified SET biosensor was used to detect streptavidin at a dilute concentration. In addition, an antibody-functionalized SET biosensor was used for immunodetection of prostate-specific antigen, demonstrating its suitability for practical use. The feasibility of ultra-sensitive detection of biomolecules for practical use by using a SET biosensor was clearly proven through this systematic study.

Isolation and partial characterization of melanoma-associated antigens identified by autologous antibody.

PubMed

Vlock, D R; Scalise, D; Meglin, N; Kirkwood, J M; Ballou, B

1988-06-01

The study of the autologous immune response to cancer avoids the difficulties encountered in the use of xenoantisera and may identify antigens of physiological relevance. However, the low titer and incidence of autologous antibody to melanoma have hampered further evaluation. By utilizing acid dissociation and ultrafiltration of serum, we have been able to augment the detectable autologous immune response to melanoma in the majority of patients studied. In autologous system Y-Mel 84:420, serum S150 demonstrated a rise in titer from 1:32 in native sera to 1:262,044 after dissociation. The antigen detected by S150 was found to be broadly represented on melanoma, glioma, renal cell carcinoma, neuroblastoma, and head and neck carcinoma cell lines. It did not react with bladder or colon carcinoma, fetal fibroblasts, pooled platelets, lymphocytes and red blood cells, or autologous cultured lymphocytes. Using polyacrylamide gel electrophoresis, S150 detects a 66,000-mol wt antigen in spent tissue culture media and serum ultrafiltrate. In cell lysate two bands between 20,000 and 30,000 mol wt are detected by S150. The 66,000-mol wt antigen is sensitive to trypsin digestion and but is resistant to pepsin and heat inactivation. Exposure of spent media to trypsin results in the development of a 24,000-mol wt band that appears to correspond to the antigen detected in the cell lysate. The difference between the antigens detected in the cell lysate as compared with spent media and serum ultrafiltrate may be due to degradation during cell lysis. We conclude that melanoma-associated antigens are present in the serum of patients with melanoma and are shed or secreted by their tumor cells.

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen.

PubMed

Dong, Huahuang; Liu, Jianli; Zhu, Hong; Ou, Chin-Yih; Xing, Wenge; Qiu, Maofeng; Zhang, Guiyun; Xiao, Yao; Yao, Jun; Pan, Pinliang; Jiang, Yan

2012-08-31

HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3-4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.

Comparison of immunodiffusion and enzyme linked immunosorbent assay in the detection of abnormal antibodies in pigeon breeder's disease.

PubMed Central

Simpson, C.; Shirodaria, P. V.; Evans, J. P.; Simpson, D. I.; Stanford, C. F.

1992-01-01

AIMS: To compare the sensitivity of two methods for the detection of serum antibodies to pigeon faecal antigens in patients with pigeon breeder's disease. METHODS: Serum samples stored at -20 degrees C from 50 patients with pigeon breeder's disease, 50 control samples from patients with other respiratory diseases, and 50 healthy blood donors were examined for the precipitating antibodies and IgG antibodies to antigens present in extract of pigeon droppings by immunodiffusion and enzyme linked immunosorbent assay (ELISA), respectively. RESULTS: Both antigen preparations of pigeon dropping extract were equally effective. A positive immunodiffusion reaction gave one or more precipitin lines and these antibodies were detected only in undiluted sera from 80% of the patients with pigeon breeder's disease. In the ELISA the sera were tested at a starting dilution of 1 in 100 because positive reactions were observed with sera from healthy blood donors at lower dilutions. All sera which gave optical density readings above 3 SD of the control value were considered to have IgG antibodies. These antibodies were detected in sera from all the patients with pigeon breeder's disease. The antibody titres were much higher in those patients who had precipitating antibodies (range 800-51,200) than those without (range 100-800). The antibodies were not detected in the sera of patients with respiratory diseases or healthy blood donors by either method. CONCLUSIONS: Antibodies to pigeon dropping antigens were detected by immunodiffusion and ELISA in sera from patients with pigeon breeder's disease but not in control sera. ELISA was a more sensitive method for detecting antibodies and therefore seems to have considerable potential as a routine technique in the serological diagnosis of pigeon breeder's disease. PMID:1624596

Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

NASA Astrophysics Data System (ADS)

Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

2012-10-01

Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

Biotin avidin amplified magnetic immunoassay for hepatitis B surface antigen detection using GoldMag nanoparticles

NASA Astrophysics Data System (ADS)

Yu, An; Geng, Tingting; Fu, Qiang; Chen, Chao; Cui, Yali

2007-04-01

Using GoldMag (Fe3O4/Au) nanoparticles as a carrier, a biotin-avidin amplified ELISA was developed to detect hepatitis B surface antigen (HBsAg). A specific antibody was labeled with biotin and then used to detect the antigen with an antibody coated on GoldMag nanoparticles by a sandwich ELISA assay. The results showed that 5 mol of biotin were surface bound per mole of antibody. The biotin-avidin amplified ELISA assay has a higher sensitivity than that of the direct ELISA assay. There is 5-fold difference between HBsAg positive and negative serum even at dilution of 1:10000, and the relative standard deviation of the parallel positive serum at dilution of 1:4000 is 5.98% (n=11).

Nano-LISA for in vitro diagnostic applications

NASA Astrophysics Data System (ADS)

Maswadi, Saher; Glickman, Randolph D.; Elliott, Rowe; Barsalou, Norman

2011-03-01

We previously reported the detection of bacterial antigen with immunoaffinity reactions using laser optoacoustic spectroscopy and antibody-coupled gold nanorods (Ab-NR) as a contrast agent specifically targeted to the antigen of interest. The Nano-LISA (Nanoparticle Linked Immunosorbent Assay) method has been adapted to detect three very common blood-borne viral infectious agents, i.e. human T-lymphotropic virus (HTLV), human immunodeficiency virus (HIV) and hepatitis-B (Hep-B). These agents were used in a model test panel to illustrate the performance of the Nano-LISA technique. A working laboratory prototype of a Nano-LISA microplate reader-sensor was assembled and tested against the panel containing specific antigens of each of the infectious viral agents. Optoacoustic (OA) responses generated by the samples were detected using the probe beam deflection technique, an all-optical, non-contact technique. A LabView graphical user interface was developed for control of the instrument and real-time display of the test results. The detection limit of Nano-LISA is at least 1 ng/ml of viral antigen, and can reach 10 pg/ml, depending on the binding affinity of the specific detection antibody used to synthesize the Ab-NR. The method has sufficient specificity, i.e. the detection reagents do not cross-react with noncomplementary antigens. Thus, the OA microplate reader, incorporating NanoLISA, has adequate detection sensitivity and specificity for use in clinical in vitro diagnostic testing.

pLDH level of clinically isolated Plasmodium vivax and detection limit of pLDH based malaria rapid diagnostic test.

PubMed

Jang, Jin Woo; Cho, Chi Hyun; Han, Eun Taek; An, Seong Soo A; Lim, Chae Seung

2013-06-03

The malaria rapid diagnostic tests (RDTs) are now widely used in the world. Compared to Plasmodium falciparum, a poor sensitivity of RDTs was reported against Plasmodium vivax based on the adopted antibody against pan-Plasmodium antigen lactate dehydrogenase (pLDH) or aldolase. Levels of pLDH were measured from patient with P. vivax, and the correlations between the levels of pLDH and the sensitivities of RDTs were analysed among Republic of Korea (ROK) isolates. Three RDTs, OptiMAL test, SD BIOLINE Malaria Ag P.f/Pan test, Humasis Malaria Pf/Pan antigen test, and the Genedia pLDH antigen ELISA were performed with blood samples from 152 febrile patients and 100 healthy controls. Three malaria RDTs revealed sensitivities between 85.5 (131/152) and 86.8% (132/152) with highest sensitivity for the detection of P.vivax by pLDH antigen ELISA test (145/152, 95.4%) in comparison to traditional microscopy using Giemsa-stained slides. None of the healthy control tested positive by three RDTs or ELISA, indicating 100% specificity in their respective test. Levels of pLDH among Korean P. vivax isolates ranged between 0 ng/mL and 22,387.2 ng/mL (mean ± standard deviation 3,917.5 ± 6,120.9 ng/mL). The lower detection limits of three RDTs were between 25 and 50 ng/mL with artificially diluted samples. The moderate degree of correlation was observed between parasitaemia and concentrations of pLDH (r = 0.4, p < 0.05). The pLDH levels of P. vivax are the main explanation for the variations in the performance of pLDH-based RDTs. Therefore, comparing sensitivities of RDT may need to include targeted biomarker value of patients.

Performance of commercial dengue NS1 ELISA and molecular analysis of NS1 gene of dengue viruses obtained during surveillance in Indonesia.

PubMed

Aryati, Aryati; Trimarsanto, Hidayat; Yohan, Benediktus; Wardhani, Puspa; Fahri, Sukmal; Sasmono, R Tedjo

2013-12-29

Early diagnosis of dengue infection is crucial for better management of the disease. Diagnostic tests based on the detection of dengue virus (DENV) Non Structural Protein 1 (NS1) antigen are commercially available with different sensitivities and specificities observed in various settings. Dengue is endemic in Indonesia and clinicians are increasingly using the NS1 detection for dengue confirmation. This study described the performance of Panbio Dengue Early NS1 and IgM Capture ELISA assays for dengue detection during our surveillance in eight cities in Indonesia as well as the genetic diversity of DENV NS1 genes and its relationship with the NS1 detection. The NS1 and IgM/IgG ELISA assays were used for screening and confirmation of dengue infection during surveillance in 2010-2012. Collected serum samples (n = 440) were subjected to RT-PCR and virus isolation, in which 188 samples were confirmed for dengue infection. The positivity of the ELISA assays were correlated with the RT-PCR results to obtain the sensitivity of the assays. The NS1 genes of 48 Indonesian virus isolates were sequenced and their genetic characteristics were studied. Using molecular data as gold standard, the sensitivity of NS1 ELISA assay for samples from Indonesia was 56.4% while IgM ELISA was 73.7%. When both NS1 and IgM results were combined, the sensitivity increased to 89.4%. The NS1 sensitivity varied when correlated with city/geographical origins and DENV serotype, in which the lowest sensitivity was observed for DENV-4 (19.0%). NS1 sensitivity was higher in primary (67.6%) compared to secondary infection (48.2%). The specificity of NS1 assay for non-dengue samples were 100%. The NS1 gene sequence analysis of 48 isolates revealed the presence of polymorphisms of the NS1 genes which apparently did not influence the NS1 sensitivity. We observed a relatively low sensitivity of NS1 ELISA for dengue detection on RT-PCR-positive dengue samples. The detection rate increased significantly when NS1 data was combined with IgM. In our study, the low sensitivity of NS1 antigen detection did not relate to NS1 genetic diversity. Rather, the performance of the NS1 antigen test was affected by the infection status of patients and geographical origin of samples.

Immunodiagnosis of Paracoccidioidomycosis due to Paracoccidioides brasiliensis Using a Latex Test: Detection of Specific Antibody Anti-gp43 and Specific Antigen gp43

PubMed Central

dos Santos, Priscila Oliveira; Rodrigues, Anderson Messias; Fernandes, Geisa Ferreira; da Silva, Silvia Helena Marques; Burger, Eva; de Camargo, Zoilo Pires

2015-01-01

Background Paracoccidioidomycosis (PCM) is a life-threatening systemic disease and is a neglected public health problem in many endemic regions of Latin America. Though several diagnostic methods are available, almost all of them present with some limitations. Method/Principle Findings A latex immunoassay using sensitized latex particles (SLPs) with gp43 antigen, the immunodominant antigen of Paracoccidioides brasiliensis, or the monoclonal antibody mAb17c (anti-gp43) was evaluated for antibody or antigen detection in sera, cerebrospinal fluid (CSF), and bronchoalveolar lavage (BAL) from patients with PCM due to P. brasiliensis. The gp43-SLPs performed optimally to detect specific antibodies with high levels of sensitivity (98.46%, 95% CI 91.7–100.0), specificity (93.94%, 95% CI 87.3–97.7), and positive (91.4%) and negative (98.9%) predictive values. In addition, we propose the use of mAb17c-SLPs to detect circulating gp43, which would be particularly important in patients with immune deficiencies who fail to produce normal levels of immunoglobulins, achieving good levels of sensitivity (96.92%, 95% CI 89.3–99.6), specificity (88.89%, 95% CI 81.0–94.3), and positive (85.1%) and negative (97.8%) predictive values. Very good agreement between latex tests and double immune diffusion was observed for gp43-SLPs (k = 0.924) and mAb17c-SLPs (k = 0.850), which reinforces the usefulness of our tests for the rapid diagnosis of PCM in less than 10 minutes. Minor cross-reactivity occurred with sera from patients with other fungal infections. We successfully detected antigens and antibodies from CSF and BAL samples. In addition, the latex test was useful for monitoring PCM patients receiving therapy. Conclusions/Significance The high diagnostic accuracy, low cost, reduced assay time, and simplicity of this new latex test offer the potential to be commercialized and makes it an attractive diagnostic assay for use not only in clinics and medical mycology laboratories, but mainly in remote locations with limited laboratory infrastructure and/or minimally trained community health workers. PMID:25679976

[Clinical microbiology laboratory and imported parasitic diseases].

PubMed

Martín-Rabadán, Pablo; Martínez-Ruiz, Rocío; Cuadros, Juan; Cañavate, Carmen

2010-12-01

Imported parasitosis represents an increasingly frequent diagnostic challenge for microbiology laboratories. A surge in immigration and international travel has led to a rise in the number of imported cases of parasitosis, and this trend is expected to continue in the future. The present article addresses this challenge by reviewing recommended diagnostic approaches and tests. Currently, microscopy is always recommended when analysing blood samples for parasites. If malaria is suspected, rapid antigen testing (including at least HRP2 antigen) should also be performed. The work-up for suspected leishmaniasis should include serology, culture, and in selected cases detection of antigen in urine. In suspected Chagas disease, two different serological tests should be performed. PCR for blood protozoa is highly sensitive, although it cannot be used to rule out Chagas disease, since this condition may be present without parasitemia. Accurate diagnosis of intestinal amebiasis usually requires PCR or antigen detection tests. In helminthiasis, traditional microscopy may need to be complemented with other tests, such as agar plate culture for strongyloidiasis, Og4C3 antigen detection for bancroftian filariasis, and antibody detection test for filariasis and schistosomiasis. Copyright © 2010 Elsevier España, S.L. All rights reserved.

[Evaluation of Mascia Brunelli rapid antigen test in the diagnosis of group A streptococcal pharyngitis].

PubMed

Barış, Ayşe; Anlıaçık, Nur; Bulut, Mehmet Emin; Deniz, Rıdvan; Yücel, Elif; Aktaş, Elif

2017-01-01

Pharyngitis in most cases is due to viral microorganisms however drug therapy without the detection of etiological agent leads to unnecessary use of antibiotics. On the other hand, when the etiologic agent is group A beta-hemolytic streptococci (GAS) it is important to identify the etiologic agent rapidly which will guide the treatment with appropriate antibiotics. The use of highly sensitive rapid tests will contribute significantly to early diagnosis and appropriate therapy. The aim of this study is to evaluate the efficacy of Mascia Brunelli rapid antigen test for the detection of GAS in throat swab samples. A total of 833 throat swab samples submitted to our laboratory with pre-diagnosis of pharyngitis were assessed between June 2016 and August 2016. The samples were simultaneously cultured and tested by rapid Mascia Brunelli Strep-A Card (Mascia Brunelli S.p.a, Italy). For identification, bacitracin sensitivity, PYR test and latex agglutination test in addition to Bruker MALDI-TOF MS (Daltonics, Germany) system were used. The density of GAS growth in the culture was noted. The samples that were false negative with Mascia Brunelli test were re-tested with QuickVue + Strep A Test (Quidel Corporation, San Diego, USA) rapid antigen test. A total of 833 patients, 376 (45.2%) female and 457 (54.8%) male were included in the study. The age range was between 0-94 years with a mean value of 7.86 ± 6.72. 125 (15%) and 94 (11.28%) of the samples were positive with culture and rapid antigen test, respectively. Mascia Brunelli antigen test gave negative results for 31 culture positive samples. Of these 31 samples, 28 were found positive by QuickVue + Strep A antigen test. As a result, the sensitivity of the test was found to be independent of the inoculum effect. The culture positivity rate in patients between 5-15 years was 18.4%. The sensitivity, specificity, positive predictive value, negative predictive value and the accuracy of Mascia Brunelli antigen test, with respect to culture, were 75.2%, 100%, 100%, 95.81% and 96.28%, respectively. In conclusion, the selection of rapid antigen tests with high sensitivity in the diagnosis of GAS pharyngitis will contribute to the prevention of resistance development by appropriate use of antibiotics as well as early diagnosis and appropriate treatment. However, confirmation of negative rapid antigen test results by culture is very important in terms of false diagnosis and prevention of incomplete treatment.

Development and Evaluation of an Immunodiffusion Test for Diagnosis of Systemic Zygomycosis (Mucormycosis): Preliminary Report

PubMed Central

Jones, Kenneth W.; Kaufman, Leo

1978-01-01

An antigen analysis with filtrate and homogenate precipitinogens of single isolates of the zygomycetes Absidia corymbifera, Mucor pusillus, Rhizopus arrhizus, and Rhizopus oryzae demonstrated the presence of common antigens among the three genera as well as antigens which permit their differentiation. Selected homogenate antigens were valuable in developing a diagnostic immunodiffusion (ID) test for systemic zygomycosis. When sera from 43 patients with various proven mycoses other than zygomycosis were tested against each of the antigens, none formed precipitin bands identical to those formed by A. cormybifera, M. pusillus, and the Rhizopus spp. rabbit reference antisera. Sera from 23 normal persons and 25 diabetics did not react with any of the antigens. Homogenate antigens detected antibody in 8 of the 11 sera (73%) from suspected or proven cases of zygomycosis, whereas ID tests with filtrate antigens detected antibody in only 2 of the 11 sera (18%). Of the eight sera that reacted with the homogenate antigens, five only reacted with a specific Rhizopus sp. antigen, two only reacted with a specific M. pusillus antigen, and one only reacted with a specific A. corymbifera antigen. Study results show the ID test with homogenate antigens to be more specific and sensitive than the ID test with filtrate antigens and indicate that the former is a promising technique for diagnosing human zygomycosis. Images PMID:75212

Recombinant antibodies and their use in biosensors.

PubMed

Zeng, Xiangqun; Shen, Zhihong; Mernaugh, Ray

2012-04-01

Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

Sensitivity and specificity enhanced enzyme-linked immunosorbent assay by rational hapten modification and heterogeneous antibody/coating antigen combinations for the detection of melamine in milk, milk powder and feed samples.

PubMed

Cao, Biyun; Yang, Hong; Song, Juan; Chang, Huafang; Li, Shuqun; Deng, Anping

2013-11-15

The adulteration of food products with melamine has led to an urgent requirement for sensitive, specific, rapid and reliable quantitative/screening methods. To enhance the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the detection of melamine in milk, milk powder and feed samples, rational hapten modification and heterogeneous antibody/coating antigen combinations were adopted. Three melamine derivatives with different length of carboxylic spacer at the end were synthesized and linked to carrier proteins for the production of immunogens and coating antigens. Monoclonal antibody against melamine was produced by hybridoma technology. Under optimal experimental conditions, the standard curves of the ELISAs for melamine were constructed in range of 0.1-100 ng mL(-1). The sensitivity was 10-300 times enhanced compared to those in the published literatures. The cross-reactivity values of the ELISAs also demonstrated the assays exhibited high specificity. Five samples were spiked with melamine at different concentrations and detected by the ELISA. The recovery rates of 72.8-123.0% and intra-assay coefficients of variation of 0.8-18.9% (n=3) were obtained. The ELISA for milk sample was confirmed by high-performance liquid chromatography with a high correlation coefficient of 0.9902 (n=6). The proposed ELISA was proven to be a feasible quantitative/screening method for melamine analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Antigen detection at atomolar concentration using optical tweezers

NASA Astrophysics Data System (ADS)

Laliberté, Mathieu; Bordeleau, François; Marceau, Normand; Sheng, Yunlong

2009-06-01

Methods that avoid intermediate amplification steps to detect protein markers of pathological disturbances would be of wide interest in the clinical environment. This is particularly the case in cancer diagnosis, where protein fragments are released into the blood by the emerging cancer cells. These fragments generate an antigen-antibody reaction, and the concentration of the antigen is known to modulate this interaction. Here we report on the development of a novel optical tweezers-based procedure to measure minute amount of antigen in a biological fluid. The force was applied on a 3μm polystyrene bead coated with Bovine Serum Albumin (BSA) attached on a 1.5 μm diameter borosilicate rod tip coated with anti-BSA antibody. First, we verified that the binding strength was dependent on the protein concentration on the bead. We then assessed the sensitivity range by finding the minimal BSA concentration in solution that can still interfere with the bead-rod linkage. On the whole, the results demonstrated that proteinous antigen present in a biological fluid could possibly be detectable at atomolar concentration through the use of an optical tweezers.

Detection and Persistence of Vi Antigen in Tissues of Actively Immunized Mice1

PubMed Central

Gaines, Sidney; Currie, Julius A.; Tully, Joseph G.

1965-01-01

Gaines, Sidney (Walter Reed Army Institute of Research, Washington, D.C.), Julius A. Currie, and Joseph G. Tully. Detection and persistence of Vi antigen in tissues of actively immunized mice. J. Bacteriol. 89:776–781. 1965.—The presence, distribution, and persistence of Vi antigen in mouse tissue was determined by means of active immunization tests with tissue extracts. Mice were injected intraperitoneally with purified Vi antigen or Vi-containing bacilli. At appropriate intervals, animals were killed, and saline extracts of their tissues were prepared. Mice were immunized with these extracts and challenged 6 days later with 10 ld50 of Salmonella typhosa Ty2. Protection was afforded by tissue extracts from Vi-injected mice, but not by normal tissue extracts. That the immunizing capacity of tissue extracts from Vi-injected mice was attributable to Vi antigen was affirmed by the demonstration that these extracts stimulated the production of Vi antibody in mice, coated erythrocytes for agglutination by Vi antiserum, and inhibited agglutination of Vi-sensitized red blood cells by known Vi antisera. Vi antigen could be detected in the liver and spleen of mice injected with as little as 1 μg. In mice given 150 μg, the antigen was still present in liver tissue 231 days later. PMID:14273660

Use of recombinant calreticulin and cercarial transformation fluid (CTF) in the serodiagnosis of Schistosoma mansoni.

PubMed

El Aswad, Bahaa El Deen Wade; Doenhoff, Michael J; El Hadidi, Abeer Shawky; Schwaeble, Wilhelm J; Lynch, Nicholas J

2011-03-01

Schistosomiasis is traditionally diagnosed by microscopic detection of ova in stool samples, but this method is labour intensive and its sensitivity is limited by low and variable egg secretion in many patients. An alternative is an ELISA using Schistosoma mansoni soluble egg antigen (SEA) to detect anti-schistosome antibody in patient samples. SEA is a good diagnostic marker in non-endemic regions but is of limited value in endemic regions, mainly because of its high cost and limited specificity. Here we assess seven novel antigens for the detection of S. mansoni antibody in an endemic region (the Northern Nile Delta). Using recombinant S. mansoni calreticulin (CRT) and fragments thereof, anti-CRT antibodies were detected in the majority of 97 patients sera. The diagnostic value of some of these antigens was, however, limited by the presence of cross-reacting antibody in the healthy controls, even those recruited in non-endemic areas. Cercarial transformation fluid (CTF), a supernatant that contains soluble material released by the cercariae upon transformation to the schistosomula, is cheaper and easier to produce than SEA. An ELISA using CTF as the detection antigen had a sensitivity of 89.7% and an estimated specificity of 100% when used in non-endemic regions, matching the performance of the established SEA ELISA. CTF was substantially more specific than SEA for diagnosis in the endemic region, and less susceptible than SEA to cross-reacting antibody in the sera of controls with other protozoan and metazoan infections. Copyright © 2010 Elsevier GmbH. All rights reserved.

Laboratory and Field Evaluation of a New Rapid Test for Detecting Wuchereria bancrofti Antigen in Human Blood

PubMed Central

Weil, Gary J.; Curtis, Kurt C.; Fakoli, Lawrence; Fischer, Kerstin; Gankpala, Lincoln; Lammie, Patrick J.; Majewski, Andrew C.; Pelletreau, Sonia; Won, Kimberly Y.; Bolay, Fatorma K.; Fischer, Peter U.

2013-01-01

Global Program to Eliminate Lymphatic Filariasis (GPELF) guidelines call for using filarial antigen testing to identify endemic areas that require mass drug administration (MDA) and for post-MDA surveillance. We compared a new filarial antigen test (the Alere Filariasis Test Strip) with the reference BinaxNOW Filariasis card test that has been used by the GPELF for more than 10 years. Laboratory testing of 227 archived serum or plasma samples showed that the two tests had similar high rates of sensitivity and specificity and > 99% agreement. However, the test strip detected 26.5% more people with filarial antigenemia (124/503 versus 98/503) and had better test result stability than the card test in a field study conducted in a filariasis-endemic area in Liberia. Based on its increased sensitivity and other practical advantages, we believe that the test strip represents a major step forward that will be welcomed by the GPELF and the filariasis research community. PMID:23690552

Utility of HLA Antibody Testing in Kidney Transplantation

PubMed Central

Konvalinka, Ana

2015-01-01

HLA antigens are polymorphic proteins expressed on donor kidney allograft endothelium and are critical targets for recipient immune recognition. HLA antibodies are risk factors for acute and chronic rejection and allograft loss. Solid-phase immunoassays for HLA antibody detection represent a major advance in sensitivity and specificity over cell-based methods and are widely used in organ allocation and pretransplant risk assessment. Post-transplant, development of de novo donor–specific HLA antibodies and/or increase in donor-specific antibodies from pretransplant levels are associated with adverse outcomes. Although single antigen bead assays have allowed sensitive detection of recipient HLA antibodies and their specificities, a number of interpretive considerations must be appreciated to understand test results in clinical and research contexts. This review, which is especially relevant for clinicians caring for transplant patients, discusses the technical aspects of single antigen bead assays, emphasizes their quantitative limitations, and explores the utility of HLA antibody testing in identifying and managing important pre- and post-transplant clinical outcomes. PMID:25804279

Evaluation of a rapid immunochromatographic ODK-0901 test for detection of pneumococcal antigen in middle ear fluids and nasopharyngeal secretions.

PubMed

Hotomi, Muneki; Togawa, Akihisa; Takei, Shin; Sugita, Gen; Sugita, Rinya; Kono, Masamitsu; Fujimaki, Yutaka; Kamide, Yosuke; Uchizono, Akihiro; Kanesada, Keiko; Sawada, Shoichi; Okitsu, Naohiro; Tanaka, Yumi; Saijo, Yoko; Yamanaka, Noboru

2012-01-01

Since the incidence of penicillin-resistant Streptococcus pneumoniae has been increasing at an astonishing rate throughout the world, the need for accurate and rapid identification of pneumococci has become increasingly important to determine the appropriate antimicrobial treatment. We have evaluated an immunochromatographic test (ODK-0901) that detects pneumococcal antigens using 264 middle ear fluids (MEFs) and 268 nasopharyngeal secretions (NPSs). A sample was defined to contain S. pneumoniae when optochin and bile sensitive alpha hemolytic streptococcal colonies were isolated by culture. The sensitivity and specificity of the ODK-0901 test were 81.4% and 80.5%, respectively, for MEFs from patients with acute otitis media (AOM). In addition, the sensitivity and specificity were 75.2% and 88.8%, respectively, for NPSs from patients with acute rhinosinusitis. The ODK-0901 test may provide a rapid and highly sensitive evaluation of the presence of S. pneumoniae and thus may be a promising method of identifying pneumococci in MEFs and NPSs.

Evaluation of a Rapid Immunochromatographic ODK-0901 Test for Detection of Pneumococcal Antigen in Middle Ear Fluids and Nasopharyngeal Secretions

PubMed Central

Hotomi, Muneki; Togawa, Akihisa; Takei, Shin; Sugita, Gen; Sugita, Rinya; Kono, Masamitsu; Fujimaki, Yutaka; Kamide, Yosuke; Uchizono, Akihiro; Kanesada, Keiko; Sawada, Shoichi; Okitsu, Naohiro; Tanaka, Yumi; Saijo, Yoko; Yamanaka, Noboru

2012-01-01

Since the incidence of penicillin-resistant Streptococcus pneumoniae has been increasing at an astonishing rate throughout the world, the need for accurate and rapid identification of pneumococci has become increasingly important to determine the appropriate antimicrobial treatment. We have evaluated an immunochromatographic test (ODK-0901) that detects pneumococcal antigens using 264 middle ear fluids (MEFs) and 268 nasopharyngeal secretions (NPSs). A sample was defined to contain S. pneumoniae when optochin and bile sensitive alpha hemolytic streptococcal colonies were isolated by culture. The sensitivity and specificity of the ODK-0901 test were 81.4% and 80.5%, respectively, for MEFs from patients with acute otitis media (AOM). In addition, the sensitivity and specificity were 75.2% and 88.8%, respectively, for NPSs from patients with acute rhinosinusitis. The ODK-0901 test may provide a rapid and highly sensitive evaluation of the presence of S. pneumoniae and thus may be a promising method of identifying pneumococci in MEFs and NPSs. PMID:22448257

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

PubMed Central

Halpern, Micah D.; Molins, Claudia R.; Schriefer, Martin

2014-01-01

A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi. PMID:24899074

Detection of target staphylococcal enterotoxin B antigen in orange juice and popular carbonated beverages using antibody-dependent antigen-capture assays.

PubMed

Principato, MaryAnn; Njoroge, Joyce M; Perlloni, Andrei; O' Donnell, Michael; Boyle, Thomas; Jones, Robert L

2010-10-01

There is a critical need for qualitative and quantitative methodologies that provide the rapid and accurate detection of food contaminants in complex food matrices. However, the sensitivity of the assay can be affected when antigen-capture is applied to certain foods or beverages that are extremely acidic. This study was undertaken to assess the effects of orange juice and popular carbonated soft drink upon the fidelity of antibody-based antigen-capture assays and to develop simple approaches that could rescue assay performance without the introduction of additional or extensive extraction procedures. We examined the effects of orange juice and a variety of popular carbonated soft drink beverages upon a quantitative Interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) assay system and a lateral flow device (LFD) adapted for the detection of staphylococcal enterotoxin B (SEB) in foods. Alterations in the performance and sensitivity of the assay were directly attributable to the food matrix, and alterations in pH were especially critical. The results demonstrate that approaches such as an alteration of pH and the use of milk as a blocking agent, either singly or in combination, will partially rescue ELISA performance. The same approaches permit lateral flow to efficiently detect antigen. Practical Application: The authors present ways to rescue an ELISA assay compromised by acidity in beverages and show that either the alteration of pH, or the use of milk as a blocking agent are not always capable of restoring the assay to its intended efficiency. However, the same methods, when employed with lateral flow technology, are rapid and extremely successful.

Antibody responses to Sarcoptes scabiei apolipoprotein in a porcine model: relevance to immunodiagnosis of recent infection.

PubMed

Rampton, Melanie; Walton, Shelley F; Holt, Deborah C; Pasay, Cielo; Kelly, Andrew; Currie, Bart J; McCarthy, James S; Mounsey, Kate E

2013-01-01

No commercial immunodiagnostic tests for human scabies are currently available, and existing animal tests are not sufficiently sensitive. The recombinant Sarcoptes scabiei apolipoprotein antigen Sar s 14.3 is a promising immunodiagnostic, eliciting high levels of IgE and IgG in infected people. Limited data are available regarding the temporal development of antibodies to Sar s 14.3, an issue of relevance in terms of immunodiagnosis. We utilised a porcine model to prospectively compare specific antibody responses to a primary infestation by ELISA, to Sar s 14.3 and to S. scabiei whole mite antigen extract (WMA). Differences in the antibody profile between antigens were apparent, with Sar s 14.3 responses detected earlier, and declining significantly after peak infestation compared to WMA. Both antigens resulted in >90% diagnostic sensitivity from weeks 8-16 post infestation. These data provide important information on the temporal development of humoral immune responses in scabies and further supports the development of recombinant antigen based immunodiagnostic tests for recent scabies infestations.

[Serological diagnosis of sporotrichosis using an antigen of Sporothrix schenckii sensu stricto mycelium].

PubMed

Alvarado, Primavera; Ostos, Ana; Franquiz, Nohelys; Roschman-González, Antonio; Zambrano, Edgar A; Mendoza, Mireya

2015-06-01

We developed and analyzed an Enzyme-Linked Immunosorbent Assay (ELISA) in order to detect antibodies in sera from sporotrichosis patients. We used a crude antigen of Sporothrix schenckii sensu stricto, obtained from the mycelial phase of the fungi. Positive sera were analyzed by other serological techniques such as double immunodiffusion (IGG) and counterimmunoelectrophoresis (CIE). The assay was validated by using sera from patients with other pathologies such as: histoplasmosis, paracoccidioidomycosis, tuberculosis, leishmaniasis, lupus and healthy individuals as negative controls. For the Sporothrix schenckii sensu stricto antigen, we found a 100% of specificity by every technique and sensitivity higher than 98% with IDD, CIE and ELISA. Our results show a high sensitivity and specificity for the Sporothrix schenckii sensu stricto antigen, so it can be used for IDD, CIE and ELISA. The results suggest that this antigen could be used in conjunction with other conventional tests for differential diagnosis and may be useful for monitoring the disease progression and response to treatment.

A new signal amplification strategy of photoelectrochemical immunoassay for highly sensitive interleukin-6 detection based on TiO2/CdS/CdSe dual co-sensitized structure.

PubMed

Fan, Gao-Chao; Ren, Xiao-Lin; Zhu, Cheng; Zhang, Jian-Rong; Zhu, Jun-Jie

2014-09-15

Dual co-sensitized structure of TiO2/CdS/CdSe was designed to develop a novel photoelectrochemical immunoassay for highly sensitive detection of human interleukin-6 (IL-6). To construct a sensing electrode, TiO2/CdS hybrid was prepared by successive adsorption and reaction of Cd(2+) and S(2-) ions on the surface of TiO2 and then was employed as matrix for immobilization of anti-IL-6 antibody, whereas CdSe QDs linked to IL-6 were used for signal amplification via the specific antibody-antigen immunoreaction between anti-IL-6 and IL-6-CdSe bioconjugate. Greatly enhanced sensitivity for IL-6 detection was derived from the new co-sensitization signal amplification strategy. First, the TiO2/CdS/CdSe co-sensitized structure extended the absorption range to long wavelength of white light, which adequately utilized the light energy. Second, the TiO2/CdS/CdSe co-sensitized structure possessed stepwise band-edge levels favoring ultrafast transfer of photogenerated electrons and significantly prompted the photoelectrochemical performance. Besides, the introduction of CdSe effectively prevented the recombination of photogenerated electrons in the conduction band of CdS, further causing an enhanced photocurrent. Accordingly, upon the co-sensitization strategy, a novel immunoassay based on the competitive binding of anti-IL-6 antibody with IL-6 antigen and IL-6-CdSe bioconjugate was developed, and it exhibited a wide linear range from 1.0 pg/mL to 100 ng/mL with a low detection limit of 0.38 pg/mL for IL-6 detection. The proposed co-sensitization strategy presented high sensitivity, reproducibility, specificity and stability, and also opened up a new promising platform for detection of other biomarkers. Copyright © 2014 Elsevier B.V. All rights reserved.

One step biofunctionalized electrospun multiwalled carbon nanotubes embedded zinc oxide nanowire interface for highly sensitive detection of carcinoma antigen-125.

PubMed

Paul, K Brince; Singh, Vikrant; Vanjari, Siva Rama Krishna; Singh, Shiv Govind

2017-02-15

Ovarian cancer is the most leading cause of cancer-related death in women . The carcinoma antigen-125, which is found on the surface of many ovarian cancer cells is known to be a gold standard clinical biomarker associated with life-threatening gynecological malignancy. In this work, we demonstrate a novel biosensor platform based on multiwalled carbon nanotubes embedded zinc oxide nanowire for the ultrasensitive detection of carcinoma antigen-125. Label free detection of the carcinoma antigen-125 was accomplished by differential voltammetry technique that demonstrated excellent sensitivity (90.14µA/(U/mL)/cm 2 ) with a detection limit of 0.00113UmL -1 concentration. The fabricated immunosensor exhibits good performance with wider detection range (0.001UmL -1 -1kUmL -1 ), reproducibility, selectivity, acceptable stability, and thus is a potential cost-effective methodology for point-of-care diagnosis. The multiwalled carbon nanotubes (MWCNTs) embedded highly oriented zinc oxide (ZnO) nanowires were synthesized by simple, low cost electrospinning technique. Compared to pure ZnO nanowires, electrochemical activity of MWCNTs embedded ZnO nanowires was found to be much higher. The calcination temperature was optimized to avoid any decomposition of the CNTs and to obtain multiwalled carbon nanotubes embedded highly crystalline ZnO nanowires. The salient feature of this biosensing platform is that one step calcination process is enough to create the functional groups on MWCNT-ZnO nanowire surface that are effective for the covalent conjugation of antibody without further surface modification. To the best of our knowledge, this is the first report on MWCNT-ZnO nanowire based immunosensor explored for the detection of cancer biomarker. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel double recognition enzyme-linked immunosorbent assay based on the nucleocapsid protein for early detection of European porcine reproductive and respiratory syndrome virus infection.

PubMed

Venteo, A; Rebollo, B; Sarraseca, J; Rodriguez, M J; Sanz, A

2012-04-01

Precise and rapid detection of porcine reproductive respiratory syndrome virus (PRRSV) infection in swine farms is critical. Improvement of control procedures, such as testing incoming gilt and surveillance of seronegative herds requires more rapid and sensitive methods. However, standard serological techniques detect mainly IgG antibodies. A double recognition enzyme-linked immunosorbent assay (DR-ELISA) was developed for detection of antibodies specific to European PRRSV. This new assay can recognize both IgM and IgG antibodies to PRSSV which might be useful for detecting in routine surveillance assays pigs that are in the very early stages of infection and missed by conventional assays detecting only IgG antibodies. DR-ELISA is based on the double recognition of antigen by antibody. In this study, the recombinant nucleocapsid protein (N) of PRRSV was used both as the coating and the enzyme-conjugated antigen. To evaluate the sensitivity of the assay at early stages of the infection, sera from 69 pigs infected with PRRSV were collected during successive days post infection (pi) and tested. While standard methods showed low sensitivity rates before day 14 pi, DR-ELISA detected 88.4% seropositive samples at day 7 showing greater sensitivity at early stages of the infection. Further studies were carried out to assess the efficiency of the new assay, and the results showed DR-ELISA to be a sensitive and accurate method for early diagnosis of EU-PRRSV infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of solid - based paper strips for rapid diagnosis of Pseudorabies infection.

PubMed

Joon Tam, Yew; Mohd Lila, Mohd Azmi; Bahaman, Abdul Rani

2004-12-01

Pseudorabies (Aujeszky's disease) is an economically significant disease of swine known to cause central nervous disorders, respiratory disease, reproductive failure and mortality in infected pigs. In attempts to eradicate the disease from becoming endemic, early detection is important to prevent further economic losses and to allow for detection and removal of infected pigs in domestic herds. Thus, a rapid and sensitive technique is necessary for the detection of the virus. For rapid and simple examination, an immuno - chromatographic lateral - flow assay system based on immunologic recognition of specific pseudorabies virus antigen was developed by utilising, as signal generator, colloidal gold conjugated to secondary antibody to detect primary or sample antibody in the sera of pseudorabies infected animals. The pseudorabies virus used as a capture antigen in the test strip was first cultivated in VERO cell culture and then purified by sucrose gradient separation to produce the viral protein concentration of 3.8 mg/ml. The standard pseudorabies antigens reacted well with the hyperimmune serum (HIS). The antibody detection system is basically composed of colloidal gold - labelled antibodies fixed on a conjugate pad, and the complementary pseudorabies antigen immobilised onto a nitrocellulose membrane forming capture zone. If the target antibody is present in a specimen, the colloidal gold-labelled antibody will form a complex with the antibody sample. Subsequently, the formed complex will migrate to the capture zone and is then bound to the solid phase via antigen - antibody interaction. As a result, a signal marker is generated by the accumulation of colloidal gold for detection confirmation. The results obtained demonstrated that the optimum combination of pseudorabies antigen needed as the capture reagent and gold conjugate as secondary antibody recognition marker was at a concentration of 0.38mg/ml and at 1:10 dilution factor respectively. The sensitivity of the solid - based test strip towards pseudorabies antibodies was high with a detection limit of 1 to 10,000 - dilution factor. The specificity of the assay was 100% with no cross - reaction being observed with other sera or antibodies. Accurate reading time needed for confirmation of the assay can be completed in 5 min with a whole blood sample of 25 microl. The colloidal gold - labelled antibody is stable at room temperature for 6 months or more (data not shown). Findings from this study indicated that the solid - based test strip assay system provided high sensitivity and specificity for the detection of pseudorabies at low levels of antibody concentration. The assay was rapid, simple, cheap, and does not require any sophisticated equipment. Thus, the solid based test strip will be a useful serological screening technique or for rapid diagnosis of an infectious disease in target populations of animals characterised by heterogeneous antibody responses.

A highly sensitive capillary electrophoresis immunoassay strategy based on dual-labeled gold nanoparticles enhancing chemiluminescence for the detection of prostate-specific antigen.

PubMed

Li, Shuting; Shi, Min; Zhao, Jingjin; Zhang, Liangliang; Huang, Yong; Zhao, Shulin

2017-07-01

An enzyme and antibody dual labeled gold nanoparticles enhancing chemiluminescence strategy was developed for highly sensitive CE immunoassay (IA) of prostate-specific antigen (PSA). In this work, gold nanoparticles were labeled with horseradish peroxidase and antiprostate specific antigen-antibody, and used as the marker (Ab * ). After PSA (antigen, Ag) was added into the system, a noncompetitive immune reaction was happen between Ab * and Ag to form an immune complex (Ag-Ab * ). Subsequently, the obtained Ag-Ab * and unreacted Ab * were separated by CE, and the chemiluminescence intensity of Ag-Ab * was used to estimate PSA concentration. The calibration curve showed a good linearity in the range of 0.25-10 ng/mL. Based on a S/N of 3, the detection limit for PAS was estimated to be 0.092 ng/mL. Proposed CE method was applied for PSA quantification in human serum samples from healthy volunteers and patients with prostate cancer. The obtained results demonstrated that the proposed CE method may serve as an alternative tool for clinical analysis of PSA. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus.

PubMed

Maifeld, Sarah V; Ro, Bodrey; Mok, Hoyin; Chu, Marla; Yu, Li; Yamagata, Ryan; Leonardson, Tansy; Chio, Vera; Parhy, Bandita; Park, Samuel; Carlson, Marcia; Machhi, Shushil; Ulbrandt, Nancy; Falsey, Ann R; Walsh, Edward E; Wang, C Kathy; Esser, Mark T; Zuo, Fengrong

2016-01-01

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized "gold standard" panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the "gold standard" diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.

Piezoelectric cantilever sensors

NASA Technical Reports Server (NTRS)

Shih, Wan Y. (Inventor); Shih, Wei-Heng (Inventor); Shen, Zuyan (Inventor)

2008-01-01

A piezoelectric cantilever with a non-piezoelectric, or piezoelectric tip useful as mass and viscosity sensors. The change in the cantilever mass can be accurately quantified by monitoring a resonance frequency shift of the cantilever. For bio-detection, antibodies or other specific receptors of target antigens may be immobilized on the cantilever surface, preferably on the non-piezoelectric tip. For chemical detection, high surface-area selective absorbent materials are coated on the cantilever tip. Binding of the target antigens or analytes to the cantilever surface increases the cantilever mass. Detection of target antigens or analytes is achieved by monitoring the cantilever's resonance frequency and determining the resonance frequency shift that is due to the mass of the adsorbed target antigens on the cantilever surface. The use of a piezoelectric unimorph cantilever allows both electrical actuation and electrical sensing. Incorporating a non-piezoelectric tip (14) enhances the sensitivity of the sensor. In addition, the piezoelectric cantilever can withstand damping in highly viscous liquids and can be used as a viscosity sensor in wide viscosity range.

Design and Preparation of Nanoparticle Dimers for SERS Detection

DTIC Science & Technology

2012-09-10

sensitivity afforded by surface enhanced Raman spectroscopy (SERS). Metal nanoparticles dimers were synthesized that incorporate SERS reporters...and antigens, based on the remarkable sensitivity afforded by surface enhanced Raman spectroscopy (SERS). Metal nanoparticles dimers were...Potma, V. A._Apkarian. High Sensitivity Surface-Enhanced Raman Scattering in Solution Using Engineered Silver Nanosphere Dimers, The Journal of

Diagnostic methods for African horsesickness virus using monoclonal antibodies to structural and non-structural proteins.

PubMed

Ranz, A I; Miguet, J G; Anaya, C; Venteo, A; Cortés, E; Vela, C; Sanz, A

1992-11-01

A panel of 32 hybridoma cell lines secreting monoclonal antibodies (MAbs) reactive with African horsesickness virus serotype 4 (AHSV-4) has been developed. Four of the MAbs recognized the major core antigen VP7, twenty recognized the outer capsid protein VP2 and eight reacted with the non-structural protein NS1. With the VP7-specific MAbs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected Vero cells and in spleen tissue from AHSV-infected horses. The sensitivity of the assay is 10 ng viral antigen per 100 microliters. The NS1-specific MAbs allowed visualization by immunofluorescence of tubule-like structures in the cytoplasm of infected Vero cells. This can be very useful as a confirmatory diagnostic procedure. The antigenic map of the outer capsid VP2 protein with MAbs is also reported.

Serodiagnosis of Tuberculosis: Comparison of Immunoglobulin A (IgA) Response to Sulfolipid I with IgG and IgM Responses to 2,3-Diacyltrehalose, 2,3,6-Triacyltrehalose, and Cord Factor Antigens

PubMed Central

Julián, Esther; Matas, Lurdes; Pérez, Andrés; Alcaide, José; Lanéelle, Marie-Antoinette; Luquin, Marina

2002-01-01

Nonpeptidic antigens from the Mycobacterium tuberculosis cell wall are the focus of extensive studies to determine their potential role as protective antigens or serological markers of tuberculous disease. Regarding this latter role and using an enzyme-linked immunosorbent assay, we have made a comparative study of the immunoglobulin G (IgG), IgM, and IgA antibody responses to four trehalose-containing glycolipids purified from M. tuberculosis: diacyltrehaloses, triacyltrehaloses, cord factor, and sulfolipid I (SL-I). Sera from 92 tuberculosis patients (taken before starting antituberculosis treatment) and a wide group of control individuals (84 sera from healthy donors, including purified protein derivative-negative, -positive, healed, and vaccinated individuals, and 52 sera from nontuberculous pneumonia patients), all from Spain, were studied. The results indicated a significantly elevated IgG and IgA antibody response in tuberculosis patients, compared with controls, with all the antigens used. SL-I was the best antigen studied, showing test sensitivities and specificities for IgG of 81 and 77.6%, respectively, and of 66 and 87.5% for IgA. Using this antigen and combining IgA and IgG antibody detection, high test specificity was achieved (93.7%) with a sensitivity of 67.5%. Currently, it is widely accepted that it is not possible to achieve sensitivities above 80% in tuberculosis serodiagnosis when using one antigen alone. Thus, we conclude that SL-I, in combination with other antigenic molecules, could be a useful antigen for tuberculosis serodiagnosis. PMID:12354881

Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation.

PubMed

Chruscinski, Andrzej; Huang, Flora Y Y; Nguyen, Albert; Lioe, Jocelyn; Tumiati, Laura C; Kozuszko, Stella; Tinckam, Kathryn J; Rao, Vivek; Dunn, Shannon E; Persinger, Michael A; Levy, Gary A; Ross, Heather J

2016-01-01

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR.

Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation

PubMed Central

Chruscinski, Andrzej; Huang, Flora Y. Y.; Nguyen, Albert; Lioe, Jocelyn; Tumiati, Laura C.; Kozuszko, Stella; Tinckam, Kathryn J.; Rao, Vivek; Dunn, Shannon E.; Persinger, Michael A.; Levy, Gary A.; Ross, Heather J.

2016-01-01

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR. PMID:26967734

Real-Time, Single-Step Bioassay Using Nanoplasmonic Resonator With Ultra-High Sensitivity

NASA Technical Reports Server (NTRS)

Zhang, Xiang (Inventor); Chen, Fanqing Frank (Inventor); Su, Kai-Hang (Inventor); Wei, Qi-Huo (Inventor); Ellman, Jonathan A. (Inventor); Sun, Cheng (Inventor)

2014-01-01

A nanoplasmonic resonator (NPR) comprising a metallic nanodisk with alternating shielding layer(s), having a tagged biomolecule conjugated or tethered to the surface of the nanoplasmonic resonator for highly sensitive measurement of enzymatic activity. NPRs enhance Raman signals in a highly reproducible manner, enabling fast detection of protease and enzyme activity, such as Prostate Specific Antigen (paPSA), in real-time, at picomolar sensitivity levels. Experiments on extracellular fluid (ECF) from paPSA-positive cells demonstrate specific detection in a complex bio-fluid background in real-time single-step detection in very small sample volumes.

Real-time, single-step bioassay using nanoplasmonic resonator with ultra-high sensitivity

DOEpatents

Zhang, Xiang; Ellman, Jonathan A; Chen, Fanqing Frank; Su, Kai-Hang; Wei, Qi-Huo; Sun, Cheng

2014-04-01

A nanoplasmonic resonator (NPR) comprising a metallic nanodisk with alternating shielding layer(s), having a tagged biomolecule conjugated or tethered to the surface of the nanoplasmonic resonator for highly sensitive measurement of enzymatic activity. NPRs enhance Raman signals in a highly reproducible manner, enabling fast detection of protease and enzyme activity, such as Prostate Specific Antigen (paPSA), in real-time, at picomolar sensitivity levels. Experiments on extracellular fluid (ECF) from paPSA-positive cells demonstrate specific detection in a complex bio-fluid background in real-time single-step detection in very small sample volumes.

Early diagnosis of dengue in travelers: comparison of a novel real-time RT-PCR, NS1 antigen detection and serology.

PubMed

Huhtamo, Eili; Hasu, Essi; Uzcátegui, Nathalie Y; Erra, Elina; Nikkari, Simo; Kantele, Anu; Vapalahti, Olli; Piiparinen, Heli

2010-01-01

The increased traveling to dengue endemic regions and the numerous epidemics have led to a rise in imported dengue. The laboratory diagnosis of acute dengue requires several types of tests and often paired samples are needed for obtaining reliable results. Although several diagnostic methods are available, proper comparative data on their performance are lacking. To compare the performance of novel methods including a novel pan-DENV real-time RT-PCR and a commercially available NS1 capture-EIA in regard to IgM detection for optimizing the early diagnosis of DENV in travelers. A panel of 99 selected early phase serum samples of dengue patients was studied by real-time RT-PCR, NS1 antigen ELISA, IgM-EIA, IgG-IFA and cell culture virus isolation. The novel real-time RT-PCR was shown specific and sensitive for detection of DENV-1-4 RNA and suitable for diagnostic use. The diagnostic rate using combination of RNA and IgM detection was 99% and using NS1 and IgM detection 95.9%. The results of RNA and NS1 antigen detection disagreed in 15.5% of samples that had only RNA or NS1 antigen detected. The diagnostic rates of early samples are higher when either RNA or NS1 antigen detection is combined with IgM detection. Besides the differences in the RNA and NS1 detection assays, the observed discrepancy of results could suggest individual variation or differences in timing of these markers in patient serum. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Performance of ARCHITECT HCV core antigen test with specimens from US plasma donors and injecting drug users.

PubMed

Mixson-Hayden, Tonya; Dawson, George J; Teshale, Eyasu; Le, Thao; Cheng, Kevin; Drobeniuc, Jan; Ward, John; Kamili, Saleem

2015-05-01

Hepatitis C virus (HCV) core antigen is a serological marker of current HCV infection. The aim of this study was mainly to evaluate the performance characteristics of the ARCHITECT HCV core antigen assay with specimens from US plasma donors and injecting drug users. A total of 551 serum and plasma samples with known anti-HCV and HCV RNA status were tested for HCV core antigen using the Abbott ARCHITECT HCV core antigen test. HCV core antigen was detectable in 100% of US plasma donor samples collected during the pre-seroconversion phase of infection (anti-HCV negative/HCV RNA positive). Overall sensitivity of the HCV core antigen assay was 88.9-94.3% in samples collected after seroconversion. The correlation between HCV core antigen and HCV RNA titers was 0.959. HCV core antigen testing may be reliably used to identify current HCV infection. Published by Elsevier B.V.

Detection of K1 antigen of Escherichia coli rods isolated from pregnant women and neonates.

PubMed

Kaczmarek, Agnieszka; Budzyńska, Anna; Gospodarek, Eugenia

2014-09-01

The K1 antigen is an important virulence determinant of Escherichia coli strains and has been shown to be associated particularly with neonatal meningitis, bacteraemia and septicaemia. Thus, its detection seems to be useful, especially in the case of E. coli strains isolated from pregnant women and newborns. In this study, the sensitivity and specificity of the latex agglutination test (Pastorex Meningitis) for identification of E. coli serogroup K1 were assessed, using PCR as the gold standard. Our results showed that consistency of results between latex agglutination test and PCR amounted to 98.5%. Therefore, Pastorex Meningitis is a good alternative to PCR and could be used for rapid K1 antigen detection, especially in local non-specialized laboratories with limited resources where PCR assay is not applied.

AuNPs/CNOs/SWCNTs/chitosan-nanocomposite modified electrochemical sensor for the label-free detection of carcinoembryonic antigen.

PubMed

Rizwan, Mohammad; Elma, Syazwani; Lim, Syazana Abdullah; Ahmed, Minhaz Uddin

2018-06-01

In this work, a nanocomposite of gold nanoparticles (AuNPs), carbon nano-onions (CNOs), single-walled carbon nanotubes (SWCNTs) and chitosan (CS) (AuNPs/CNOs/SWCNTs/CS) was prepared for the development of highly sensitive electrochemical immunosensor for the detection of carcinoembryonic antigen (CEA), clinical tumor marker. Firstly, layer-by-layer fabrication of the CEA-immunosensors was studied using cyclic voltammetry (CV) and square wave voltammetry (SWV). By combining the advantages of large surface area and electronic properties of AuNPs, CNOs, SWCNTs, and film forming properties of CS, AuNPs/CNOs/SWCNTs/CS-nanocomposite-modified glassy carbon electrode showed a 200% increase in effective surface area and electronic conductivity. The calibration plot gave a negative linear relationship between log[concentration] of CEA and electrical current with a correlation coefficient of 0.9875. The CEA-immunosensor demonstrated a wide linear detection range of 100 fg mL -1 to 400 ng mL -1 with a low detection limit of 100 fg mL -1 . In addition to high sensitivity, reproducibility and large stability, CEA-immunosensor provided an excellent selectivity and resistant-to-interference in the presence of other antigens in serum and hence a potential to be used with real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

PubMed Central

Patra, Kailash P.; Saito, Mayuko; Atluri, Vidya L.; Rolán, Hortensia G.; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N.; Gotuzzo, Eduardo; Gilman, Robert H.; Tsolis, Renee M.; Vinetz, Joseph M.

2014-01-01

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

Nanostructured materials detect epidermal growth factor receptor, neuron specific enolase and carcinoembryonic antigen

NASA Astrophysics Data System (ADS)

Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Badulescu, Marius

2015-09-01

New nanostructured materials based on thin films of Cu and Ni deposited on textile material (veil), as well as gold nanostructured microspheres were used for the design of new stochastic sensors. The stochastic sensors were able to detect simultaneously a panel of biomarkers comprising epidermal growth factor receptor, neuron specific enolase, and carcinoembryonic antigen from whole blood samples with high reliabilities - recovery tests higher than 97.00%, with a RSD (%) lower than 0.1%. The stochastic sensors had shown high sensitivities and low determination levels for the detection of the proposed panel of biomarkers making early detection of lung cancer possible by fast screening of whole blood.

Direct covalent attachment of small peptide antigens to enzyme-linked immunosorbent assay plates using radiation and carbodiimide activation.

PubMed

Dagenais, P; Desprez, B; Albert, J; Escher, E

1994-10-01

Direct adsorption of small peptide antigens to unaltered, commercially available polystyrene surfaces may be too weak to permit suitable assay by ELISA. We therefore developed a simple method for the covalent attachment of small, potentially single epitope antigens to polystyrene surfaces. Chemical activation of polystyrene plates with carbodiimide considerably improves the total and covalent attachment of radioactive octapeptides. The covalent attachment was demonstrated by washing with hot detergent. A 3.5 Mrad gamma-irradiation of plates also increases total binding, particularly in combination with chemical activation. The covalent attachment presumably occurs through formation and chemical activation of carboxylate functions on the polystyrene surface which form amide bonds with peptides. ELISA test was performed with CGRP and successive smaller CGRP fragments. Covalent attachment of C-terminal peptide fragments as detection antigens allows optimal recognition and sensitivity even for hexapeptides, while decapeptide antigens were already poorly recognized using a conventional antigen plating technique. Repetitive detergent washes and/or prolonged storage of plates with covalently bound antigens did not reduce their ELISA sensitivity. The method with storage and reutilization capacities that we present here will be useful for the development of preplated antibody screening test.

Evaluation of an enzyme immunoassay for detection of dengue virus NS1 antigen in human serum.

PubMed

Dussart, Philippe; Labeau, Bhety; Lagathu, Gisèle; Louis, Philippe; Nunes, Marcio R T; Rodrigues, Sueli G; Storck-Herrmann, Cécile; Cesaire, Raymond; Morvan, Jacques; Flamand, Marie; Baril, Laurence

2006-11-01

We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

Circulating antigen tests and urine reagent strips for diagnosis of active schistosomiasis in endemic areas.

PubMed

Ochodo, Eleanor A; Gopalakrishna, Gowri; Spek, Bea; Reitsma, Johannes B; van Lieshout, Lisette; Polman, Katja; Lamberton, Poppy; Bossuyt, Patrick M M; Leeflang, Mariska M G

2015-03-11

Point-of-care (POC) tests for diagnosing schistosomiasis include tests based on circulating antigen detection and urine reagent strip tests. If they had sufficient diagnostic accuracy they could replace conventional microscopy as they provide a quicker answer and are easier to use. To summarise the diagnostic accuracy of: a) urine reagent strip tests in detecting active Schistosoma haematobium infection, with microscopy as the reference standard; and b) circulating antigen tests for detecting active Schistosoma infection in geographical regions endemic for Schistosoma mansoni or S. haematobium or both, with microscopy as the reference standard. We searched the electronic databases MEDLINE, EMBASE, BIOSIS, MEDION, and Health Technology Assessment (HTA) without language restriction up to 30 June 2014. We included studies that used microscopy as the reference standard: for S. haematobium, microscopy of urine prepared by filtration, centrifugation, or sedimentation methods; and for S. mansoni, microscopy of stool by Kato-Katz thick smear. We included studies on participants residing in endemic areas only. Two review authors independently extracted data, assessed quality of the data using QUADAS-2, and performed meta-analysis where appropriate. Using the variability of test thresholds, we used the hierarchical summary receiver operating characteristic (HSROC) model for all eligible tests (except the circulating cathodic antigen (CCA) POC for S. mansoni, where the bivariate random-effects model was more appropriate). We investigated heterogeneity, and carried out indirect comparisons where data were sufficient. Results for sensitivity and specificity are presented as percentages with 95% confidence intervals (CI). We included 90 studies; 88 from field settings in Africa. The median S. haematobium infection prevalence was 41% (range 1% to 89%) and 36% for S. mansoni (range 8% to 95%). Study design and conduct were poorly reported against current standards. Tests for S. haematobium Urine reagent test strips versus microscopyCompared to microscopy, the detection of microhaematuria on test strips had the highest sensitivity and specificity (sensitivity 75%, 95% CI 71% to 79%; specificity 87%, 95% CI 84% to 90%; 74 studies, 102,447 participants). For proteinuria, sensitivity was 61% and specificity was 82% (82,113 participants); and for leukocyturia, sensitivity was 58% and specificity 61% (1532 participants). However, the difference in overall test accuracy between the urine reagent strips for microhaematuria and proteinuria was not found to be different when we compared separate populations (P = 0.25), or when direct comparisons within the same individuals were performed (paired studies; P = 0.21).When tests were evaluated against the higher quality reference standard (when multiple samples were analysed), sensitivity was marginally lower for microhaematuria (71% vs 75%) and for proteinuria (49% vs 61%). The specificity of these tests was comparable. Antigen assayCompared to microscopy, the CCA test showed considerable heterogeneity; meta-analytic sensitivity estimate was 39%, 95% CI 6% to 73%; specificity 78%, 95% CI 55% to 100% (four studies, 901 participants). Tests for S. mansoni Compared to microscopy, the CCA test meta-analytic estimates for detecting S. mansoni at a single threshold of trace positive were: sensitivity 89% (95% CI 86% to 92%); and specificity 55% (95% CI 46% to 65%; 15 studies, 6091 participants) Against a higher quality reference standard, the sensitivity results were comparable (89% vs 88%) but specificity was higher (66% vs 55%). For the CAA test, sensitivity ranged from 47% to 94%, and specificity from 8% to 100% (4 studies, 1583 participants). Among the evaluated tests for S. haematobium infection, microhaematuria correctly detected the largest proportions of infections and non-infections identified by microscopy.The CCA POC test for S. mansoni detects a very large proportion of infections identified by microscopy, but it misclassifies a large proportion of microscopy negatives as positives in endemic areas with a moderate to high prevalence of infection, possibly because the test is potentially more sensitive than microscopy.

Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis.

PubMed

Khalid, Ruqyya; Afzal, Madeeha; Khurshid, Sana; Paracha, Rehan Zafar; Khan, Imran H; Akhtar, Muhammad Waheed

Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that α-helical and β-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli.

Simultaneous human platelet antigen genotyping and detection of novel single nucleotide polymorphisms by targeted next-generation sequencing.

PubMed

Davey, Sue; Navarrete, Cristina; Brown, Colin

2017-06-01

Twenty-nine human platelet antigen systems have been described to date, but the majority of current genotyping methods are restricted to the identification of those most commonly associated with alloantibody production in a clinical context. This can result in a protracted investigation if causative human platelet antigens are rare or novel. A targeted next-generation sequencing approach was designed to detect all known human platelet antigens with the additional capability of identifying novel mutations in the encoding genes. A targeted enrichment, high-sensitivity HaloPlex assay was designed to sequence all exons and flanking regions of the six genes known to encode human platelet antigens. Indexed DNA libraries were prepared from 47 previously human platelet antigen-genotyped samples and subsequently combined into one of three pools for sequencing on an Illumina MiSeq platform. The generated FASTQ files were aligned and scrutinized for each human platelet antigen polymorphism using SureCall data analysis software. Forty-six samples were successfully genotyped for human platelet antigens 1 through 29bw, with an average per base coverage depth of 1144. Concordance with historical human platelet antigen genotypes was 100%. A putative novel mutation in Exon 10 of the integrin β-3 (ITGB3) gene from an unsolved case of fetal neonatal alloimmune thrombocytopenia was also detected. A next-generation sequencing-based method that can accurately define all known human platelet antigen polymorphisms was developed. With the ability to sequence up to 96 samples simultaneously, our HaloPlex design could be used for high-throughput human platelet antigen genotyping. This method is also applicable for investigating fetal neonatal alloimmune thrombocytopenia when rare or novel human platelet antigens are suspected. © 2017 AABB.

Development of an Enzyme-Linked Immunosorbent Assay Based on Fusion VP2332-452 Antigen for Detecting Antibodies against Aleutian Mink Disease Virus.

PubMed

Chen, Xiaowei; Song, Cailing; Liu, Yun; Qu, Liandong; Liu, Dafei; Zhang, Yun; Liu, Ming

2016-02-01

For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2332-452 protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2332-452 ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2332-452 ELISA may be a preferable method for detecting antibodies against AMDV. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

pH responsive label-assisted click chemistry triggered sensitivity amplification for ultrasensitive electrochemical detection of carbohydrate antigen 24-2.

PubMed

Zheng, Yun; Zhao, Lihua; Ma, Zhanfang

2018-05-15

Sensitivity amplification strategy by implementing click chemistry in the construction of biosensing interface can efficiently improve the performance of immunosensor. Herein, we developed a sandwich-type amperometric immunosensor for ultrasensitive detection of carbohydrate antigen 24-2 (CA 242) based on pH responsive label-assisted click chemistry triggered sensitivity amplification strategy. The sensitivity of amperometric immunosensor relies on the current response differences (ΔI) caused by per unit concentration target analyte. The pH responsive Cu 2+ -loaded polydopamine (CuPDA) particles conjugated with detection antibodies were employed as labels, which can release Cu(II) ions by regulating pH. In the presence of ascorbic acid (reductant), Cu(II) ions were reduced to Cu(I) ions. Azide-functionalized double-stranded DNA (dsDNA) as signal enhancer was immobilized on the substrate through Cu + -catalyzed azide/alkyne cycloaddition reaction. With the help of the click reaction, the ΔI caused by target was elevated prominently, resulting in sensitivity amplification of the immunosensor. Under optimal condition, the proposed immunosensor exhibited excellent performance with linear range from 0.0001 to 100 U mL -1 and ultralow detection limit of 20.74 μU mL -1 . This work successfully combines click chemistry with pH-responsive labels in sandwich-type amperometric immunosensor, providing a promising sensitivity amplification strategy to construct immunosensing platform for analysis of other tumor marker. Copyright © 2018 Elsevier B.V. All rights reserved.

Benefit of hepatitis C virus core antigen assay in prediction of therapeutic response to interferon and ribavirin combination therapy.

PubMed

Takahashi, Masahiko; Saito, Hidetsugu; Higashimoto, Makiko; Atsukawa, Kazuhiro; Ishii, Hiromasa

2005-01-01

A highly sensitive second-generation hepatitis C virus (HCV) core antigen assay has recently been developed. We compared viral disappearance and first-phase kinetics between commercially available core antigen (Ag) assays, Lumipulse Ortho HCV Ag (Lumipulse-Ag), and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor test, version 2 (Amplicor M), to estimate the predictive benefit of a sustained viral response (SVR) and non-SVR in 44 genotype 1b patients treated with interferon (IFN) and ribavirin. HCV core Ag negativity could predict SVR on day 1 (sensitivity = 100%, specificity = 85.0%, accuracy = 86.4%), whereas RNA negativity could predict SVR on day 7 (sensitivity = 100%, specificity = 87.2%, accuracy = 88.6%). None of the patients who had detectable serum core Ag or RNA on day 14 achieved SVR (specificity = 100%). The predictive accuracy on day 14 was higher by RNA negativity (93.2%) than that by core Ag negativity (75.0%). The combined predictive criterion of both viral load decline during the first 24 h and basal viral load was also predictive for SVR; the sensitivities of Lumipulse-Ag and Amplicor-M were 45.5 and 47.6%, respectively, and the specificity was 100%. Amplicor-M had better predictive accuracy than Lumipulse-Ag in 2-week disappearance tests because it had better sensitivity. On the other hand, estimates of kinetic parameters were similar regardless of the detection method. Although the correlations between Lumipulse-Ag and Amplicor-M were good both before and 24 h after IFN administration, HCV core Ag seemed to be relatively lower 24 h after IFN administration than before administration. Lumipulse-Ag seems to be useful for detecting the HCV concentration during IFN therapy; however, we still need to understand the characteristics of the assay.

Accurate antemortem diagnosis of equine protozoal myeloencephalitis (EPM) based on detecting intrathecal antibodies against Sarcocystis neurona using the SnSAG2 and SnSAG4/3 ELISAs.

PubMed

Reed, S M; Howe, D K; Morrow, J K; Graves, A; Yeargan, M R; Johnson, A L; MacKay, R J; Furr, M; Saville, W J A; Williams, N M

2013-01-01

Recent work demonstrated the value of antigen-specific antibody indices (AI and C-value) to detect intrathecal antibody production against Sarcocystis neurona for antemortem diagnosis of equine protozoal myeloencephalitis (EPM). The study was conducted to assess whether the antigen-specific antibody indices can be reduced to a simple serum : cerebrospinal fluid (CSF) titer ratio to achieve accurate EPM diagnosis. Paired serum and CSF samples from 128 horses diagnosed by postmortem examination. The sample set included 44 EPM cases, 35 cervical-vertebral malformation (CVM) cases, 39 neurologic cases other than EPM or CVM, and 10 non-neurologic cases. Antibodies against S. neurona were measured in serum and CSF pairs using the SnSAG2 and SnSAG4/3 (SnSAG2, 4/3) ELISAs, and the ratio of each respective serum titer to CSF titer was determined. Likelihood ratios and diagnostic sensitivity and specificity were calculated based on serum titers, CSF titers, and serum : CSF titer ratios. Excellent diagnostic sensitivity and specificity was obtained from the SnSAG2, 4/3 serum : CSF titer ratio. Sensitivity and specificity of 93.2 and 81.1%, respectively, were achieved using a ratio cutoff of ≤100, whereas sensitivity and specificity were 86.4 and 95.9%, respectively, if a more rigorous cutoff of ≤50 was used. Antibody titers in CSF also provided good diagnostic accuracy. Serum antibody titers alone yielded much lower sensitivity and specificity. The study confirms the value of detecting intrathecal antibody production for antemortem diagnosis of EPM, and they further show that the antigen-specific antibody indices can be reduced in practice to a simple serum : CSF titer ratio. Copyright © 2013 by the American College of Veterinary Internal Medicine.

Radioimmunoassay of human Hageman factor (factor XII). [/sup 125/I tracer technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saito, H.; Ratnoff, O.D.; Pensky, J.

A specific, sensitive, and reproducible radioimmunoassay for human Hageman factor (HF, factor XII) has been developed with purified human HF and monospecific rabbit antibody. Precise measurements of HF antigen were possible for concentrations as low as 0.1 percent of that in normal pooled plasma. A good correlation (correlation coefficient = 0.82) existed between the titers of HF measured by clot-promoting assays and radioimmunoassays among 42 normal adults. Confirming earlier studies, HF antigen was absent in Hageman trait plasma, but other congenital deficient plasmas, including those of individuals with Fletcher trait and Fitzgerald trait, contained normal amounts of HF antigen. HFmore » antigen was reduced in the plasmas of patients with disseminated intravascular coagulation or advanced liver cirrhosis, but it was normal in those of patients with chronic renal failure or patients under treatment with warfarin. HF antigen was detected by this assay in plasmas of primates, but not detectable in plasmas of 11 nonprimate mammalian and one avian species.« less

Comparison of two antigen assays for rapid intrapartum detection of vaginal group B streptococcal colonization.

PubMed

Green, M; Dashefsky, B; Wald, E R; Laifer, S; Harger, J; Guthrie, R

1993-01-01

As part of a clinical investigation evaluating the efficacy of intrapartum antigen detection for screening for heavy vaginal colonization with group B streptococci (GBS), we compared the performance of modified Bactigen and Directigen GBS latex particle agglutination (LPA) kits. Paired vaginal swabs obtained from women in labor were rapidly transported to the laboratory and used for culturing (both swabs) and LPA testing (one swab by each method). GBS growth was estimated semiquantitatively and further designated as light or heavy growth. Performance specifications for each method were determined by comparing LPA and culture results from the same swab. A total of 4,251 paired swabs were evaluated during the study period. The performance specifications for detecting GBS growth of any degree for Bactigen and Directigen, respectively, were as follows: sensitivity, 20 and 24%; specificity, 99 and 99%. The performance specifications for heavy colonization for Bactigen and Directigen, respectively, were as follows: sensitivity, 57 and 62%; specificity, 99 and 99%. Neither LPA kit was a sensitive indicator of vaginal colonization with GBS or neonatal infection.

Broad spectrum reactivity versus subtype specificity-trade-offs in serodiagnosis of influenza A virus infections by competitive ELISA.

PubMed

Postel, A; Ziller, M; Rudolf, M; Letzel, T; Ehricht, Ralf; Pourquier, P; Dauber, M; Grund, C; Beer, Martin; Harder, T C

2011-04-01

Avian influenza viruses (AIVs) of the H5 and H7 subtypes can cause substantial economic losses in the poultry industry and are a potential threat to public health. Serosurveillance of poultry populations is an important monitoring tool and can also be used for control of vaccination campaigns. The purpose of this study was to develop broadly reactive, yet subtype-specific competitive ELISAs (cELISAs) for the specific detection of antibodies to the notifiable AIV subtypes H5 and H7 as an alternative to the gold standard haemagglutination inhibition assay (HI). Broadly reacting monoclonal competitor antibodies (mAbs) and genetically engineered subtype H5 or H7 haemagglutinin antigen, expressed and in vivo biotinylated in insect cells, were used to develop the cELISAs. Sera from galliform species and water fowl (n=793) were used to evaluate the performance characteristics of the cELISAs. For the H5 specific cELISA, 98.1% test sensitivity and 91.5% test specificity (97.7% and 90.2% for galliforms; 98.9% and 92.6% for waterfowl), and for the H7 cELISA 97.3% sensitivity and 91.8% specificity (95.3% and 98.9% for galliforms; 100% and 82.7% for waterfowl) were reached when compared to HI. The use of competitor mAbs with broad spectrum reactivity within an AIV haemagglutinin subtype allowed for homogenous detection with high sensitivity of subtype-specific antibodies induced by antigenically widely distinct isolates including antigenic drift variants. However, a trade-off regarding sensitivity versus nonspecific detection of interfering antibodies induced by phylo- and antigenically closely related subtypes, e.g., H5 versus H2 and H7 versus H15, must be considered. The observed intersubtype antibody cross-reactivity remains a disturbance variable in AIV subtype-specific serodiagnosis which negatively affects specificity. Copyright © 2011 Elsevier B.V. All rights reserved.

Detection of Helicobacter pylori urease antigen in saliva in patients with different gastric H. pylori status.

PubMed

El Khadir, Mounia; Alaoui Boukhris, Samia; Benajah, Dafr-Allah; El Rhazi, Karima; Ibrahimi, Sidi Adil; El Abkari, Mohamed; Harmouch, Taoufiq; Nejjari, Chakib; Mahmoud, Mustapha; Benlemlih, Mohamed; Bennani, Bahia

2016-07-01

Finding a simple, accurate, and noninvasive diagnosis method is a substantial challenge for the detection of Helicobacter pylori. The aim of the present study was to compare the presence of H. pylori urease antigen in saliva with the presence of this bacterium in gastric mucosa. Saliva samples and gastric biopsies were taken from 153 consenting Moroccan patients. Saliva samples were analyzed using an immunochromatographic test for urease antigen H. pylori detection. Thereafter, the gastric biopsies were analyzed by histology and polymerase chain reaction (PCR) to detect this bacterium. From a total of 153 recruited Moroccan patients, H. pylori was detected in 28 (18.30%), 87 (57.24%), and 69 (45.10%) cases by saliva test, histology, and PCR, respectively. A significant association was observed between the presence of H. pylori antigen in saliva and age. However, no association was found with sex, H. pylori virulence factors, gastric disease outcome, and density of the bacterium on the gastric mucosa. Considering that only 90 patients presented concordant results on H. pylori diagnosis (positive or negative) by both histology and PCR, the immunochromatographic test showed very low sensitivity (29.79%) and high specificity (90.70%). Of these two tests, the positive and negative predictive values were 77.78% and 54.17%, respectively. The accuracy of the test for salivary detection of urease antigen H. pylori was 58.89%. This study demonstrated a low detection rate of H. pylori antigens in saliva compared with the presence of this bacterium in gastric mucosa, suggesting that saliva cannot be used as a suitable sample for the diagnosis of H. pylori in our study population. Copyright © 2016. Published by Elsevier Taiwan LLC.

Malaria prevalence defined by microscopy, antigen detection, DNA amplification and total nucleic acid amplification in a malaria-endemic region during the peak malaria transmission season.

PubMed

Waitumbi, John N; Gerlach, Jay; Afonina, Irina; Anyona, Samuel B; Koros, Joseph N; Siangla, Joram; Ankoudinova, Irina; Singhal, Mitra; Watts, Kate; Polhemus, Mark E; Vermeulen, Nicolaas M; Mahoney, Walt; Steele, Matt; Domingo, Gonzalo J

2011-07-01

To determine the malaria prevalence by microscopy, antigen detection and nucleic acid detection in a defined subpopulation in a Plasmodium falciparum-endemic region during the peak transmission season. Blood specimens were collected in a cross-sectional study involving children aged 5-10 years (n = 195) presenting with acute fever to two clinics in Western Kenya. All specimens underwent microscopy, HRP2 and aldolase antigen detection by enzyme immunoassay (EIA), parasite-specific DNA and total nucleic acid (RNA and DNA) by real-time PCR (qPCR) and reverse-transcriptase PCR (qRT-PCR). Microscopy detected 65/195 cases of malaria infection [95% confidence interval (CI) 52-78]. HRP2 and aldolase EIA had similar sensitivity levels detecting antigen in 65/195 (95% CI, 52-78) and 57/195 (95% CI, 45-70) cases. Discordants in antigen detection vs. microscopy occurred at <470 parasites/μl and <4900 parasites/μl for HRP2 and aldolase, respectively. Detection of total nucleic acid allowed a 3 log lower limit of detection than just DNA detection by real-time PCR in vitro. In clinical specimens, 114/195 (95% CI, 100-127) were qPCR positive (DNA), and 187/195 (95% CI, 179-191) were qRT-PCR positive (DNA plus RNA). The prevalence of submicroscopic malaria infection was significantly higher when detecting total nucleic acid than just DNA in this outpatient population during the high transmission season. Defining standards for submicroscopic infection will be important for control programmes, diagnostics development efforts and molecular epidemiology studies. © 2011 Blackwell Publishing Ltd.

Unlabeled multi tumor marker detection system based on bioinitiated light addressable potentiometric sensor.

PubMed

Jia, Yun-Fang; Gao, Chun-Ying; He, Jia; Feng, Dao-Fu; Xing, Ke-Li; Wu, Ming; Liu, Yang; Cai, Wen-Sheng; Feng, Xi-Zeng

2012-08-21

Multi biomarkers' assays are of great significance in clinical diagnosis. A label-free multi tumor markers' parallel detection system was proposed based on a light addressable potentiometric sensor (LAPS). Arrayed LAPS chips with basic structure of Si(3)N(4)-SiO(2)-Si were prepared on silicon wafers, and the label-free parallel detection system for this component was developed with user friendly controlling interfaces. Then the l-3,4-dihydroxyphenyl-alanine (L-Dopa) hydrochloric solution was used to initiate the surface of LAPS. The L-Dopa immobilization state was investigated by the theoretical calculation. L-Dopa initiated LAPS' chip was biofunctionalized respectively by the antigens and antibodies of four tumor markers, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9) and Ferritin. Then unlabeled antibodies and antigens of these four biomarkers were detected by the proposed detection systems. Furthermore physical and measuring principles in this system were described, and qualitative understanding for experimental data were given. The measured response ranges were compared with their clinical cutoff values, and sensitivities were calculated by OriginLab. The results indicate that this bioinitiated LAPS based label-free detection system may offer a new choice for the realization of unlabeled multi tumor markers' clinical assay.

Issues encountered in development of enzyme-linked immunosorbent assay for use in detecting Influenza A virus subtype H5N1 exposure in swine.

PubMed

Buehler, Jason; Lager, Kelly; Vincent, Amy; Miller, Cathy; Thacker, Eileen; Janke, Bruce

2014-03-01

A potential mechanism by which highly pathogenic avian Influenza A virus subtype H5N1 could more readily infect human beings is through the infection of and adaptation in pigs. To detect the occurrence of such infection, monitoring of pig populations through serological screening would be highly desirable. In the current study, hemagglutination inhibition assays were able to detect antibodies against H5N1 developed in pigs, but because of antigenic variation between clades, the use of multiple virus strains were required. Whole recombinant virus and recombinant hemagglutinin antigen enzyme-linked immunosorbent assays (ELISAs) were generated that could detect antibody against multiple H5N1 strains, but which also detected antibody against endemic swine influenza viruses. A recombinant hemagglutinin antigen-based ELISA was as effective as the whole virus antigen ELISAs in detecting antibody against the H5N1 virus strains used and eliminated nearly all of the cross-reactivity with non-H5N1 virus antibody. The current study also highlighted the difficulty in establishing a decision (cutoff) value that would effectively counterbalance nonspecific reactivity against sensitivity. The results provide important information and considerations for the development of serological screening assays for highly pathogenic avian H5N1 viruses.

Detection of early stage prostate cancer by using a simple carbon nanotube@paper biosensor.

PubMed

Ji, Sungkyung; Lee, Myeongsoon; Kim, Don

2018-04-15

This study is an investigation for an inexpensive, simple and sensitive biosensor to detect prostate cancer using bioactivated-multi wall carbon nanotubes (MWCNTs, diameter of 20nm, length of 5µm) and a micro-pore filter paper (pore size of 0.45µm). For the immunoassay of prostate specific antigen (PSA), which is a biomarker of prostate cancer, MWCNTs were activated with PSA antibody (monoclonal antibody of the prostate specific antigen) by using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium salt (NHSS). The activated MWCNTs were deposited on the micro-pore filter paper to use as a biosensor. The prepared biosensor can assay from 0 to 500ng/mL of PSA level within 2h with the detection limit of 1.18ng/mL by the measurement of resistance change. The resistance change was caused by site selective interaction between PSA and PSA-antigen with an inexpensive bench top digital multimeter (5 1/2 digits). The detection range and sensitivity of the prepared sensor are good enough to diagnose the early stage of prostate cancer (> 4ng/mL of PSA). This paper-based biosensor is about 20 times cheaper (fabricated biosensor price: 2.4 $) and over 10 times faster than enzyme-linked immunosorbent assay (ELISA), which is a general method for the detection of a specific protein in the modernized hospitals. Furthermore, the maximum detection limit is about 50 times higher than ELISA. Copyright © 2017 Elsevier B.V. All rights reserved.

Experimental support for an immunological approach to the search for life on other planets.

PubMed

Schweitzer, Mary Higby; Wittmeyer, Jennifer; Avci, Recep; Pincus, Seth

2005-02-01

We propose a three-phase approach to test for evidence of life in extraterrestrial samples. The approach capitalizes on the flexibility, sensitivity, and specificity of antibody-antigen interactions. Data are presented to support the first phase, in which various extraction protocols are compared for efficiency, and in which a preliminary suite of antibodies are tested against various antigens. The antigens and antibodies were chosen on the basis of criteria designed to optimize the detection of extraterrestrial biomarkers unique to living or once-living organisms.

Optoacoustic detection of viral antigens using targeted gold nanorods

NASA Astrophysics Data System (ADS)

Maswadi, Saher; Woodward, Lee; Glickman, Randolph D.; Barsalou, Norman

2009-02-01

We are detecting antigens (Ag), isolated from infectious organisms, utilizing laser optoacoustic spectroscopy and antibody-coupled gold nanorod (NR) contrast agents specifically targeted to the antigen of interest. We have detected, in clinical ocular samples, both Herpes Simplex Virus Type 1 and 2 (HSV-1 and HSV-2) . A monoclonal antibody (Ab) specific to both HSV-1 and HSV-2 was conjugated to gold nanorods to produce a targeted contrast agent with a strong optoacoustic signal. Elutions obtained from patient corneal swabs were adsorbed in standard plastic micro-wells. An immunoaffinity reaction was then performed with the functionalized gold nanorods, and the results were probed with an OPO laser, emitting wavelengths at the peak absorptions of the nanorods. Positive optoacoustic responses were obtained from samples containing authentic (microbiologically confirmed) HSV-1 and HSV-2. To obtain an estimate of the sensitivity of the technique, serial dilutions from 1 mg/ml to 1 pg/ml of a C. trachomatis surface Ag were prepared, and were probed with a monoclonal Ab, specific to the C. trachomatis surface Ag, conjugated to gold nanorods. An optoacoustic response was obtained, proportional to the concentration of antigen, and with a limit of detection of about 5 pg/ml. The optoacoustic signals generated from micro-wells containing albumin or saline were similar to those from blank wells. The potential benefit of this method is identify viral agents more rapidly than with existing techniques. In addition, the sensitivity of the assay is comparable or superior to existing colorimetric- or fluorometric-linked immunoaffinity assays.

Comparison of ELISA and Microscopy for detection of Cryptosporidium in stool

PubMed Central

Sharma, Madhu; Chaudhary, Uma; Yadav, Aparna

2014-01-01

Background: Cryptosporidiosis, a diarrheal disease caused by the protozoan parasite Cryptosporidium spp. has become recognized as one of the most common causes of water borne diseases in humans. Aims and Objectives: To compare the sensitivity of ELISA and Microscopy for detection of Cryptosporidium in stool samples Materials and Methods: The study was conducted in the Department of Microbiology of PT. B.D. Sharma PGIMS Rohtak, between January 2011 to june 2011 on 50 stool samples, which were processed for detection of cryptosporidial antigen by ELISA and detection of cysts by microscopy (Modified Ziehl and Nelsen staining). Study and Design: This was a prospective study conducted in the Department of Microbiology in PT. BD Sharma, PGIMS, Rohtak, India. Result: Out of total, 50 stool samples eighteen (36%) samples were found positive for Cryptosporidium cysts by microscopy in comparison to 3(6%) stool samples which were found positive for cryptosporidial antigen by ELISA. Samples found positive with ELISA were also positive with microscopy. Sensitivity, specificity, positive predictive value and negative predictive value for ELISA was 16.7%, 100%, 100% and 68% respectively. Conclusion: The study concludes that stool microscopic Modified acid fast staining is more sensitive method than ELISA for detection of Cryptosporidium in stool samples but the specificity of ELISA was more than microscopy. PMID:25584216

Plasmonic nanosensors: Inverse sensitivity

NASA Astrophysics Data System (ADS)

Käll, Mikael

2012-07-01

Enzyme-modified plasmonic nanoparticles that generate a signal that is larger when the concentration of the target molecule is lower can detect ultralow levels of the cancer biomarker prostate-specific antigen in whole serum.

Application of crude and recombinant ELISAs and immunochromatographic test for serodiagnosis of animal trypanosomosis in the Umkhanyakude district of KwaZulu-Natal province, South Africa

PubMed Central

NGUYEN, Thu-Thuy; MOTSIRI, Mono Sophie; TAIOE, Moeti Oriel; MTSHALI, Moses Sibusiso; GOTO, Yasuyuki; KAWAZU, Shin-Ichiro; THEKISOE, Oriel Matlhahane Molifi; INOUE, Noboru

2014-01-01

A total of 231 serum samples were collected from sheep (n=9), goats (n=99) and cattle (n=123) in northeastern KwaZulu-Natal, South Africa. Trypanosome infection was detected using Trypanosoma brucei brucei crude antigen (TbbCA) and T. congolense crude antigen (TcoCA) ELISA assays. Recombinant antigen (T. evansi GM6 which consisted of 4 repeat domains, TeGM6-4r) ELISA and immunochromatographic test (ICT) were also used. Crude antigen ELISA, TeGM6-4r-ELISA and ICT detected 27.3%, 29% and 19.9% of trypanosome seropositive samples, respectively. Trypanosome infection prevalence in cattle and goats was 35.8–46.3% and 0–9.1%, respectively. Out of 9 sheep serum samples, 2–4 sera (22.2–44.4%) were positive. The detection performance of crude and recombinant antigen ELISAs was relatively similar (K=0.6–0.7); both are recommended for reference diagnosis and large scale epidemiological surveys. There is potential application for ICT in on-site diagnosis, but its sensitivity should be improved. PMID:25342634

Immunocytochemical detection of Mycobacterium Tuberculosis complex specific antigen, MPT64, improves diagnosis of tuberculous lymphadenitis and tuberculous pleuritis.

PubMed

Tadele, Agerie; Beyene, Demissew; Hussein, Jemal; Gemechu, Tuffa; Birhanu, Asaye; Mustafa, Tehmina; Tsegaye, Aster; Aseffa, Abraham; Sviland, Lisbet

2014-11-25

A rapid, sensitive and accurate laboratory diagnosis is of prime importance in suspected extrapulmonary tuberculosis (EPTB) cases. However, traditional techniques for the detection of acid-fast bacilli have limitations. The aim of the study was to evaluate the diagnostic value of immunocytochemical staining for detection of Mycobacterium tuberculosis complex specific antigen, MPT64, in aspirates from pleural effusions and lymph nodes, the most common presentations of EPTB. A cross-sectional study was conducted by including patients at Tikur Anbessa Specialized Hospital and the United Vision Medical Services from December 2011 to June 2012. Lymph node aspirates and pleural fluid samples were collected and analyzed from a total of 51 cases (26 tuberculous (TB) pleuritis and 25 TB lymphadenitis) and 67 non-TB controls. Each specimen was subjected to Ziehl-Neelsen (ZN) staining, culture on Lowenstein- Jensen (LJ) medium, cytological examination, Polymerase Chain Reaction (PCR) using IS1081gene sequence as a primer and immunocytochemistry (ICC) with polyclonal anti-MPT64 antibody. All patients were screened for HIV. ICC was positive in 38 of 51 cases and in the 7 of 67 controls giving an overall sensitivity and specificity of 74.5% and 89.5%, respectively. Using IS1081-PCR as a reference method, the sensitivity and specificity, positive and negative predictive value of ICC was 88.1%, 89.5%, 82.2% and 93.2%, respectively. The case detection rate increased from 13.7% by ZN stain to 19.6% by LJ culture, to 66.7% by cytology and 74.5% by ICC. Immunocytochemistry with anti-MPT64 antigen improved detection of TB in pleural effusion and lymph node aspirates. Further studies using monoclonal antibodies on samples from other sites of EPTB is recommended to validate this relatively simple diagnostic method for EPTB.

Multiplex detection of pathogen biomarkers in human blood, serum, and saliva using silicon photonic microring resonators

NASA Astrophysics Data System (ADS)

Estrada, I. A.; Burlingame, R. W.; Wang, A. P.; Chawla, K.; Grove, T.; Wang, J.; Southern, S. O.; Iqbal, M.; Gunn, L. C.; Gleeson, M. A.

2015-05-01

Genalyte has developed a multiplex silicon photonic chip diagnostics platform (MaverickTM) for rapid detection of up to 32 biological analytes from a drop of sample in just 10 to 20 minutes. The chips are manufactured with waveguides adjacent to ring resonators, and probed with a continuously variable wavelength laser. A shift in the resonant wavelength as mass binds above the ring resonators is measured and is directly proportional to the amount of bound macromolecules. We present here the ability to multiplex the detection of hemorrhagic fever antigens in whole blood, serum, and saliva in a 16 minute assay. Our proof of concept testing of a multiplex antigencapture chip has the ability to detect Zaire Ebola (ZEBOV) recombinant soluble glycoprotein (rsGP), Marburg virus (MARV) Angola recombinant glycoprotein (rGP) and dengue nonstructural protein I (NS1). In parallel, detection of 2 malaria antigens has proven successful, but has yet to be incorporated into multiplex with the others. Each assay performs with sensitivity ranging from 1.6 ng/ml to 39 ng/ml depending on the antigen detected, and with minimal cross-reactivity.

Use of a Dual-Antigen Rapid Diagnostic Test to Screen Children for Severe Plasmodium falciparum Malaria in a High-Transmission, Resource-Limited Setting.

PubMed

Boyce, Ross; Reyes, Raquel; Matte, Michael; Ntaro, Moses; Mulogo, Edgar; Siedner, Mark J

2017-10-16

In rural areas, many patients with malaria seek care at peripheral health facilities or community case management programs. While this strategy is effective for the management of uncomplicated malaria, severe malaria necessitates prompt detection and referral to facilities with adequate resources. In this prospective, observational cohort study, we assessed the accuracy of a dual-band (histidine-rich protein-2/pan-lactate dehydrogenase [HRP2/pLDH]) rapid diagnostic test (RDT) to differentiate uncomplicated from severe malaria. We included children aged <12 years who presented to a rural clinic in western Uganda with a positive HRP2 or HRP2/pLDH RDT. We estimated the test characteristics of a dual-antigen (HRP2+/pLDH+) band positive RDT compared to World Health Organization-defined clinical and laboratory criteria to detect severe malaria. A total of 2678 children underwent testing for malaria with an RDT, and 83 (9.0%) satisfied criteria for severe malaria. The sensitivity and specificity of a HRP2+/pLDH+ result for severe malaria was 97.6% (95% confidence interval [CI], 90.8%-99.6%) and 75.6% (95% CI, 73.8%-77.4%), respectively. An HRP2+/pLDH+ result was significantly more sensitive (97.6% vs 68.7%, P < .001) for the detection of severe malaria compared to algorithms that incorporate screening for danger signs. A positive dual-antigen (HRP2/pLDH) RDT has higher sensitivity than the use of clinical manifestations to detect severe malaria, making it a promising tool in the triage of children with malaria in low-resource settings. Additional work is needed to operationalize diagnostic and treatment algorithms that include dual-antigen RDTs to avoid over referral. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch] proteins: effects of thermal treatments and in vitro digestion.

PubMed

Venkatachalam, Mahesh; Teuber, Suzanne S; Peterson, W Rich; Roux, Kenneth H; Sathe, Shridhar K

2006-02-22

Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods.

Identification of new antigen candidates for the early diagnosis of Mycobacterium avium subsp. paratuberculosis infection in goats.

PubMed

Souriau, Armel; Freret, Sandrine; Foret, Benjamin; Willemsen, Peter T J; Bakker, Douwe; Guilloteau, Laurence A

2017-12-01

Currently Mycobacterium avium subsp. paratuberculosis (MAP) infection is diagnosed through indirect tests based on the immune response induced by the infection. The antigens commonly used in IFN-γ release assays (IGRA) are purified protein derivative tuberculins (PPD). However, PPDs, lack both specificity (Sp) and sensitivity (Se) in the early phase of infection. This study investigated the potential of 16 MAP recombinant proteins and five lipids to elicit the release of IFN-γ in goats from herds with or without a history of paratuberculosis. Ten recombinant proteins were selected as potential candidates for the detection of MAP infection in young goats. They were found to detect 25 to 75% of infected shedder (IS) and infected non-shedder (INS) kids younger than 10months of age. In comparison, PPD was shown to detect only 10% of INS and no IS kids. For seven antigens, Se (21-33%) and Sp (≥90%) of IGRA were shown to be comparable with PPD at 20months old. Only three antigens were suitable candidates to detect IS adult goats, although Se was lower than that obtained with PPD. In paratuberculosis-free herds, IGRA results were negative in 97% of indoor goats and 86% of outdoor goats using the 10 antigens. However, 22 to 44% of one-year-old outdoor goats were positive suggesting that they may be infected. In conclusion, this study showed that ten MAP recombinant proteins are potential candidates for early detection of MAP infected goats. Combining these antigens could form a possible set of MAP antigens to optimize the Se of caprine IGRA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sandwich enzyme-linked immunosorbent assay (ELISA) for measuring the concentration of, and detection of antibodies to, Aujeszky's disease virus.

PubMed

Kardi, V; Szegletes, E; Perényi, T; Pergel, I; Smal, Z

1990-01-01

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for measuring Aujeszky's disease virus (ADV) antigen concentration and an inhibition technique based on the former was developed for detection of antibodies to ADV. The results were checked by determining the cytopathic and serum neutralization titres. The correlation was satisfactory in both cases, with correlation coefficients above 0.8. When measuring ADV antigen concentration, the lower limit of detection was 10(3) TCID 50/0.2 ml. The sensitivity of ELISA in detecting antibodies to ADV was found to be superior to that of the serum neutralization test and, thus, enabled the testing of rabbit and guinea-pig sera.

Detecting Tie2, an endothelial growth factor receptor, by using immunohistochemistry in mouse lungs.

PubMed

Guha, Prajna P; David, Sascha A; Ghosh, Chandra C

2014-01-01

Immunohistochemical (IHC) staining is an invaluable, sensitive, and effective method to detect the presence and localization of proteins in the cellular compartment in tissues. The basic concept of IHC is detecting the antigen in tissues by means of specific antibody binding, which is then demonstrated with a colored histochemical reaction that can be observed under a light microscope. The most challenging aspect of IHC techniques is optimizing the precise experimental conditions that are required to get a specific and a strong signal. The critical steps of IHC are specimen acquisition, fixation, permeabilization, detection system, and selection of the antigen specific antibody and its optimization. Here, we elaborate the technique using the endothelial growth factor binding receptor Tie2 in mouse lungs.

[Dengue fever cases in Czech workers returning from the Maldives].

PubMed

Trojánek, Milan; Tomíčková, Dora; Roháčová, Hana; Kosina, Pavel; Gebouský, Jan; Dvořák, Jan; Chmelik, Vaclav; Batistová, Květoslava; Husa, Petr; Maixner, Jan; Sojková, Naděžda; Zelená, Hana; Marešová, Vilma; Stejskal, František

2013-09-01

The objective of this study is to present epidemiological characteristics and clinical symptoms of dengue fever cases in Czech workers who acquired the infection while working on the island of Fushivelavaru, Maldives. Furthermore, the study compares the sensitivity of novel direct detection assays, i.e. Real-Time Polymerase Chain Reaction (RT-PCR) and detection of Dengue NS1 antigen. The retrospective study evaluated the clinical course of dengue fever cases in Czech workers returning from a job in the Maldives who were diagnosed with dengue virus infection from September 1 to October 31, 2012. The laboratory diagnosis of dengue fever was based on the serological detection of IgM and IgG antibodies, detection of dengue NS1 antigen by enzyme-linked immunosobent assay (ELISA), and detection of dengue virus DNA by RT-PCR. The infection with dengue virus was confirmed in 18 males with a median age of 40 years (IQR 36-47) who returned from a job in the Maldives. Only one patient required admission to the hospital while the others were treated on an outpatient basis. The most frequently observed symptoms were fever (18), headache (9), muscle and joint pain (8 and 7, respectively), and rash (9). Typical laboratory findings were leukocytopenia and thrombocytopenia, a low CRP level, and elevated aminotransferase activity. The clinical course was uncomplicated in all patients. The dengue NS1 antigen detection (positive in all 10 patients with acute dengue fever) showed significantly higher sensitivity than the detection of viral RNA using RT-PCR (positive in 4 patients), p = 0.011. Although the vast majority of dengue fever cases are diagnosed among travellers returning from the tropics, the presented study points out the risks posed by dengue fever to long-term workers in endemic areas. The infection in the serologically naïve hosts is usually uncomplicated; however, infected persons are at significant risk of developing a severe complicated clinical course if challenged by another serotype. Furthermore, sick leaves or premature departures cause a considerable economic burden to employers. In the diagnosis of acute dengue fever, preference should be given to highly sensitive and specific tests for the direct detection of dengue virus (NS1 antigen and RT-PCR assays).

Surface-enhanced Raman scattering study of carcinoembryonic antigen in serum from patients with colorectal cancers

NASA Astrophysics Data System (ADS)

Chen, Gang; Chen, Yanping; Zheng, Xiongwei; He, Cheng; Lu, Jianping; Feng, Shangyuan; Chen, Rong; Zeng, Haisan

2013-12-01

In this work, we developed a SERS platform for quantitative detection of carcinoembryonic antigen (CEA) in serum of patients with colorectal cancers. Anti-CEA-functionalized 4-mercaptobenzoic acid-labeled Au/Ag core-shell bimetallic nanoparticles were prepared first and then used to analyze CEA antigen solutions of different concentrations. A calibration curve was established in the range from 5 × 10-3 to 5 × 105 ng/mL. Finally, this new SERS probe was applied for quantitative detection of CEA in serum obtained from 26 colorectal cancer patients according to the calibration curve. The results were in good agreement with that obtained by electrochemical luminescence method, suggesting that SERS immunoassay has high sensitivity and specificity for CEA detection in serum. A detection limit of 5 pg/ml was achieved. This study demonstrated the feasibility and great potential for developing this new technology into a clinical tool for analysis of tumor markers in the blood.

Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries

PubMed Central

Zimmerman, Sandra G; Peters, Nathaniel C; Altaras, Ariel E; Berg, Celeste A

2014-01-01

In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)–RNA FISH double labeling (IF/FISH). Each procedure balances conflicting requirements for permeabilization, fixation and preservation of antigenicity to detect RNA and protein expression with high resolution and sensitivity. The ISH protocol uses alkaline phosphatase–conjugated digoxigenin antibodies followed by a color reaction, whereas FISH detection involves tyramide signal amplification (TSA). To simultaneously preserve antigens for protein detection and enable RNA probe penetration for IF/FISH, we perform IF before FISH and use xylenes and detergents to permeabilize the tissue rather than proteinase K, which can damage the antigens. ISH and FISH take 3 d to perform, whereas IF/FISH takes 5 d. Probe generation takes 1 or 2 d to perform. PMID:24113787

A NEW SENSITIVE ASSAY FOR ANTIBODY AGAINST CELL SURFACE ANTIGENS BASED ON INHIBITION OF CELL-DEPENDENT ANTIBODY-MEDIATED CYTOTOXICITY

PubMed Central

Halloran, Phil; Schirrmacher, Volker; Festenstein, Hilliard

1974-01-01

Inhibition of cell-dependent antibody-mediated cytotoxicity has been investigated as a new assay for antibody against cell surface antigens. The cytotoxicity system consisted of effector cells (normal mouse spleen cells), target cells (61Cr-labeled chicken erythrocytes), and antitarget cell antibody. Addition of antibody against cell surface antigens in the effector cell population regularly inhibited the cytotoxicity measured in this system. This cytotoxicity inhibition assay (CIA) detected antibody with a variety of specificities: anti-H-2, anti-Thy 1.2, anti-immunoglobulin, and antimouse bone marrow-derived lymphocyte antigen. When the inhibition by anti-H-2 sera was analyzed using effector cells from congenic mice, the activity was found to be directed against specificities mapping in the H-2K, H-2D, and I regions of the H-2 complex, correlating well with the specificities characterized by complement-dependent assays. A comparison between the sensitivity of the CIA and complement-dependent lysis revealed that the CIA was 2–11 times more sensitive for anti-H-2 antisera and 20–780 times more sensitive for certain antisera against subpopulations of the spleen cells (i.e., T cells or B cells). The CIA proved to be precise, sensitive, and reliable. It may become a very useful antibody assay in various species including man. PMID:4547657

Novel Shear-horizontal Surface Acoustic Wave Based Immunosensors Using SiO2Waveguiding Layers And Flow Injection Analysis.

PubMed

Guo, X S; Chen, Y Q; Yang, X L; Wang, L R

2005-01-01

Surface acoustic wave (SAW) devices based on shear-horizontal (SH) waves can be used as mass-sensitive immunosensors. This paper presents a novel SH-SAW sensor to detect anti-immunoglobulin (IgG) molecules by means of the antibody-antigen binding mechanism. The sensor system comprising dual delay lines was fabricated on 36° Y-X LiTaO3substrate. A SiO2layer was used as love mode waveguiding layers, well as insulating and chemically resistant protective layer. Moreover, flow injection analysis (FIA) method was used for continuous detection the protein molecules. The protein A was immobilized on the optional surface of the gold layer, then coupled with IgG to adsorb the antigens to be measured in the protein solution. The operational frequency of the system changed due to the interaction of antibody-antigen binding. The experimental result demonstrates the sensor has stable frequency response to the mass loading effect of the various anti-IgG concentrations with the sensitivity up to 3.3ng/ml/Hz.

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

PubMed

López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

2007-11-01

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.

Antibody Responses to Sarcoptes scabiei Apolipoprotein in a Porcine Model: Relevance to Immunodiagnosis of Recent Infection

PubMed Central

Rampton, Melanie; Walton, Shelley F.; Holt, Deborah C.; Pasay, Cielo; Kelly, Andrew; Currie, Bart J.; McCarthy, James S.; Mounsey, Kate E.

2013-01-01

No commercial immunodiagnostic tests for human scabies are currently available, and existing animal tests are not sufficiently sensitive. The recombinant Sarcoptes scabiei apolipoprotein antigen Sar s 14.3 is a promising immunodiagnostic, eliciting high levels of IgE and IgG in infected people. Limited data are available regarding the temporal development of antibodies to Sar s 14.3, an issue of relevance in terms of immunodiagnosis. We utilised a porcine model to prospectively compare specific antibody responses to a primary infestation by ELISA, to Sar s 14.3 and to S. scabiei whole mite antigen extract (WMA). Differences in the antibody profile between antigens were apparent, with Sar s 14.3 responses detected earlier, and declining significantly after peak infestation compared to WMA. Both antigens resulted in >90% diagnostic sensitivity from weeks 8–16 post infestation. These data provide important information on the temporal development of humoral immune responses in scabies and further supports the development of recombinant antigen based immunodiagnostic tests for recent scabies infestations. PMID:23762351

Nano-particle enhanced impedimetric biosensor for detedtion of foodborne pathogens

NASA Astrophysics Data System (ADS)

Kim, G.; Om, A. S.; Mun, J. H.

2007-03-01

Recent outbreaks of foodborne illness have been increased the need for rapid and sensitive methods for detection of these pathogens. Conventional methods for pathogens detection and identification involve prolonged multiple enrichment steps. Even though some immunological rapid assays are available, these assays still need enrichment steps result in delayed detection. Biosensors have shown great potential for rapid detection of foodborne pathogens. They are capable of direct monitoring the antigen-antibody reactions in real time. Among the biosensors, impedimetric biosensors have been widely adapted as an analysis tool for the study of various biological binding reactions because of their high sensitivity and reagentless operation. In this study a nanoparticle-enhanced impedimetric biosensor for Salmonella enteritidis detection was developed which detected impedance changes caused by the attachment of the cells to the anti-Salmonella antibodies immobilized on interdigitated gold electrodes. Successive immobilization of neutravidin followed by anti-Salmonella antibodies was performed to the sensing area to create a biological detection surface. To enhance the impedance responses generated by antigen-antibody reactions, anti-Salmonella antibody conjugated nanoparticles were introduced on the sensing area. Using a portable impedance analyzer, the impedance across the interdigital electrodes was measured after the series of antigen-antibody bindings. Bacteria cells present in solution attached to capture antibodies and became tethered to the sensor surface. Attached bacteria cells changed the dielectric constant of the media between the electrodes thereby causing a change in measured impedance. Optimum input frequency was determined by analyzing frequency characteristics of the biosensor over ranges of applied frequencies from 10 Hz to 400 Hz. At 100 Hz of input frequency, the biosensor was most sensitive to the changes of the bacteria concentration and this frequency was used for the detection experiments. The biosensor was able to detect 106 CFU/mL in phosphate buffered saline (PBS) with a detection time of 3 minutes. Additional use of nanoparticles significantly enhanced the detection performance. By using the nanoparticles the biosensor could detect 104 CFU/mL of Salmonella enteritidis in PBS and 105 CFU/mL of cells in milk.

Urine Antigen Detection as an Aid to Diagnose Invasive Aspergillosis.

PubMed

Marr, Kieren A; Datta, Kausik; Mehta, Seema; Ostrander, Darin B; Rock, Michelle; Francis, Jesse; Feldmesser, Marta

2018-04-19

Establishing rapid diagnoses of invasive aspergillosis (IA) is priority, given poor outcomes of late therapy. Non-culture based tests that detect galactomannan and β-D glucan are available, but are technically cumbersome and rely on invasive sampling (blood or bronchoalveolar lavage). We optimized a lateral flow dipstick assay using the galactofuranose -specific monoclonal antibody (mAb476), which was previously shown to recognize urine antigens after Aspergillus fumigatus pulmonary infection in an`imals. Urine samples were obtained from a cohort of 78 subjects undergoing clinical evaluation for suspected invasive fungal infections, and stored frozen until testing. Urine was processed by centrifugation through desalting columns and exposed to dipsticks. Reviewers blinded to EORTC/MSG clinical diagnoses graded results. Western blots were performed on urines from two subjects to characterize mAb476-reactive antigens. Per-patient sensitivity and specificity for diagnosis of proven or probable IA in the overall cohort was 80% (95% CI: 61.4-92.3) and 92% (95% CI 74-99). In the sub-group with cancer, sensitivity was 89.5% (95% CI 66.7-98.7) and specificity was 90.9% (95% CI 58.7 - 99.8); amongst all others, sensitivity and specificity were 63.6 (95% CI 30.8 - 89.1) and 92.9 (66.1 - 99.8), respectively. Eliminating lung transplant recipients with airway disease increased sensitivity in the non-cancer cohort (85.7%, 95% CI 42.1-99.6%). Semi-quantitative urine assay results correlated with serum galactomannan indices. Western blots demonstrated mAb476-reactive antigens in urine from cases, ranging between 26 - 35kDa in size. Urine testing using mAb476 may be used as an aid to diagnose IA in high-risk patients.

Development and application of an indirect immunoperoxidase assay for the detection of Duck swollen head hemorrhagic disease virus antigen in Pekin ducks (Anas platyrhynchos).

PubMed

Li, Chuanfeng; Shen, Chanjuan; Cheng, Anchun; Wang, Mingshu; Zhang, Na; Zhou, Yi; Zhu, Dekang; Jia, Renyong; Luo, Qihui; Chen, Xiaoyue

2010-01-01

An improved indirect immunoperoxidase assay (IPA) was developed to detect antigens of Duck swollen head hemorrhagic disease virus (DSHDV) in paraformaldehyde-fixed, paraffin-embedded tissues of Pekin ducks (Anas platyrhynchos). This technique used an indirect streptavidin-alkaline phosphatase labeling system with polyclonal antiserum developed against purified DSHDV antigens. Specimens from the experimentally inoculated Pekin ducks with DSHDV and archived paraffin-embedded tissues from natural cases of Duck viral swollen head hemorrhagic disease (DVSHD) were examined by clinical and histological criteria. Positive staining was most widely observed in the cytoplasm of the following organs: immune, digestive, and urinary organs, heart, lung, and trachea, which corresponded to the intracellular distribution of reovirus. The DSHDV antigens were first detected at 4 hr postinoculation in the bursa of Fabricius of infected ducks. Therefore, this method was suitable for the early diagnosis of DVSHD. Immunoperoxidase staining was not present in tissues and organs of sham-inoculated ducks (negative control). The IPA developed in the current study is a convenient, sensitive, and specific means of detecting DSHDV and is applicable to routine diagnosis, retrospective studies, and prospective studies of DSHDV infection in ducks.

CdSe/ZnS Quantum Dot-Labeled Lateral Flow Strips for Rapid and Quantitative Detection of Gastric Cancer Carbohydrate Antigen 72-4

NASA Astrophysics Data System (ADS)

Yan, Xinyu; Wang, Kan; Lu, Wenting; Qin, Weijian; Cui, Daxiang; He, Jinghua

2016-03-01

Carbohydrate antigen 72-4 (CA72-4) is an important biomarker associated closely with diagnosis and prognosis of early gastric cancer. How to realize quick, sensitive, specific, and quantitative detection of CA72-4 in clinical specimens has become a great requirement. Herein, we reported a CdSe/ZnS quantum dot-labeled lateral flow test strip combined with a charge-coupled device (CCD)-based reader was developed for rapid, sensitive, and quantitative detection of CA72-4. Two mouse monoclonal antibodies (mAbs) against CA72-4 were employed. One of them was coated as a test line, while another mAb was labeled with quantum dots and coated onto conjugate pad. The goat anti-mouse IgG was immobilized as a control line. After sample was added, a sandwich structure was formed with CA72-4 and these two mAbs. The fluorescent signal from quantum dots (QD)-labeled mAb in sandwich structure was related to the amount of detected CA72-4. A CCD-based reader was used to realize quantitative detection of CA72-4. Results showed that developed QD-labeled lateral flow strips to detect CA72-4 biomarker with the sensitivity of 2 IU/mL and 10 min detection time. One hundred sera samples from clinical patients with gastric cancer and healthy people were used to confirm specificity of this strip method; results showed that established strip method own 100 % reproducibility and 100 % specificity compared with Roche electrochemiluminescence assay results. In conclusion, CdSe/ZnS quantum dot-labeled lateral flow strips for detection of CA72-4 could realize rapid, sensitive, and specific detection of clinical samples and could own great potential in clinical translation in near future.

Immunodiagnosis of Human Fascioliasis: An Update of Concepts and Performances of the Serological Assays

PubMed Central

Khabisi, Samaneh Abdolahi

2017-01-01

Human Fascioliasis (HF) is a foodborne neglected parasitic disease caused by Fasciola hepatica and Fasciola gigantica. New epidemiological data suggest that the endemic areas of the disease are expanding and HF is being reported from areas where it was previously not observed. Diagnosis of HF is challenging. Performances of parasitological approaches, based on the detection of parasite’s egg in the stool, are not satisfactory. Currently serological methods for the diagnosis of HF are mainly based on detection of anti-Fasciola antibodies in serum. Although, there have been some improvement in the development of immunological diagnostic tests for the diagnosis of HF, yet these tests suffer from insufficiency in sensitivity or/and specificity. Detection of antigens, rather than antibodies, seems to be a suitable approach in the diagnosis of HF. Antigen can be detected in sera or stool of the fascioliasis patients. Circulating antigen in serum disappears within a short time and most of the circulating antigens are in immune complex forms which are not freely available to be detected. Therefore, antigenemia might not be an appropriate method for the diagnosis of HF. Detection of antigen in stool (coproantigens) seems to be a suitable alternative method for the diagnosis of HF. Recent data provided convincing evidence that detection of coproantigen improved and simplified the diagnosis of HF. The present review highlights the new achievements in designing and improvement of diagnostic approaches for the immunodiagnosis of HF. Moreover, current status of the available immunodiagnostic techniques for the diagnosis of HF, their strengths and weaknesses has been discussed. PMID:28764235

[The use of the macrophage disappearance reaction for detecting delayed hypersensitivity to Yersinia pestis antigens].

PubMed

Vasil'eva, G I; Doroshenko, E P; Kiseleva, A K; Pustovalov, V L

1990-12-01

The possibility of using the reaction of macrophage disappearance (RMD) for the detection of delayed hypersensitivity (DH) to Y. pestis has been studied. As the result of these studies, RMD has been found suitable, in principle, for use in the quantitative evaluation of DH to Y. pestis. High sensitivity and specificity of this reaction have been established. The presence of DH in the process of the formation of immunity after immunization with Y. pestis antigen FIA has been shown. RMD can be observed during 28 days after immunization (the term of observation).

Diagnosis of lyme borreliosis.

PubMed

Aguero-Rosenfeld, Maria E; Wang, Guiqing; Schwartz, Ira; Wormser, Gary P

2005-07-01

A large amount of knowledge has been acquired since the original descriptions of Lyme borreliosis (LB) and of its causative agent, Borrelia burgdorferi sensu stricto. The complexity of the organism and the variations in the clinical manifestations of LB caused by the different B. burgdorferi sensu lato species were not then anticipated. Considerable improvement has been achieved in detection of B. burgdorferi sensu lato by culture, particularly of blood specimens during early stages of disease. Culturing plasma and increasing the volume of material cultured have accomplished this. Further improvements might be obtained if molecular methods are used for detection of growth in culture and if culture methods are automated. Unfortunately, culture is insensitive in extracutaneous manifestations of LB. PCR and culture have high sensitivity on skin samples of patients with EM whose diagnosis is based mostly on clinical recognition of the lesion. PCR on material obtained from extracutaneous sites is in general of low sensitivity, with the exception of synovial fluid. PCR on synovial fluid has shown a sensitivity of up to >90% (when using four different primer sets) in patients with untreated or partially treated Lyme arthritis, making it a helpful confirmatory test in these patients. Currently, the best use of PCR is for confirmation of the clinical diagnosis of suspected Lyme arthritis in patients who are IgG immunoblot positive. PCR should not be used as the sole laboratory modality to support a clinical diagnosis of extracutaneous LB. PCR positivity in seronegative patients suspected of having late manifestations of LB most likely represents a false-positive result. Because of difficulties in direct methods of detection, laboratory tests currently in use are mainly those detecting antibodies to B. burgdorferi sensu lato. Tests used to detect antibodies to B. burgdorferi sensu lato have evolved from the initial formats as more knowledge on the immunodominant antigens has been collected. The recommendation for two-tier testing was an attempt to standardize testing and improve specificity in the United States. First-tier assays using whole-cell sonicates of B. burgdorferi sensu lato need to be standardized in terms of antigen composition and detection threshold of specific immunoglobulin classes. The search for improved serologic tests has stimulated the development of recombinant protein antigens and the synthesis of specific peptides from immunodominant antigens. The use of these materials alone or in combination as the source of antigen in a single-tier immunoassay may someday replace the currently recommended two-tier testing strategy. Evaluation of these assays is currently being done, and there is evidence that certain of these antigens may be broadly cross-reactive with the B. burgdorferi sensu lato species causing LB in Europe.

The evaluation of three diagnostic tests for the detection of equine influenza nucleoprotein in nasal swabs.

PubMed

Galvin, Pamela; Gildea, Sarah; Nelly, Maura; Quinlivan, Michelle; Arkins, Sean; Walsh, Cathal; Cullinane, Ann

2014-05-01

Equine influenza (EI) is a highly contagious respiratory disease of horses. The aim of this study was to evaluate two rapid antigen detection kits (Directigen or DFA, and Espline) and a commercial ELISA for the detection of EI nucleoprotein in nasal swabs. Nasal swab samples from naturally and experimentally infected horses were used to compare the sensitivity and specificity of these assays to virus isolation (VI) and real-time RT-PCR. If real-time RT-PCR was considered as the gold standard, the sensitivity of the other tests in field samples was 68% (DFA), 35% (ELISA), 29% (Espline), and 9% (VI). These tests had 100% specificity when compared to real-time RT-PCR. A receiver operating characteristic (ROC) curve indicated that decreasing the cutoff of the ELISA would increase sensitivity with some loss of specificity. In samples from experimentally infected horses, the sensitivity of the tests compared with real-time RT-PCR was 69% (VI), 27% (DFA), 6% (Espline), and 2% (ELISA). The specificity was 100% for Espline and ELISA and 95% for VI and DFA. This study illustrated that DFA is the most sensitive antigen detection test evaluated for the diagnosis of EI and that it can detect virus in some subclinical infected and vaccinated horses. The results suggest that DFA is a useful adjunct to laboratory tests and may be effective as a screening test in a quarantine station or similar facility where horses are monitored daily. © 2014 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Development of a novel lateral flow assay for detection of African swine fever in blood.

PubMed

Sastre, P; Gallardo, C; Monedero, A; Ruiz, T; Arias, M; Sanz, A; Rueda, P

2016-09-15

African swine fever (ASF) is a viral infectious disease of domestic and wild suids of all breeds and ages, causing a wide range of hemorrhagic syndromes and frequently characterized by high mortality. The disease is endemic in Sub-Saharan Africa and Sardinia. Since 2007, it has also been present in different countries of Eastern Europe, where control measures have not been effective so far. The continued spread poses a serious threat to the swine industry worldwide. In the absence of vaccine, early detection of infected animals is of paramount importance for control of the outbreak, to prevent the transmission of the virus to healthy animals and subsequent spreading of the disease. Current laboratory diagnosis is mainly based on virological methods (antigen and genome detection) and serodiagnosis. In the present work, a Lateral Flow Assay (LFA) for antigen detection has been developed and evaluated. The test is based on the use of a MAb against VP72 protein of ASFV, the major viral capsid protein and highly immunogenic. First experiments using VP72 viral and recombinant protein or inactivated culture virus showed promising results with a sensitivity similar to that of a commercially available Antigen-ELISA. Moreover, these strips were tested with blood from experimentally infected pigs and field animals and the results compared with those of PCR and Antigen-ELISA. For the experimentally infected samples, there was an excellent correlation between the LFA and the ELISA, while the PCR always showed to be more sensitive (38 % positive samples by PCR versus 27 % by LFA). The LFA was demonstrated to be positive for animals with circulating virus levels exceeding 10(4) HAU. With the field samples, once again, the PCR detected more positives than either the Antigen-ELISA or LFA, although here the number of positive samples scored by the LFA exceeded the values obtained with the Antigen-ELISA, showing 60 % positivity vs 48 % for the ELISA. For the two groups of sera, the specificity was close to 100 % indicating that hardly any false positive samples were found. The newly developed LFA allows rapid and reliable detection of ASFV, at field and laboratory level, providing a new useful tool for control programs and in situations where laboratory support and skilled personnel are limited.

Prostate Specific Membrane Antigen Positron Emission Tomography May Improve the Diagnostic Accuracy of Multiparametric Magnetic Resonance Imaging in Localized Prostate Cancer.

PubMed

Rhee, H; Thomas, P; Shepherd, B; Gustafson, S; Vela, I; Russell, P J; Nelson, C; Chung, E; Wood, G; Malone, G; Wood, S; Heathcote, P

2016-10-01

Positron emission tomography using ligands targeting prostate specific membrane antigen has recently been introduced. Positron emission tomography imaging with (68)Ga-PSMA-HBED-CC has been shown to detect metastatic prostate cancer lesions at a high rate. In this study we compare multiparametric magnetic resonance imaging and prostate specific membrane antigen positron emission tomography of the prostate with whole mount ex vivo prostate histopathology to determine the true sensitivity and specificity of these imaging modalities for detecting and locating tumor foci within the prostate. In a prospective clinical trial setting 20 patients with localized prostate cancer and a planned radical prostatectomy were recruited. All patients underwent multiparametric magnetic resonance imaging and positron emission tomography before surgery, and whole mount histopathology slides were directly compared to the images. European Society of Urogenital Radiology guidelines for reporting magnetic resonance imaging were used as a template for regional units of analysis. The uropathologist and radiologists were blinded to individual components of the study, and the final correlation was performed by visual and deformable registration analysis. A total of 50 clinically significant lesions were identified from the whole mount histopathological analysis. Based on regional analysis the sensitivity, specificity, positive predictive value and negative predictive value for multiparametric magnetic resonance imaging were 44%, 94%, 81% and 76%, respectively. With prostate specific membrane antigen positron emission tomography the sensitivity, specificity, positive predictive value and negative predictive value were 49%, 95%, 85% and 88%, respectively. Prostate specific membrane antigen positron emission tomography yielded a higher specificity and positive predictive value. A significant proportion of cancers are potentially missed and underestimated by both imaging modalities. Prostate specific membrane antigen positron emission tomography may be used in addition to multiparametric magnetic resonance imaging to help improve local staging in those patients undergoing retropubic radical prostatectomy. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Serodiagnostic potential of immuno-PCR using a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor in tuberculosis patients.

PubMed

Singh, Netrapal; Sreenivas, Vishnubhatla; Sheoran, Abhishek; Sharma, Suman; Gupta, Krishna B; Khuller, Gopal K; Mehta, Promod K

2016-01-01

A novel indirect immuno-polymerase chain reaction (I-PCR) assay was developed for the detection of circulating anti-Ag85B (antigen 85B, Rv1886c), anti-ESAT-6 (early secretory antigenic target-6, Rv3875) and anti-cord factor (trehalose 6,6'-dimycolate) antibodies from the sera samples of pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients and the results were compared with an analogous enzyme-linked immunosorbent assay (ELISA). We covalently attached the amino-modified reporter DNA to the dithiothreitol (DTT)-reduced anti-human IgG antibody through a chemical linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The detection of cocktail of anti-Ag85B, anti-ESAT-6 and anti-cord factor antibodies was found to be superior to the detection of individual antibodies. The sensitivities of 89.5% and 77.5% with I-PCR and 70.8% and 65% with ELISA were observed in smear-positive and smear-negative PTB cases, respectively with high specificity (90.9%). On the other hand, a sensitivity of 77.5% with I-PCR and 65% with ELISA was observed in EBTB cases. The detection of cocktail of antibodies by I-PCR is likely to improve the utility of existing algorithms for TB diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Analytical sensitivity of current best-in-class malaria rapid diagnostic tests.

PubMed

Jimenez, Alfons; Rees-Channer, Roxanne R; Perera, Rushini; Gamboa, Dionicia; Chiodini, Peter L; González, Iveth J; Mayor, Alfredo; Ding, Xavier C

2017-03-24

Rapid diagnostic tests (RDTs) are today the most widely used method for malaria diagnosis and are recommended, alongside microscopy, for the confirmation of suspected cases before the administration of anti-malarial treatment. The diagnostic performance of RDTs, as compared to microscopy or PCR is well described but the actual analytical sensitivity of current best-in-class tests is poorly documented. This value is however a key performance indicator and a benchmark value needed to developed new RDTs of improved sensitivity. Thirteen RDTs detecting either the Plasmodium falciparum histidine rich protein 2 (HRP2) or the plasmodial lactate dehydrogenase (pLDH) antigens were selected from the best performing RDTs according to the WHO-FIND product testing programme. The analytical sensitivity of these products was evaluated using a range of reference materials including P. falciparum and Plasmodium vivax whole parasite samples as well as recombinant proteins. The best performing HRP2-based RDTs could detect all P. falciparum cultured samples at concentrations as low as 0.8 ng/mL of HRP2. The limit of detection of the best performing pLDH-based RDT specifically detecting P. vivax was 25 ng/mL of pLDH. The analytical sensitivity of P. vivax and Pan pLDH-based RDTs appears to vary considerably from product to product, and improvement of the limit-of-detection for P. vivax detecting RDTs is needed to match the performance of HRP2 and Pf pLDH-based RDTs for P. falciparum. Different assays using different reference materials produce different values for antigen concentration in a given specimen, highlighting the need to establish universal reference assays.

Metal-organic gel enhanced fluorescence anisotropy for sensitive detection of prostate specific antigen

NASA Astrophysics Data System (ADS)

Zhao, Ting Ting; Peng, Zhe Wei; Yuan, Dan; Zhen, Shu Jun; Huang, Cheng Zhi; Li, Yuan Fang

2018-03-01

In this contribution, we demonstrated that Cu-based metal-organic gel (Cu-MOG) was able to serve as a novel amplification platform for fluorescence anisotropy (FA) assay for the first time, which was confirmed by the sensitive detection of a common cancer biomarker, prostate specific antigen (PSA). The dye-labeled probe aptamer (PA) product was adsorbed onto the benzimidazole derivative-containing Cu-MOG via electrostatic incorporation and strong π-π stacking interactions, which significantly increased the FA value due to the enlargement of the molecular volume of the PA/Cu-MOG complex. With the introduction of target PSA, the FA value was obviously decreased on account of the specific recognition between PSA and PA which resulted in the detachment of PA from the surface of MOG. The linear range was from 0.5-8 ng/mL, with a detection limit of 0.33 ng/mL. Our work has thus helped to demonstrate promising application of MOG material in the fields of biomolecules analysis and disease diagnosis.

Field validation of recombinant antigen immunoassays for diagnosis of Lassa fever.

PubMed

Boisen, Matthew L; Hartnett, Jessica N; Shaffer, Jeffrey G; Goba, Augustine; Momoh, Mambu; Sandi, John Demby; Fullah, Mohamed; Nelson, Diana K S; Bush, Duane J; Rowland, Megan M; Heinrich, Megan L; Koval, Anatoliy P; Cross, Robert W; Barnes, Kayla G; Lachenauer, Anna E; Lin, Aaron E; Nekoui, Mahan; Kotliar, Dylan; Winnicki, Sarah M; Siddle, Katherine J; Gbakie, Michael; Fonnie, Mbalu; Koroma, Veronica J; Kanneh, Lansana; Kulakosky, Peter C; Hastie, Kathryn M; Wilson, Russell B; Andersen, Kristian G; Folarin, Onikepe O; Happi, Christian T; Sabeti, Pardis C; Geisbert, Thomas W; Saphire, Erica Ollmann; Khan, S Humarr; Grant, Donald S; Schieffelin, John S; Branco, Luis M; Garry, Robert F

2018-04-12

Lassa fever, a hemorrhagic fever caused by Lassa virus (LASV), is endemic in West Africa. It is difficult to distinguish febrile illnesses that are common in West Africa from Lassa fever based solely on a patient's clinical presentation. The field performance of recombinant antigen-based Lassa fever immunoassays was compared to that of quantitative polymerase chain assays (qPCRs) using samples from subjects meeting the case definition of Lassa fever presenting to Kenema Government Hospital in Sierra Leone. The recombinant Lassa virus (ReLASV) enzyme-linked immunosorbant assay (ELISA) for detection of viral antigen in blood performed with 95% sensitivity and 97% specificity using a diagnostic standard that combined results of the immunoassays and qPCR. The ReLASV rapid diagnostic test (RDT), a lateral flow immunoassay based on paired monoclonal antibodies to the Josiah strain of LASV (lineage IV), performed with 90% sensitivity and 100% specificity. ReLASV immunoassays performed better than the most robust qPCR currently available, which had 82% sensitivity and 95% specificity. The performance characteristics of recombinant antigen-based Lassa virus immunoassays indicate that they can aid in the diagnosis of LASV Infection and inform the clinical management of Lassa fever patients.

Parasite detection in patients with post kala-azar dermal leishmaniasis in India: a comparison between molecular and immunological methods

PubMed Central

Salotra, P; Sreenivas, G; Beena, K R; Mukherjee, A; Ramesh, V

2003-01-01

Aims: To evaluate the sensitivity and specificity of serological, immunohistochemical, and molecular methods in the diagnosis of post kala-azar dermal leishmaniasis (PKDL). Methods: Twenty five patients with confirmed PKDL and 25 controls were included in the study. G2D10, a monoclonal antibody against Leishmania, was used for the immunohistochemical (IHC) staining of lesion sections to visualise anti-Leishmania donovani antibodies. The diagnostic usefulness of IHC was compared with enzyme linked immunosorbent assay (ELISA) with a recombinant (rk39) antigen, and a species specific polymerase chain reaction (PCR) assay, amplifying a kinetoplast minicircle DNA sequence. Results: IHC detected 22 of 25 PKDL cases, giving a sensitivity of 88%. The diagnostic sensitivity of both the ELISA and PCR tests was higher (96%). All of the 25 controls examined were negative in PCR, indicating 100% specificity of the test, whereas ELISA showed 96% specificity. Conclusions: IHC with G2D10 significantly enhances the sensitivity of detection of PKDL over routine haematoxylin and eosin staining. ELISA with a recombinant antigen is an economical and practical assay. PCR is the most sensitive and specific diagnostic method for PKDL. The tests described would facilitate the recognition of patients with PKDL, enabling timely treatment, which would contribute greatly to the control of kala-azar. PMID:14600129

Development of a Sensitive Luciferase-Based Sandwich ELISA System for the Detection of Human Extracellular Matrix 1 Protein.

PubMed

Li, Ya; Li, Yanqing; Zhao, Junli; Zheng, Xiaojing; Mao, Qinwen; Xia, Haibin

2016-12-01

Enzyme-linked immunosorbent assay (ELISA) has been one of the main methods for detecting an antigen in an aqueous sample for more than four decades. Nowadays, one of the biggest concerns for ELISA is still how to improve the sensitivity of the assay, and the luciferase-luciferin reaction system has been noticed as a new detection method with high sensitivity. In this study, a luciferin-luciferase reaction system was used as the detection method for a sandwich ELISA system. It was shown that this new system led to an increase in the detection sensitivity of at least two times when compared with the traditional horseradish peroxidase (HRP) detection method. Lastly, the serum levels of the human extracellular matrix 1 protein of breast cancer patients were determined by the new system, which were overall similar to the HRP chemiluminescent system. Furthermore, this new luciferase reporter can be implemented into other ELISA systems for the purpose of increasing the assay sensitivity.

Differential antigenic protein recovery from Taenia solium cyst tissues using several detergents.

PubMed

Navarrete-Perea, José; Orozco-Ramírez, Rodrigo; Moguel, Bárbara; Sciutto, Edda; Bobes, Raúl J; Laclette, Juan P

2015-07-01

Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). The protein extracts of T. solium cysts are complex mixtures including cyst's and host proteins. Little is known about the influence of using different detergents in the efficiency of solubilization-extraction of these proteins, including relevant antigens. Here, we describe the use of CHAPS, ASB-14 and Triton X-100, alone or in combination in the extraction buffers, as a strategy to notably increase the recovery of proteins that are usually left aside in insoluble fractions of cysts. Using buffer with CHAPS alone, 315 protein spots were detected through 2D-PAGE. A total of 255 and 258 spots were detected using buffers with Triton X-100 or ASB-14, respectively. More protein spots were detected when detergents were combined, i.e., 2% CHAPS, 1% Triton X-100 and 1% ASB-14 allowed detection of up to 368 spots. Our results indicated that insoluble fractions of T. solium cysts were rich in antigens, including several glycoproteins that were sensitive to metaperiodate treatment. Host proteins, a common component in protein extracts of cysts, were present in larger amounts in soluble than insoluble fractions of cysts proteins. Finally, antigens present in the insoluble fraction were more appropriate as a source of antigens for diagnostic procedures. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of Prototype Filovirus Recombinant Antigen Immunoassays

PubMed Central

Boisen, Matt L.; Oottamasathien, Darin; Jones, Abigail B.; Millett, Molly M.; Nelson, Diana S.; Bornholdt, Zachary A.; Fusco, Marnie L.; Abelson, Dafna M.; Oda, Shun-ichiro; Hartnett, Jessica N.; Rowland, Megan M.; Heinrich, Megan L.; Akdag, Marjan; Goba, Augustine; Momoh, Mambu; Fullah, Mohammed; Baimba, Francis; Gbakie, Michael; Safa, Sadiki; Fonnie, Richard; Kanneh, Lansana; Cross, Robert W.; Geisbert, Joan B.; Geisbert, Thomas W.; Kulakosky, Peter C.; Grant, Donald S.; Shaffer, Jeffery G.; Schieffelin, John S.; Wilson, Russell B.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.; Khan, S. Humarr; Pitts, Kelly R.

2015-01-01

Background. Throughout the 2014–2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. Methods. Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. Results. Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus–specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. Conclusions. The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins. PMID:26232440

Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans

PubMed Central

Cevallos, William; Calvopiña, Manuel; Nipáz, Victoria; Vicente-Santiago, Belén; López-Albán, Julio; Fernández-Soto, Pedro; Guevara, Ángel; Muro, Antonio

2017-01-01

BACKGROUND Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing. OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera. METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections. PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana. MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections. PMID:28443982

Evaluation of a dengue NS1 antigen detection assay sensitivity and specificity for the diagnosis of acute dengue virus infection.

PubMed

Hermann, Laura L; Thaisomboonsuk, Butsaya; Poolpanichupatam, Yongyuth; Jarman, Richard G; Kalayanarooj, Siripen; Nisalak, Ananda; Yoon, In-Kyu; Fernandez, Stefan

2014-10-01

Currently, no dengue NS1 detection kit has regulatory approval for the diagnosis of acute dengue fever. Here we report the sensitivity and specificity of the InBios DEN Detect NS1 ELISA using a panel of well characterized human acute fever serum specimens. The InBios DENV Detect NS1 ELISA was tested using a panel composed of 334 serum specimens collected from acute febrile patients seeking care in a Bangkok hospital in 2010 and 2011. Of these patients, 314 were found to have acute dengue by either RT-PCR and/or anti-dengue IgM/IgG ELISA. Alongside the InBios NS1 ELISA kit, we compared the performance characteristics of the BioRad Platelia NS1 antigen kit. The InBios NS1 ELISA Ag kit had a higher overall sensitivity (86% vs 72.8%) but equal specificity (100%) compared to the BioRad Platelia kit. The serological status of the patient significantly influenced the outcome. In primary infections, the InBios NS1 kit demonstrated a higher sensitivity (98.8%) than in secondary infections (83.5%). We found significant variation in the sensitivity of the InBios NS1 ELISA kit depending on the serotype of the dengue virus and also found decreasing sensitivity the longer after the onset of illness, showing 100% sensitivity early during illness, but dropping below 50% by Day 7. The InBios NS1 ELISA kit demonstrated high accuracy when compared to the initial clinical diagnosis with greater than 85% agreement when patients were clinically diagnosed with dengue illness. Results presented here suggest the accurate detection of circulating dengue NS1 by the InBios DENV Detect NS1 ELISA can provide clinicians with a useful tool for diagnosis of early dengue infections.

Immunolocalization and immunodetection of the excretory/secretory (ES) antigens of Fasciola gigantica.

PubMed

Khan, M A Hannan; Ullah, Rizwan; Rehman, Abdur; Rehman, Lubna; P A, Ahammed Shareef; Abidi, S M A

2017-01-01

The digenetic trematode Fasciola gigantica is a parasite of great agricultural and economic importance. Along with Fasciola hepatica, F. gigantica incurs huge economic losses to the agricultural sector. Because of unavailability of an effective and commercial vaccine, the earliest diagnosis of the disease is the only way to control the disease. The conventional coprological techniques are able to detect the disease only after the parasites get matured and starts releasing their eggs with the faeces of host, therefore prepatent infection remain undiagnosed. The alternative method is by serological tests that uses circulatory antigens. Despite high sensitivity, their reliability is quite low because of the common antigens shared between different helminth parasites. To overcome this, investigation was shifted to identify the copro-antigens which could be more sensitive and reliable. In the present study, we tried to identify some of the immunodominant proteins from the Excretory Secretory (ES) product of F. gigantica which can be further characterized and used for early detection of infection and also as drug and vaccine candidates. The ES products of F. gigantica were collected and used for raising the polyclonal antibody in rabbit. The polypeptide profile was generated as well as immunogenic polypeptides were identified. The Source of ES antigen was immunolocalized using confocal microscopy and dot blot assay was performed to diagnose field infection. The polypeptide profile of ES products revealed a total of 24 polypeptides out of which 12 immunogenic polypeptides were identified by western blotting. Confocal micrographs showed the immunolocalization of antigens in the intestinal caecae, vitalline glands, gonads as well as in the tegument of the worm. The dot blot assay confirmed the utility of ES products for the detection of field infection. Subsequently, cross reactivity was found negative with Gigantocotyle explanatum; an amphitome parasite of same habitat. However, the cross reactivity with other helminths needs to be worked out.

Immunolocalization and immunodetection of the excretory/secretory (ES) antigens of Fasciola gigantica

PubMed Central

Ullah, Rizwan; Rehman, Abdur; Rehman, Lubna; P. A., Ahammed Shareef; Abidi, S. M. A.

2017-01-01

The digenetic trematode Fasciola gigantica is a parasite of great agricultural and economic importance. Along with Fasciola hepatica, F. gigantica incurs huge economic losses to the agricultural sector. Because of unavailability of an effective and commercial vaccine, the earliest diagnosis of the disease is the only way to control the disease. The conventional coprological techniques are able to detect the disease only after the parasites get matured and starts releasing their eggs with the faeces of host, therefore prepatent infection remain undiagnosed. The alternative method is by serological tests that uses circulatory antigens. Despite high sensitivity, their reliability is quite low because of the common antigens shared between different helminth parasites. To overcome this, investigation was shifted to identify the copro-antigens which could be more sensitive and reliable. In the present study, we tried to identify some of the immunodominant proteins from the Excretory Secretory (ES) product of F. gigantica which can be further characterized and used for early detection of infection and also as drug and vaccine candidates. The ES products of F. gigantica were collected and used for raising the polyclonal antibody in rabbit. The polypeptide profile was generated as well as immunogenic polypeptides were identified. The Source of ES antigen was immunolocalized using confocal microscopy and dot blot assay was performed to diagnose field infection. The polypeptide profile of ES products revealed a total of 24 polypeptides out of which 12 immunogenic polypeptides were identified by western blotting. Confocal micrographs showed the immunolocalization of antigens in the intestinal caecae, vitalline glands, gonads as well as in the tegument of the worm. The dot blot assay confirmed the utility of ES products for the detection of field infection. Subsequently, cross reactivity was found negative with Gigantocotyle explanatum; an amphitome parasite of same habitat. However, the cross reactivity with other helminths needs to be worked out. PMID:28973017

Improved porous silicon (P-Si) microarray based PSA (prostate specific antigen) immunoassay by optimized surface density of the capture antibody

PubMed Central

Lee, SangWook; Kim, Soyoun; Malm, Johan; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas

2014-01-01

Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA - prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5ngmL−1, 80pgmL−1, and 800fgmL−1 when arraying the PSA antibody, H117 at the concentration 15µgmL−1, 35µgmL−1 and 154µgmL−1, respectively. We further investigated PSA spiked into human female serum in the range of 800fgmL−1 to 500ngmL−1. The microarray showed a LOD of 800fgmL−1 and a dynamic range of 800 fgmL−1 to 80ngmL−1 in serum spiked samples. PMID:24016590

A Theory of Self- Nonself Discrimination

ERIC Educational Resources Information Center

Bretscher, Peter; Cohn, Melvin

1970-01-01

Theoretical model suggests antigen-sensitive cells produce antibodies only when two antigenic determinants are recognized, and explains self-nonself discrimination by antibody system. Specific antigen-sensitive cells accumulate in absence of foreign antigens and co-operate to induce antibodies if antigen enter the body; but antigen-sensitive cells…

Value of Different Assays for Detection of Human Cytomegalovirus (HCMV) in Predicting the Development of HCMV Disease in Human Immunodeficiency Virus-Infected Patients

PubMed Central

Blank, Brian S. N.; Meenhorst, Pieter L.; Mulder, Jan Willem; Weverling, Gerrit Jan; Putter, Hein; Pauw, Wouter; van Dijk, Willemien C.; Smits, Paul; Lie-A-Ling, Sonja; Reiss, Peter; Lange, Joep M. A.

2000-01-01

In the present prospective study, five blood tests for detection of human cytomegalovirus (HCMV), nucleic acid sequence-based amplification (NASBA) for detection of early (immediate-early antigen) and late (pp67) mRNA, PCR for detection of HCMV DNA (DNA PCR), culture, and pp65 antigenemia assay, and culture and DNA PCR of urine and throat swab specimens were compared for their abilities to predict the development of disease caused by HCMV (HCMV disease). Of 101 human immunodeficiency virus (HIV)-infected patients with ≤100 CD4+ lymphocytes per mm3, 25 patients developed HCMV disease. The pp65 antigenemia assay (sensitivity, 50%; specificity, 89%) and DNA PCR of blood (sensitivity, 69%; specificity, 75%) were most accurate in predicting the development of HCMV disease within the next 12 months. Both blood culture and late pp67 mRNA NASBA had high specificities (91 and 90%, respectively) but low sensitivities (25 and 13%, respectively). The sensitivities of urine culture, DNA PCR, throat swab specimen culture, DNA PCR, and NASBA of blood for detection of the immediate-early antigen were 73, 87, 53, 67, and 63%, respectively, and the specificities were 58, 46, 76, 60, and 72%, respectively. The positive predictive values of all tests however, were low and did not exceed 50%. In conclusion, virological screening by these qualitative assays for detection of HCMV is of limited value for prediction of the development of HCMV disease in HIV-infected patients. PMID:10655346

Hybrid Synthetic Receptors on MOSFET Devices for Detection of Prostate Specific Antigen in Human Plasma.

PubMed

Tamboli, Vibha K; Bhalla, Nikhil; Jolly, Pawan; Bowen, Chris R; Taylor, John T; Bowen, Jenna L; Allender, Chris J; Estrela, Pedro

2016-12-06

The study reports the use of extended gate field-effect transistors (FET) for the label-free and sensitive detection of prostate cancer (PCa) biomarkers in human plasma. The approach integrates for the first time hybrid synthetic receptors comprising of highly selective aptamer-lined pockets (apta-MIP) with FETs for sensitive detection of prostate specific antigen (PSA) at clinically relevant concentrations. The hybrid synthetic receptors were constructed by immobilizing an aptamer-PSA complex on gold and subjecting it to 13 cycles of dopamine electropolymerization. The polymerization resulted in the creation of highly selective polymeric cavities that retained the ability to recognize PSA post removal of the protein. The hybrid synthetic receptors were subsequently used in an extended gate FET setup for electrochemical detection of PSA. The sensor was reported to have a limit of detection of 0.1 pg/mL with a linear detection range from 0.1 pg/mL to 1 ng/mL PSA. Detection of 1-10 pg/mL PSA was also achieved in diluted human plasma. The present apta-MIP sensor developed in conjunction with FET devices demonstrates the potential for clinical application of synthetic hybrid receptors for the detection of clinically relevant biomarkers in complex samples.

A new bead-based human platelet antigen antibodies detection assay versus the monoclonal antibody immobilization of platelet antigens assay.

PubMed

Porcelijn, Leendert; Huiskes, Elly; Comijs-van Osselen, Ilona; Chhatta, Aniska; Rathore, Vipul; Meyers, Matthew; de Haas, Masja

2014-06-01

The performance of a newly developed Luminex bead-based platelet (PLT) antibody detection method (PAKLx) was compared with the monoclonal antibody immobilization of PLT antigens (MAIPA) assay and the LifeScreen Deluxe Luminex bead-based HLA Class I antibody detection method (LMX). Six sera containing anti-human PLT antigen (HPA)-1a (n=2), HPA-1b, HPA-2b, HPA-3a, or HPA-5b were tested in titration. A total of 194 sera, including HPA-1a, -1b, -2a, -2b, -3a, -5a, and -5b antibodies with or without HLA antibodies (n=63); glycoprotein (GP) IV antibodies (n=1); PLT autoantibodies (n=3); HLA antibodies (n=45); and samples with no PLT-reactive antibodies (n=82), were tested in both assays. Comparable levels of sensitivity were obtained for the MAIPA and PAKLx. The PAKLx showed four (6%) false-negative results in 67 sera with HPA or GP-reactive antibodies: anti-HPA-3a (n=1) or anti-HPA-5b (n=3). The PAKLx showed in 10 of the total 194 samples (5%) the presence of antibodies not detected by the MAIPA. This concerned broadly GP-reactive antibodies (n=7), anti-GPIIb/IIIa combined with anti-HPA-3a (n=1), anti-HPA-1a (borderline, n=1), and anti-GPIV (n=1). Testing 175 sera for anti-HLA Class I antibodies in the PAKLx and LMX showed four discrepant results: PAKLx negative and LMX positive, n=3 and n=1, respectively. For the vast majority of the specimens tested (93%) the results of the PAKLx were in concordance with the MAIPA. The PAKLx is a fast, easy to perform, and sensitive PLT antibody screening method. © 2013 AABB.

Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum.

PubMed

Bosch, Irene; de Puig, Helena; Hiley, Megan; Carré-Camps, Marc; Perdomo-Celis, Federico; Narváez, Carlos F; Salgado, Doris M; Senthoor, Dewahar; O'Grady, Madeline; Phillips, Elizabeth; Durbin, Ann; Fandos, Diana; Miyazaki, Hikaru; Yen, Chun-Wan; Gélvez-Ramírez, Margarita; Warke, Rajas V; Ribeiro, Lucas S; Teixeira, Mauro M; Almeida, Roque P; Muñóz-Medina, José E; Ludert, Juan E; Nogueira, Mauricio L; Colombo, Tatiana E; Terzian, Ana C B; Bozza, Patricia T; Calheiros, Andrea S; Vieira, Yasmine R; Barbosa-Lima, Giselle; Vizzoni, Alexandre; Cerbino-Neto, José; Bozza, Fernando A; Souza, Thiago M L; Trugilho, Monique R O; de Filippis, Ana M B; de Sequeira, Patricia C; Marques, Ernesto T A; Magalhaes, Tereza; Díaz, Francisco J; Restrepo, Berta N; Marín, Katerine; Mattar, Salim; Olson, Daniel; Asturias, Edwin J; Lucera, Mark; Singla, Mohit; Medigeshi, Guruprasad R; de Bosch, Norma; Tam, Justina; Gómez-Márquez, Jose; Clavet, Charles; Villar, Luis; Hamad-Schifferli, Kimberly; Gehrke, Lee

2017-09-27

The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

[Evaluation of modern epizootic activity of natural tularemia foci in Voronezh region using immune-serological and molecular-genetic study of main carriers of the disease].

PubMed

Meshcheriakova, I S; Trankvilevskiĭ, D V; Kvasov, D A; Mikhaĭlova, T V; Kormilitsina, M I; Demidova, T N; Stepkin, Iu I; Zhukov, V I

2015-01-01

Improvement of monitoring and prognosis of epidemic manifestations of natural foci of tularemia on the territory of Voronezh region using immune-serological and molecular-genetic study of main carriers of the disease. 539 small mammals captured during summer period of 2011 in 4 districts of North-Eastern part of Voronezh region were studied. Animal organs were studied by serologic (search for Francisella tularensis antigens) and molecular-biologic (detection of F. tularensis DNA) methods. Tularemia antigen was detected using passive hemagglutination reaction (PHAR) with erythrocytic tularemia immunoglobulin diagnosticum. Real-time polymerase chain reaction (RT-PCR) was applied for detection of tularemia causative agent DNA. Complex study revealed epizootic activity of natural foci of tularemia in the examined territory. F. tularensis antigen and/or DNA were detected in 82 objects (15.2%). Use of RT-PCR allowed to additionally detect samples with relatively low content of F. tularensis DNA substrate, when antigen was not detected in samples. High sensitivity and specificity of the RT-PCR was ensured by inclusion of specific probes (tu14-PR2 and ISFTu2P). The results obtained give evidence on functioning and epizootic activity of natural foci of tularemia in Voronezh region that requires constant monitoring of the territory and prophylaxis measures, first of all vaccination of risk groups by live tularemia vaccine.

Comparison of Antibody Responses to a Potential Combination of Specific Glycolipids and Proteins for Test Sensitivity Improvement in Tuberculosis Serodiagnosis

PubMed Central

Julián, Esther; Matas, Lurdes; Alcaide, José; Luquin, Marina

2004-01-01

The humoral response to different proteinaceous antigens of Mycobacterium tuberculosis is heterogeneous among patients with active disease, and this has originated in the proposal to use a combination of several specific antigens to find an efficient serodiagnostic test for tuberculosis (TB). However, to date, comparisons of antibody responses to several antigens in the same population have been carried out without consideration of antigenic cell wall glycolipids. In the present study the presence of immunoglobulin G (IgG), IgM, and IgA antibodies to M. tuberculosis glycolipids (sulfolipid I, diacyltrehaloses, triacyltrehaloses, and cord factor) was compared with the response to four commercially available tests based on the 38-kDa protein mixed with the 16-kDa protein or lipoarabinomannan. Fifty-two serum samples from TB patients and 83 serum samples from control individuals (48 healthy individuals and 35 non-TB pneumonia patients) were studied. Three relevant results were obtained. (i) Smear-negative TB patients presented low humoral responses, but the sera which did react principally showed IgA antibodies to some glycolipidic antigens. (ii) TB patients exhibit heterogeneous humoral responses against glycolipidic antigens. (iii) Finally, test sensitivity is improved (from 23 to 62%) when IgG and IgA antibodies are detected together in tests based on different antigens (proteins and glycolipids). We conclude that it is possible to include glycolipidic antigens in a cocktail of specific antigens from M. tuberculosis to develop a serodiagnostic test. PMID:14715547

Evaluation of two new commercial tests for the diagnosis of acute dengue virus infection using NS1 antigen detection in human serum.

PubMed

Dussart, Philippe; Petit, Laure; Labeau, Bhety; Bremand, Laetitia; Leduc, Alexandre; Moua, David; Matheus, Séverine; Baril, Laurence

2008-08-20

We compared the performance of two new commercial tests for the detection of dengue NS1 protein during the clinical phase of dengue virus (DENV) infection-an immunochromatographic test allowing rapid detection of the NS1 antigen, Dengue NS1 Ag STRIP (Bio-Rad Laboratories - Marnes La Coquette, France), and a two-step sandwich-format microplate enzyme-linked immunosorbent assay (ELISA), pan-E Dengue Early ELISA (Panbio - Brisbane, Australia)-with a one-step sandwich-format microplate ELISA, the Platelia Dengue NS1 Ag test (Bio-Rad). We tested 272 serum samples from patients with dengue disease. Of these, 222 were from patients with acute infection of one of the four dengue serotypes, detected by RT-PCR and/or virus isolation. Forty-eight acute-phase serum samples from patients not infected with dengue virus were also included. The sensitivity of the Platelia Dengue NS1 Ag test on acute serum samples (n = 222) was 87.4% (95% confidence interval: 82.3% to 91.5%); that of Dengue NS1 Ag STRIP was 81.5% (95% CI: 75.8% to 86.4%) after 15 minutes and 82.4% (95% CI: 76.8% to 87.2%) after 30 minutes. Both tests had a specificity of 100% (97.5% CI, one-sided test: 92.6% to 100.0%). The pan-E Dengue Early ELISA had a sensitivity of 60.4% (95% CI: 53.4% to 66.8%) and a specificity of 97.9% (95% CI: 88.9% to 99.9%). Our findings support the use of diagnostic tools based on the NS1 antigen detection for the diagnosis of acute DENV infection. The immunochromatographic test, Dengue NS1 Ag STRIP-the first rapid diagnostic test for DENV infection-was highly sensitive and specific, and would therefore be a suitable first-line test in the field. The pan-E Dengue Early ELISA was less sensitive than the Platelia test; this two-step ELISA should be combined with DENV IgM antibody detection for the diagnosis of DENV infection.

Rapid detection of EBOLA VP40 in microchip immunofiltration assay

NASA Astrophysics Data System (ADS)

Miethe, Peter; Gary, Dominik; Hlawatsch, Nadine; Gad, Anne-Marie

2015-05-01

In the spring of 2014, the Ebola virus (EBOV) strain Zaire caused a dramatic outbreak in several regions of West Africa. The RT-PCR and antigen capture diagnostic proved to be effective for detecting EBOV in blood and serum. In this paper, we present data of a rapid antigen capture test for the detection of VP40. The test was performed in a microfluidic chip for immunofiltration analysis. The chip integrates all necessary assay components. The analytical sensitivity of the rapid test was 8 ng/ml for recombinant VP40. In serum and whole blood samples spiked with virus culture material, the detection limit was 2.2 x 102 PFU/ml. The performance data of the rapid test (15 min) are comparable to that of the VP40 laboratory ELISA.

Evaluation of pH-sensitive fusogenic polymer-modified liposomes co-loaded with antigen and α-galactosylceramide as an anti-tumor vaccine

PubMed Central

OKAZAKI, Seiji; IWASAKI, Tadashi; YUBA, Eiji; WATARAI, Shinobu

2017-01-01

pH-Sensitive fusogenic polymer-modified (pH-sensitive) liposomes co-loaded with tumor model antigen, ovalbumin (OVA), and adjuvant, α-galactosylceramide (α-GalCer) were fabricated and administered subcutaneously into mice. The ability of pH-sensitive liposomes containing OVA and α-GalCer to stimulate cellular and humoral immune responses in vivo was compared with OVA-encapsulating pH-sensitive liposomes as well as with OVA alone. After immunization, significant OVA-specific antibodies were detected in the serum. When sera were analyzed for isotype distribution, antigen-specific IgG1 antibody responses were noted in mice immunized with OVA alone, whereas immunization with OVA-containing pH-sensitive liposomes and with pH-sensitive liposomes containing OVA and α-GalCer resulted in the induction of OVA-specific IgG1 and IgG2b antibody responses. Moreover, more substantial production of IFN-γ and IL-4 was demonstrated in spleen cells from mice immunized with pH-sensitive liposomes having OVA and α-GalCer than OVA-containing pH-sensitive liposomes in vitro. Spleen cells from the immunized mice showed strong cytotoxic activity against E.G7-OVA tumor cells. In addition, prophylactic vaccination efficacy against tumor formation was evaluated. In all mice immunized with pH-sensitive liposomes having OVA and α-GalCer, immunization provided substantial protection from tumor formation. The therapeutic efficacy of pH-sensitive liposomes containing OVA and α-GalCer against already established E.G7-OVA tumors was also investigated. Tumor growth was reduced significantly in all mice treated with pH-sensitive liposomes having OVA and α-GalCer. The provided evidence on the advantage of antigen and α-GalCer co-encapsulation into pH-sensitive liposomes should be considered in the design of future cancer vaccines for prophylactic and therapeutic purposes. PMID:29311431

Evaluation of pH-sensitive fusogenic polymer-modified liposomes co-loaded with antigen and α-galactosylceramide as an anti-tumor vaccine.

PubMed

Okazaki, Seiji; Iwasaki, Tadashi; Yuba, Eiji; Watarai, Shinobu

2018-02-09

pH-Sensitive fusogenic polymer-modified (pH-sensitive) liposomes co-loaded with tumor model antigen, ovalbumin (OVA), and adjuvant, α-galactosylceramide (α-GalCer) were fabricated and administered subcutaneously into mice. The ability of pH-sensitive liposomes containing OVA and α-GalCer to stimulate cellular and humoral immune responses in vivo was compared with OVA-encapsulating pH-sensitive liposomes as well as with OVA alone. After immunization, significant OVA-specific antibodies were detected in the serum. When sera were analyzed for isotype distribution, antigen-specific IgG1 antibody responses were noted in mice immunized with OVA alone, whereas immunization with OVA-containing pH-sensitive liposomes and with pH-sensitive liposomes containing OVA and α-GalCer resulted in the induction of OVA-specific IgG1 and IgG2b antibody responses. Moreover, more substantial production of IFN-γ and IL-4 was demonstrated in spleen cells from mice immunized with pH-sensitive liposomes having OVA and α-GalCer than OVA-containing pH-sensitive liposomes in vitro. Spleen cells from the immunized mice showed strong cytotoxic activity against E.G7-OVA tumor cells. In addition, prophylactic vaccination efficacy against tumor formation was evaluated. In all mice immunized with pH-sensitive liposomes having OVA and α-GalCer, immunization provided substantial protection from tumor formation. The therapeutic efficacy of pH-sensitive liposomes containing OVA and α-GalCer against already established E.G7-OVA tumors was also investigated. Tumor growth was reduced significantly in all mice treated with pH-sensitive liposomes having OVA and α-GalCer. The provided evidence on the advantage of antigen and α-GalCer co-encapsulation into pH-sensitive liposomes should be considered in the design of future cancer vaccines for prophylactic and therapeutic purposes.

Diagnostic sensitivity and specificity of a participatory disease surveillance method for highly pathogenic avian influenza in household chicken flocks in Indonesia.

PubMed

Robyn, M; Priyono, W B; Kim, L M; Brum, E

2012-06-01

A study was conducted to assess the diagnostic sensitivity and specificity of a disease surveillance method for diagnosis of highly pathogenic avian influenza (HPAI) outbreaks in household chicken flocks used by participatory disease surveillance (PDS) teams in Yogyakarta Province, Indonesia. The Government of Indonesia, in partnership with the Food and Agriculture Organization of the United Nations, has implemented a PDS method for the detection of HPAI outbreaks in poultry since 2006. The PDS method in Indonesia utilizes both a clinical case definition (CD) and the result of a commercial rapid antigen test kit Yogyakarta 55611, to diagnose HPAI outbreaks, primarily in backyard chicken flocks. The following diagnostic sensitivities and specificities were obtained relative to real-time reverse transcription-PCR as the gold standard diagnostic test: 1) 89% sensitivity (CI95: 75%-97%) and 96% specificity (CI95: 89%-99%) for the PDS CD alone; 2) 86% sensitivity (CI95: 71%-95%) and 99% specificity (CI95: 94%-100%) for the rapid antigen test alone; and 3) 84% sensitivity (CI95: 68%-94%) and 100% specificity (CI95: 96%-100%) for the PDS CD result combined with the rapid antigen test result. Based on these results, HPAI outbreaks in extensively raised household chickens can be diagnosed with sufficient sensitivity and specificity using the PDS method as implemented in Indonesia. Subject to further field evaluation, data from this study suggest that the diagnostic sensitivity of the PDS method may be improved by expanding the PDS CD to include more possible clinical presentations of HPAI and by increasing the number of rapid antigen tests to three different birds with HPAI-compatible signs of same flock.

Antisperm antibodies in prepubertal boys and their reactivity with antigenic determinants on differentiated spermatozoa.

PubMed

Domagała, A; Kamieniczna, M; Kowalczyk, D; Kurpisz, M

1998-09-01

Antisperm antibodies induced in prepubertal boys with testicular failures were characterized by using four techniques of antibody detection. The reactivity of circulating antisperm antibodies in prepubertal boys and the reactivity of antibodies in sera samples of adult fertile and infertile males were compared against the same sperm antigenic pools (live or fixed spermatozoa, or sperm antigenic extracts). The incidence of antisperm antibodies in sera samples of 69 prepubertal boys with testicular failures and 21 samples obtained from adult, male individuals was assessed by indirect immunobead binding test (IDIBT), flow cytometry measurement, enzyme-linked immunosorbent assay, and Western blotting. Immunoblot analysis was performed by using sperm extracts of glycosylated and deglycosylated solubilized membrane antigens. Sera samples were studied in a group composed of healthy prepubertal boys (n = 7) and prepubertal boys with testicular failures (n = 69). Applied tests of antibody detection revealed striking differences in a group of boys with testicular pathology. With IDIBT, 7% of the sera samples were found positive, whereas with flow cytometry measurement, 48% of the sera samples were positive. Immunosorbent assay (fixed sperm) indicated 32% positive cases in the same group. The sera samples were found to be positive in 65% of immunoblotting reactions with glycosylated antigens and in 70% of immunoblotting reactions with deglycosylated antigens. All applied detection assays were clearly negative on sera samples from fertile, adult males. Western immunoblotting indicated an immunodominant antigenic determinant of 58 kDa. Tests of antibody detection with the use of live sperm (IDIBT and flow cytometry measurements) presented low sensitivity (8% and 48%, respectively) in a group of prepubertal boys. This observation underlines the difficulties in assigning the prospective prognosis of future fertility status in prepubertal boys with antisperm antibodies.

False-positive cerebrospinal fluid cryptococcus antigen in Libman-Sacks endocarditis.

PubMed

Isseh, Iyad N; Bourgi, Kassem; Nakhle, Asaad; Ali, Mahmoud; Zervos, Marcus J

2016-12-01

Cryptococcus meningoencephalitis is a serious opportunistic infection associated with high morbidity and mortality in immunocompromised hosts, particularly patients with advanced AIDS disease. The diagnosis is established through cerebrospinal fluid (CSF) cryptococcus antigen detection and cultures. Cryptococcus antigen testing is usually the initial test of choice due its high sensitivity and specificity along with the quick availability of the results. We hereby report a case of a false-positive CSF cryptococcus antigen assay in a patient with systemic lupus erythematosus presenting with acute confusion. While initial CSF evaluation revealed a positive cryptococcus antigen assay, the patient's symptoms were inconsistent with cryptococcus meningoencephalitis. A repeat CSF evaluation, done 3 days later, revealed a negative CSF cryptococcus antigen assay. Given the patient's active lupus disease and the elevated antinuclear antibody titers, we believe that the initial positive result was a false positive caused by interference from autoantibodies.

A New MIC1-MAG1 Recombinant Chimeric Antigen Can Be Used Instead of the Toxoplasma gondii Lysate Antigen in Serodiagnosis of Human Toxoplasmosis

PubMed Central

Holec-Gąsior, Lucyna; Ferra, Bartłomiej; Drapała, Dorota; Lautenbach, Dariusz

2012-01-01

This study presents an evaluation of the MIC1 (microneme protein 1)-MAG1 (matrix antigen 1) Toxoplasma gondii recombinant chimeric antigen for the serodiagnosis of human toxoplasmosis for the first time. The recombinant MIC1-MAG1 antigen was obtained as a fusion protein containing His tags at the N- and C-terminal ends using an Escherichia coli expression system. After purification by metal affinity chromatography, the chimeric protein was tested for usefulness in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-T. gondii immunoglobulin G (IgG). One hundred ten sera from patients at different stages of infection and 40 sera from seronegative patients were examined. The results obtained for the MIC1-MAG1 chimeric antigen were compared with those of IgG ELISAs using a Toxoplasma lysate antigen (TLA), a combination of recombinant antigens (rMIC1ex2-rMAG1) and single recombinant proteins (rMIC1ex2 and rMAG1). The sensitivity of the IgG ELISA calculated from all of the positive serum samples was similar for the MIC1-MAG1 chimeric antigen (90.8%) and the TLA (91.8%), whereas the sensitivities of the other antigenic samples used were definitely lower, at 69.1% for the mixture of antigens, 75.5% for the rMIC1ex2, and 60% for rMAG1. This study demonstrates that the MIC1-MAG1 recombinant chimeric antigen can be used instead of the TLA in the serodiagnosis of human toxoplasmosis. PMID:22116686

Development of a Peptide ELISA for the Diagnosis of Aleutian Mink Disease

PubMed Central

Wang, Yang; Lu, Rongguang; Hu, Bo; Lv, Shuang; Xue, Xianghong; Li, Xintong; Ling, Mingyu; Fan, Sining; Zhang, Hailing; Yan, Xijun

2016-01-01

Aleutian disease (AD) is a common immunosuppressive disease in mink farms world-wide. Since the 1980s, counterimmunoelectrophoresis (CIEP) has been the main detection method for infection with the Aleutian Mink Disease Virus (AMDV). In this study, six peptides derived from the AMDV structural protein VP2 were designed, synthesized, and used as ELISA antigens to detect anti-AMDV antibodies in the sera of infected minks. Serum samples were collected from 764 minks in farms from five different provinces, and analyzed by both CIEP (a gold standard) and peptide ELISA. A peptide designated P1 (415 aa–433 aa) exhibited good antigenicity. A novel ELISA was developed using ovalbumin-linked peptide P1 to detect anti-AMDV antibodies in mink sera. The sensitivity and specificity of the peptide ELISA was 98.0% and 97.5%, respectively. Moreover, the ELISA also detected 342 early-stage infected samples (negative by CIEP and positive by PCR), of which 43.6% (149/342) were true positives. These results showed that the peptide ELISA had better sensitivity compared with CIEP, and therefore could be preferable over CIEP for detecting anti-AMDV antibodies in serological screening. PMID:27802320

Development of Specific Sequence-Characterized Amplified Region Markers for Detecting Histoplasma capsulatum in Clinical and Environmental Samples

PubMed Central

Frías De León, María Guadalupe; Arenas López, Gabina; Taylor, Maria Lucia; Acosta Altamirano, Gustavo

2012-01-01

Sequence-characterized amplified region (SCAR) markers, generated by randomly amplified polymorphic DNA (RAPD)-PCR, were developed to detect Histoplasma capsulatum selectively in clinical and environmental samples. A 1,200-bp RAPD-PCR-specific band produced with the 1281-1283 primers was cloned, sequenced, and used to design two SCAR markers, 1281-1283220 and 1281-1283230. The specificity of these markers was confirmed by Southern hybridization. To evaluate the relevance of the SCAR markers for the diagnosis of histoplasmosis, another molecular marker (M antigen probe) was used for comparison. To validate 1281-1283220 and 1281-1283230 as new tools for the identification of H. capsulatum, the specificity and sensitivity of these markers were assessed for the detection of the pathogen in 36 clinical (17 humans, as well as 9 experimentally and 10 naturally infected nonhuman mammals) and 20 environmental (10 contaminated soil and 10 guano) samples. Although the two SCAR markers and the M antigen probe identified H. capsulatum isolates from different geographic origins in America, the 1281-1283220 SCAR marker was the most specific and detected the pathogen in all samples tested. In contrast, the 1281-1283230 SCAR marker and the M antigen probe also amplified DNA from Aspergillus niger and Cryptococcus neoformans, respectively. Both SCAR markers detected as little as 0.001 ng of H. capsulatum DNA, while the M antigen probe detected 0.5 ng of fungal DNA. The SCAR markers revealed the fungal presence better than the M antigen probe in contaminated soil and guano samples. Based on our results, the 1281-1283220 marker can be used to detect and identify H. capsulatum in samples from different sources. PMID:22189121

Simple Tests for Rapid Detection of Canine Parvovirus Antigen and Canine Parvovirus-Specific Antibodies▿ †

PubMed Central

Marulappa, Shashidhara Y.; Kapil, Sanjay

2009-01-01

Canine parvovirus (CPV) is the number one viral cause of enteritis, morbidity, and mortality in 8-week-old young puppies. We have developed twin assays (slide agglutination test [SAT] for CPV antigen and slide inhibition test [SIT] for CPV antibody) that are sensitive, specific, cost-effective, generic for all genotypes of CPV, and provide instant results for CPV antigen detection in feces and antibody quantification in serum. We found these assays to be useful for routine applications in kennels with large numbers of puppies at risk. The results of these assays are available in 1 min and do not require any special instrumentation. SAT-SIT technology will find applications in rapid screening of samples for other hemagglutinating emerging viruses of animals and humans (influenza virus and severe acute respiratory syndrome coronavirus). PMID:18987166

MEMS reagent and sample handling procedure: Feasibility of viral antibody detection by passive immune agglutination

NASA Technical Reports Server (NTRS)

Bailey, G. D.; Tenoso, H. J.

1975-01-01

An attempt was made to develop a test requiring no preadsorption steps for the assessment of antibodies to rubella and mumps viruses using the passive immune agglutination (PIA) method. Both rubella and mumps antigens and antibodies were prepared. Direct PIA tests, using rubella antigen-coated beads, and indirect PIA tests, using rubella antibody-coated beads, were investigated. Attempts, using either method, were unsuccessful. Serum interference along with nonspecific agglutination of beads by the rubella antigen resulted in no specific response under the test conditions investigated. A new, highly sensitive approach, the enzyme immunoassay (EIA) test system, is recommended to overcome the nonspecificity. This system is a logical outgrowth of some of the solid phase work done on MEMS and represents the next generation tests system that can be directly applied to early disease detection and monitoring.

Evaluation of two 4th generation point-of-care assays for the detection of Human Immunodeficiency Virus infection.

PubMed

Stafylis, Chrysovalantis; Klausner, Jeffrey D

2017-01-01

Fourth generation assays detect simultaneously antibodies for HIV and the p24 antigen, identifying HIV infection earlier than previous generation tests. Previous studies have shown that the Alere Determine HIV-1/2 Combo has lower than anticipated performance in detecting antibodies for HIV and the p24 antigen. Furthermore, there are currently very few studies evaluating the performance of Standard Diagnostics BIOLINE HIV Ag/Ab Combo. To evaluate the performance of the Alere Determine HIV-1/2 Combo and the Standard Diagnostics BIOLINE HIV Ag/Ab Combo in a panel of frozen serum samples. The testing panel included 133 previously frozen serum specimens from the UCLA Clinical Microbiology & Immunoserology laboratory. Reference testing included testing for HIV antibodies by a 3rd generation enzyme immunoassay followed by HIV RNA detection. Antibody negative and RNA positive sera were also tested by a laboratory 4th generation HIV Ab/Ag enzyme immunoassay. Reference testing yielded 97 positives for HIV infection and 36 negative samples. Sensitivity of the Alere test was 95% (88-98%), while the SD Bioline sensitivity was 91% (83-96%). Both assays showed 100% (90-100%) specificity. No indeterminate or invalid results were recorded. Among 13 samples with acute infection (HIV RNA positive, HIV antibody negative), 12 were found positive by the first assay and 8 by the second. The antigen component of the Alere assay detected 10 acute samples, while the SD Bioline assay detected only one. Both rapid assays showed very good overall performance in detecting HIV infection in frozen serum samples, but further improvements are required to improve the performance in acute infection.

Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

PubMed

López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

2009-09-01

A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used.

Development and evaluation of porcine cysticercosis QuickELISA in Triturus EIA analyzer.

PubMed

Handali, Sukwan; Pattabhi, Sowmya; Lee, Yeuk-Mui; Silva-Ibanez, Maria; Kovalenko, Victor A; Levin, Andrew E; Gonzalez, Armando E; Roberts, Jacquelin M; Garcia, Hector H; Gilman, Robert H; Hancock, Kathy; Tsang, Victor C W

2010-01-01

We evaluated three diagnostic antigens (recombinant GP50, recombinant T24H, and synthetic Ts18var1) for cysticercosis and found that all three performed well in detecting cysticercosis in humans and pigs in several assay formats. These antigens were adapted to a new antibody detection format (QuickELISA). With one single incubation step which involves all reactants except the enzyme substrate, the QuickELISA is particularly suited for automation. We formatted the QuickELISA for the Triturus EIA analyzer for testing large numbers of samples. We found that in QuickELISA formats rGP50 and rT24H have better sensitivity and specificity than sTs18var1 for detecting porcine cysticercosis.

First field trial of an immunoradiometric assay for the detection of malaria sporozoites in mosquitoes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, F.H.; Zavala, F.; Graves, P.M.

1984-07-01

An immunoradiometric assay (IRMA) using a monoclonal antibody to the major surface protein of Plasmodium falciparum sporozoites was used to assess the P. falciparum sporozoite rate in a West African population of Anopheles gambiae (s.1.). Unlike current dissection techniques, the IRMA could detect sporozoite antigen in dried as well as fresh mosquitoes. In a controlled comparison, the sensitivity of the IRMA was comparable that of the dissection technique. Additionally, the IRMA was species specific and quantitative. Sensitivity of the assay was sufficient to detect sporozoite infections resulting from the development of a single oocyst.

Diagnostic accuracy of three monoclonal stool tests in a large series of untreated Helicobacter pylori infected patients.

PubMed

Lario, Sergio; Ramírez-Lázaro, María José; Montserrat, Antònia; Quílez, María Elisa; Junquera, Félix; Martínez-Bauer, Eva; Sanfeliu, Isabel; Brullet, Enric; Campo, Rafael; Segura, Ferran; Calvet, Xavier

2016-06-01

Immunochromatographic tests need to be improved in order to enhance their reliability. Recently, several new kits have appeared on the market. The objective was to evaluate the diagnostic accuracy of three monoclonal rapid stool tests - the new Uni-Gold™ H.pylori Antigen (Trinity Biotech, Ireland), the RAPID Hp StAR (Oxoid Ltd., UK) and the ImmunoCard STAT! HpSA (Meridian Diagnostics, USA) - for detecting H. pylori infection prior to eradication treatment. Diagnostic accuracy (sensitivity and specificity) and reliability (concordance between observers) were evaluated in 250 untreated consecutive dyspeptic patients. The gold standard for diagnosing H. pylori infection was defined as the concordance of two or more of rapid urease test (RUT), histopathology and urease breath test (UBT) or positive culture in isolation. Readings of immunochromatographic tests were performed by two different observers. Sensitivity, specificity, positive and negative predictive values and 95% confidence intervals were calculated. Sensitivity and specificity were compared using the McNemar test. The three tests showed a good correlation, with Kappa values>0.9. RAPID Hp StAR had a sensitivity of 91%-92% and a specificity ranging from 77% to 85%. Its sensitivity was higher than that of Uni-Gold™ H.pylori Antigen and ImmunoCard STAT! HpSA (p<0.01). Uni-Gold™ H.pylori Antigen kit showed a sensitivity of 83%, similar to ImmunoCard STAT! HpSA. Specificity of Uni-Gold™ H.pylori Antigen approached 90% (87-89%) and was superior to that of RAPID Hp StAR (p<0.01). Uni-Gold™ H.pylori Antigen and ImmunoCard STAT! HpSA present similar levels of diagnostic accuracy. RAPID Hp StAR was the most sensitive but less reliable of the three immunochromatographic stool tests. None are as accurate and reliable as UBT, RUT and histology. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Diagnostic potential of low molecular weight excretory secretory proteins of Paramphistomum epiclitum for caprine amphistomosis.

PubMed

Jaiswal, Amit Kumar; Shanker, Daya; Sudan, Vikrant; Singh, Amit

2018-06-15

In the present study, the 75% alcoholic fractionation of excretory-secretory (ES) antigen isolated from 200 to 300 live P. epiclitum was assessed for its diagnostic potential for the detection of caprine amphistomosis by using antibody detection enzyme immunoassay. Prior to enzyme immunoassay, 75% alcoholic fractionation of excretory-secretory (ES) antigen was subjected to SDS- PAGE and western blot analysis for the presence of immunoreactive polypeptides. SDS-PAGE analysis of ES antigen resolved a total 7 polypeptides bands of size 56, 27, 25, 22.5, 12, 11 and 10 kDa. Western blot analysis revealed only two immunoreactive polypeptides (11 kDa and 12 kDa) when polypeptides resolved in SDS-PAGE were probed with known positive pooled serum. None of the polypeptides showed reactions with pooled known negative serum. The working dilutions of antigen, sera and conjugates were determined by checkerboard titration for employing ELISA and cut-off O.D. was calculated 0.616 by using the mean absorbance value of 11 negative kid sera. The sensitivity and specificity of ELISA was found to be 100% and 86.76%, respectively. As per kappa value estimation, the strength of agreement was found to be good. Antibodies to 75% alcoholic fractionation of ES antigen was detected in 20% goats (n = 160) of either sex, although faecal examination detected 10.6% goats to be infected with amphistomosis. The study confirmed that 75% alcoholic fractionation of ES antigen of P. epiclitum based ELISA had good value for serodiagnosis of caprine amphistomosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Benefit of Hepatitis C Virus Core Antigen Assay in Prediction of Therapeutic Response to Interferon and Ribavirin Combination Therapy

PubMed Central

Takahashi, Masahiko; Saito, Hidetsugu; Higashimoto, Makiko; Atsukawa, Kazuhiro; Ishii, Hiromasa

2005-01-01

A highly sensitive second-generation hepatitis C virus (HCV) core antigen assay has recently been developed. We compared viral disappearance and first-phase kinetics between commercially available core antigen (Ag) assays, Lumipulse Ortho HCV Ag (Lumipulse-Ag), and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor test, version 2 (Amplicor M), to estimate the predictive benefit of a sustained viral response (SVR) and non-SVR in 44 genotype 1b patients treated with interferon (IFN) and ribavirin. HCV core Ag negativity could predict SVR on day 1 (sensitivity = 100%, specificity = 85.0%, accuracy = 86.4%), whereas RNA negativity could predict SVR on day 7 (sensitivity = 100%, specificity = 87.2%, accuracy = 88.6%). None of the patients who had detectable serum core Ag or RNA on day 14 achieved SVR (specificity = 100%). The predictive accuracy on day 14 was higher by RNA negativity (93.2%) than that by core Ag negativity (75.0%). The combined predictive criterion of both viral load decline during the first 24 h and basal viral load was also predictive for SVR; the sensitivities of Lumipulse-Ag and Amplicor-M were 45.5 and 47.6%, respectively, and the specificity was 100%. Amplicor-M had better predictive accuracy than Lumipulse-Ag in 2-week disappearance tests because it had better sensitivity. On the other hand, estimates of kinetic parameters were similar regardless of the detection method. Although the correlations between Lumipulse-Ag and Amplicor-M were good both before and 24 h after IFN administration, HCV core Ag seemed to be relatively lower 24 h after IFN administration than before administration. Lumipulse-Ag seems to be useful for detecting the HCV concentration during IFN therapy; however, we still need to understand the characteristics of the assay. PMID:15634970

Advantages of bioconjugated silica-coated nanoparticles as an innovative diagnosis for human toxoplasmosis.

PubMed

Aly, Ibrahim; Taher, Eman E; El Nain, Gehan; El Sayed, Hoda; Mohammed, Faten A; Hamad, Rabab S; Bayoumy, Elsayed M

2018-01-01

Nanotechnology is a promising arena for generating new applications in Medicine. To successfully functionalised nanoparticles for a given biomedical application, a wide range of chemical, physical and biological factors have to be taken into account. Silica-coated nanoparticles, (SiO2NP) exhibit substantial diagnostic activity owing to their large surface to volume ratios and crystallographic surface structure. This work aimed to evaluate the advantage of bioconjugation of SiO2NP with PAb against Toxoplasma lyzate antigen (TLA) as an innovative diagnostic method for human toxoplasmosis. This cross-sectional study included 120 individuals, divided into Group I: 70 patients suspected for Toxoplasma gondii based on the presence of clinical manifestation. Group II: 30 patients harboring other parasites than T. gondii Group III: 20 apparently healthy individuals free from toxoplasmosis and other parasitic infections served as negative control. Detection of circulating Toxoplasma antigen was performed by Sandwich ELISA and Nano-sandwich ELISA on sera and pooled urine of human samples. Using Sandwich ELISA, 10 out of 70 suspected Toxoplasma-infected human serum samples showed false negative and 8 out of 30 of other parasites groups were false positive giving 85.7% sensitivity and 84.0% specificity, while the sensitivity and specificity were 78.6% and 70% respectively in urine samples. Using Nano-Sandwich ELISA, 7 out of 70 suspected Toxoplasma-infected human samples showed false negative results and the sensitivity of the assay was 90.0%, while 4 out of 30 of other parasites groups were false positive giving 92.0% specificity, while the sensitivity and specificity were 82.6% and 80% respectively in urine samples. In conclusion, our data demonstrated that loading SiO2 nanoparticles with pAb increased the sensitivity and specificity of Nano-sandwich ELISA for detection of T.gondii antigens in serum and urine samples, thus active (early) and light infections could be easily detected. Copyright © 2017 Elsevier B.V. All rights reserved.

Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

PubMed Central

Kamel, Hanan H.; Saad, Ghada A.

2013-01-01

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable. PMID:23710084

Dot-blot immunoassay of Fasciola gigantica infection using 27 kDa and adult worm regurge antigens in Egyptian patients.

PubMed

Kamel, Hanan H; Saad, Ghada A; Sarhan, Rania M

2013-04-01

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.

Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves.

PubMed

Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

2016-01-01

The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.

Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves

PubMed Central

Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

2016-01-01

The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings. PMID:26930597

Evaluation of the Alexon-Trend ProSpecT Campylobacter Microplate Assay

PubMed Central

Tolcin, Rita; LaSalvia, Margaret M.; Kirkley, Barbara A.; Vetter, Emily A.; Cockerill, Franklin R.; Procop, Gary W.

2000-01-01

We evaluated stool specimens known to contain or be free of Campylobacter by traditional culture, using the ProSpecT Campylobacter microplate assay (Alexon-Trend, Ramsey, Minn.). This rapid enzyme immunoassay for the detection of Campylobacter-specific antigens demonstrated 96% sensitivity and 99% specificity and is an acceptable alternative method of Campylobacter detection. PMID:11015419

Detection of egg yolk antibodies reflecting Salmonella enteritidis infections using a surface plasmon resonance biosensor.

PubMed

Thomas, Ekelijn; Bouma, Annemarie; van Eerden, Ellen; Landman, Wil J M; van Knapen, Frans; Stegeman, Arjan; Bergwerff, Aldert A

2006-08-31

A surface plasmon resonance (SPR) biosensor assay was developed on the basis of a lipopolysaccharide antigen of Salmonella enterica serovar enteritidis (S. enterica serovar enteritidis) to detect egg yolk antibodies against S. enterica serovar enteritidis. This biosensor assay was compared to two commercial ELISA kits based on LPS antigen and flagellar antigen. A number of 163 egg yolk and combined egg white and yolk samples from chickens experimentally infected with S. enterica serovar enteritidis and 90 egg yolk and combined egg white and yolk samples from uninfected chickens were analyzed. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 82% and a diagnostic specificity of 100%. The within-day coefficient of variation of a positive internal-control egg yolk was 1%. The SPR biosensor assay was able to detect antibodies in a significantly higher percentage of known positive samples than the commercial ELISA's. The anticipated use of the SPR biosensor assay is to determine the S. enterica serovar enteritidis serostatus of non-vaccinated layer hens.

Evaluation of Mycobacterium tuberculosis Early Secreted Antigenic Target 6 Recombinant Protein as a Diagnostic Marker in Skin Test.

PubMed

Moradi, Jale; Mosavari, Nader; Ebrahimi, Mahmoud; Arefpajohi, Reza; Tebianian, Majid

2015-02-01

Tuberculosis (TB) is the leading infectious disease in the developing world. Delayed-type hypersensitivity skin test diagnoses TB using tuberculin purified protein derivative (PPD), but this test is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacillus Calmette-Guérin (BCG) vaccination or an infection caused by nontuberculous mycobacteria (NTM). This study was performed to evaluate the use of recombinant early secretory antigenic target 6 (rESAT-6), a secretory protein found only in MTB, Mycobacterium bovis, and few other mycobacterial species, as a skin marker for MTB in guinea pigs. We prepared recombinant MTB ESAT-6 and evaluated its use as a specific antigen for MTB in guinea pigs. Our results show that the purified MTB rESAT-6 antigen is capable of inducing a positive reaction only in guinea pigs sensitized to MTB. No such reaction was observed in the animals sensitized to M. bovis, BCG vaccination, or NTM (Mycobacterium avium). Our study results confirm that the ESAT-6 antigen is more specific to MTB infection than PPD and could be used in more specific skin tests for detection of MTB in large animals and in humans.

Serodiagnosis of toxocariasis by ELISA using crude antigen of Toxocara canis larvae.

PubMed

Jin, Yan; Shen, Chenghua; Huh, Sun; Sohn, Woon-Mok; Choi, Min-Ho; Hong, Sung-Tae

2013-08-01

Toxocariasis is a worldwide zoonosis caused by larvae of ascarid nematodes of dogs or cats, Toxocara canis or T. cati. Diagnosis of human toxocariasis currently relies on serology that uses T. canis excretory-secretory antigen to detect specific IgG antibodies by ELISA. We investigated the serodiagnostic efficacy of ELISA using crude antigen of T. canis larvae (TCLA). Serum specimens of 64 clinically confirmed toxocariasis, 115 healthy controls, and 119 other tissue-invading helminthiases were screened by ELISA using TCLA. The ELISA using TCLA showed 92.2% (59/64 patient samples) sensitivity and 86.6% (103/119) specificity. Its positive diagnostic predictivity was 78.7% and negative predictivity was 97.8%. No serum of healthy controls reacted but that of anisakiasis (45.5%), gnathostomiasis (19.2%), clonorchiasis (15.8%), sparganosis (11.1%), and cysticercosis (6.3%) cross-reacted. Immunoblot analysis on TCLA recognized antigenic proteins of 28- and 30-kDa bands in their dominant protein quantity and strong blotting reactivity. The present results indicate that the ELISA using our TCLA antigen is acceptable by the sensitivity and specificity for serodiagnosis of human toxocariasis. ELISA with TCLA is recommended to make differential diagnosis for patients with any sign of organ infiltration and eosinophilia.

Electrochemical Quartz Crystal Nanobalance (EQCN) Based Biosensor for Sensitive Detection of Antibiotic Residues in Milk.

PubMed

Bhand, Sunil; Mishra, Geetesh K

2017-01-01

An electrochemical quartz crystal nanobalance (EQCN), which provides real-time analysis of dynamic surface events, is a valuable tool for analyzing biomolecular interactions. EQCN biosensors are based on mass-sensitive measurements that can detect small mass changes caused by chemical binding to small piezoelectric crystals. Among the various biosensors, the piezoelectric biosensor is considered one of the most sensitive analytical techniques, capable of detecting antigens at picogram levels. EQCN is an effective monitoring technique for regulation of the antibiotics below the maximum residual limit (MRL). The analysis of antibiotic residues requires high sensitivity, rapidity, reliability and cost effectiveness. For analytical purposes the general approach is to take advantage of the piezoelectric effect by immobilizing a biosensing layer on top of the piezoelectric crystal. The sensing layer usually comprises a biological material such as an antibody, enzymes, or aptamers having high specificity and selectivity for the target molecule to be detected. The biosensing layer is usually functionalized using surface chemistry modifications. When these bio-functionalized quartz crystals are exposed to a particular substance of interest (e.g., a substrate, inhibitor, antigen or protein), binding interaction occurs. This causes a frequency or mass change that can be used to determine the amount of material interacted or bound. EQCN biosensors can easily be automated by using a flow injection analysis (FIA) setup coupled through automated pumps and injection valves. Such FIA-EQCN biosensors have great potential for the detection of different analytes such as antibiotic residues in various matrices such as water, waste water, and milk.

Diagnosis of Plasmodium falciparum infection in man: detection of parasite antigens by ELISA*

PubMed Central

Mackey, L. J.; McGregor, I. A.; Paounova, N.; Lambert, P. H.

1982-01-01

An ELISA method has been developed for the diagnosis of Plasmodium falciparum infection in man. Parasites from in vitro cultures of P. falciparum were used as source of antigen for the solid phase and the source of specific antibody was immune Gambian sera; binding of antibody in antigen-coated wells was registered by means of alkaline phosphatase-conjugated anti-human IgG. Parasites were detected on the basis of inhibition of antibody-binding. The test was applied to the detection of parasites in human red blood cells (RBC) from in vitro cultures of P. falciparum and in RBC from infected Gambians; RBC from 100 Geneva blood donors served as normal, uninfected controls. In titration experiments, the degree of antibody-binding inhibition correlated with the number of parasites in the test RBC. Parasites were detected at a level of 8 parasites/106 RBC. Samples of RBC were tested from 126 Gambians with microscopically proven infection; significant antibody-binding inhibition was found in 86% of these cases, where parasitaemia ranged from 10 to 125 000/μl of blood. The presence of high-titre antibody in the test preparations was found to reduce the sensitivity of parasite detection in infected RBC from in vitro cultures mixed with equal volumes of different antibody-containing sera. The sensitivity was restored in most cases by recovering the RBC by centrifugation before testing. In a preliminary experiment, there was no significant difference in antibody-binding inhibition using fresh infected RBC and RBC dried on filter-paper and recovered by elution, although there was greater variation in the latter samples. PMID:7044589

Evaluation of ICT filariasis card test using whole capillary blood: comparison with Knott's concentration and counting chamber methods.

PubMed

Njenga, S M; Wamae, C N

2001-10-01

An immunochromatographic card test (ICT) that uses fingerprick whole blood instead of serum for diagnosis of bancroftian filariasis has recently been developed. The card test was validated in the field in Kenya by comparing its sensitivity to the combined sensitivity of Knott's concentration and counting chamber methods. A total of 102 (14.6%) and 117 (16.7%) persons was found to be microfilaremic by Knott's concentration and counting chamber methods, respectively. The geometric mean intensities (GMI) were 74.6 microfilariae (mf)/ml and 256.5 mf/ml by Knott's concentration and counting chamber methods, respectively. All infected individuals detected by both Knott's concentration and counting chamber methods were also antigen positive by the ICT filariasis card test (100% sensitivity). Further, of 97 parasitologically amicrofilaremic persons, 24 (24.7%) were antigen positive by the ICT. The overall prevalence of antigenemia was 37.3%. Of 100 nonendemic area control persons, none was found to be filarial antigen positive (100% specificity). The results show that the new version of the ICT filariasis card test is a simple, sensitive, specific, and rapid test that is convenient in field settings.

Toxoplasma gondii Recombinant Antigens as Tools for Serodiagnosis of Human Toxoplasmosis: Current Status of Studies

PubMed Central

2013-01-01

Toxoplasma gondii is a parasitic protozoan which is the cause of toxoplasmosis. Although human toxoplasmosis in healthy adults is usually asymptomatic, serious disease can occur in the case of congenital infections and immunocompromised individuals. Furthermore, despite the exact recognition of its etiology, it still presents a diagnostic problem. Diagnosis of toxoplasmosis is mainly based on the results of serological tests detecting anti-T. gondii-specific antibodies in the patient's serum sample. The specificities and sensitivities of serology tests depend mostly on the diagnostic antigen(s) used. Most of the commercial serological kits currently available are based on Toxoplasma lysate antigens (TLAs). In recent years, many studies showed that recombinant antigenic proteins of T. gondii may be an alternative source of antigens which are very useful for the serodiagnosis of toxoplasmosis. This article presents a review of current studies on the application and usefulness of different T. gondii recombinant antigens in serological tests for the diagnosis of human toxoplasmosis. PMID:23784855

Application of Protein Microarrays for Multiplexed Detection of Antibodies to Tumor Antigens in Breast Cancer

PubMed Central

Anderson, Karen S.; Ramachandran, Niroshan; Wong, Jessica; Raphael, Jacob V.; Hainsworth, Eugenie; Demirkan, Gokhan; Cramer, Daniel; Aronzon, Diana; Hodi, F. Stephen; Harris, Lyndsay; Logvinenko, Tanya; LaBaer, Joshua

2012-01-01

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum, may exist in greater concentrations than their cognate antigens, and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies. PMID:18311903

Characterization of an F1 deletion mutant of Yersinia pestis CO92, pathogenic role of F1 antigen in bubonic and pneumonic plague, and evaluation of sensitivity and specificity of F1 antigen capture-based dipsticks.

PubMed

Sha, Jian; Endsley, Janice J; Kirtley, Michelle L; Foltz, Sheri M; Huante, Matthew B; Erova, Tatiana E; Kozlova, Elena V; Popov, Vsevolod L; Yeager, Linsey A; Zudina, Irina V; Motin, Vladimir L; Peterson, Johnny W; DeBord, Kristin L; Chopra, Ashok K

2011-05-01

We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37 °C, although the mutant's growth dropped slightly during the late phase at 37 °C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 10(5) to 5 × 10(5) CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 10(8) CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.

Characterization of an F1 Deletion Mutant of Yersinia pestis CO92, Pathogenic Role of F1 Antigen in Bubonic and Pneumonic Plague, and Evaluation of Sensitivity and Specificity of F1 Antigen Capture-Based Dipsticks▿

PubMed Central

Sha, Jian; Endsley, Janice J.; Kirtley, Michelle L.; Foltz, Sheri M.; Huante, Matthew B.; Erova, Tatiana E.; Kozlova, Elena V.; Popov, Vsevolod L.; Yeager, Linsey A.; Zudina, Irina V.; Motin, Vladimir L.; Peterson, Johnny W.; DeBord, Kristin L.; Chopra, Ashok K.

2011-01-01

We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37°C, although the mutant's growth dropped slightly during the late phase at 37°C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 105 to 5 × 105 CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 108 CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague. PMID:21367990

Detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology.

PubMed

Zijlmans, H J; Bonnet, J; Burton, J; Kardos, K; Vail, T; Niedbala, R S; Tanke, H J

1999-02-01

A novel luminescent reporter for the sensitive detection of antigens in tissue sections or on cell membranes is described. It consists of submicron-size phosphor crystals (0.2-0.4 microm), which are surface labeled with avidin or antibodies and capable of binding specifically to antigens on intact cells or in tissue sections. These phosphor reporters exhibit two-photon, anti-Stokes luminescence by up-converting infrared to visible light and are named Up-converting Phosphor Technology (UPT). They typically consist of yttriumoxysulfides doped with two different lanthanides exhibiting photostable, strong emission in the visible (blue, green, and red) upon excitation in the infrared. This report describes the conjugation of phosphor particles to NeutrAvidin with the subsequent use of this conjugate in a model system consisting of prostate-specific antigen in tissue sections and the CD4 membrane antigen on human lymphocytes. An epi-illumination fluorescence microscope was adapted to provide near-IR excitation using a xenon lamp for visualization of the visible emission. Advantages of UPT are (i) permanent, strong, anti-Stokes emission of discrete wavelengths; (ii) unmatched contrast in biological specimens due to the absence of autofluorescence upon excitation with IR light; (iii) simultaneous detection of multiple target analytes; and (iv) low-cost microscope modifications. The new methodology has not only high potential value in diagnostic pathology as described here, but may offer advantages for the detection of proteins or nucleic acids when applied to molecular biology, genomic research, virology, and microbiology. Copyright 1999 Academic Press.

Preliminary Validation of Direct Detection of Foot-And-Mouth Disease Virus within Clinical Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Simple Lateral Flow Device for Detection

PubMed Central

Waters, Ryan A.; Fowler, Veronica L.; Armson, Bryony; Nelson, Noel; Gloster, John; Paton, David J.; King, Donald P.

2014-01-01

Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD). Current approaches involve either; 1) Detection of FMD virus (FMDV) with immuochromatographic antigen lateral flow devices (LFD), which have relatively low analytical sensitivity, or 2) portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP) may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription–LAMP (RT-LAMP) assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing “proof of concept” for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health. PMID:25165973

Single-molecule detection of proteins with antigen-antibody interaction using resistive-pulse sensing of submicron latex particles

NASA Astrophysics Data System (ADS)

Takakura, T.; Yanagi, I.; Goto, Y.; Ishige, Y.; Kohara, Y.

2016-03-01

We developed a resistive-pulse sensor with a solid-state pore and measured the latex agglutination of submicron particles induced by antigen-antibody interaction for single-molecule detection of proteins. We fabricated the pore based on numerical simulation to clearly distinguish between monomer and dimer latex particles. By measuring single dimers agglutinated in the single-molecule regime, we detected single human alpha-fetoprotein molecules. Adjusting the initial particle concentration improves the limit of detection (LOD) to 95 fmol/l. We established a theoretical model of the LOD by combining the reaction kinetics and the counting statistics to explain the effect of initial particle concentration on the LOD. The theoretical model shows how to improve the LOD quantitatively. The single-molecule detection studied here indicates the feasibility of implementing a highly sensitive immunoassay by a simple measurement method using resistive-pulse sensing.

Development of a highly sensitive bioluminescent enzyme immunoassay for hepatitis B virus surface antigen capable of detecting divergent mutants.

PubMed

Minekawa, Takayuki; Takehara, Shizuka; Takahashi, Masaharu; Okamoto, Hiroaki

2013-08-01

Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals.

Evaluation of twenty-two rapid antigen detection tests in the diagnosis of Equine Influenza caused by viruses of H3N8 subtype.

PubMed

Yamanaka, Takashi; Nemoto, Manabu; Bannai, Hiroshi; Tsujimura, Koji; Kondo, Takashi; Matsumura, Tomio; Gildea, Sarah; Cullinane, Ann

2016-03-01

Equine influenza (EI) is a highly contagious disease caused by viruses of the H3N8 subtype. The rapid diagnosis of EI is essential to reduce the disease spread. Many rapid antigen detection (RAD) tests for diagnosing human influenza are available, but their ability to diagnose EI has not been systematically evaluated. The aim of this study was to compare the performance of 22 RAD tests in the diagnosis of EI. The 22 RAD tests were performed on fivefold serial dilutions of EI virus to determine their detection limits. The four most sensitive RAD tests (ImmunoAce Flu, BD Flu examan, Quick chaser Flu A, B and ESPLINE Influenza A&B-N) were further evaluated using nasopharyngeal samples collected from experimentally infected and naturally infected horses. The results were compared to those obtained using molecular tests. The detection limits of the 22 RAD tests varied hugely. Even the four RAD tests showing the best sensitivity were 125-fold less sensitive than the molecular techniques. The duration of virus detection in the experimentally infected horses was shorter using the RAD tests than using the molecular techniques. The RAD tests detected between 27% and 73% of real-time RT-PCR-positive samples from naturally infected horses. The study demonstrated the importance of choosing the right RAD tests as only three of 22 were fit for diagnosing EI. It was also indicated that even RAD tests with the highest sensitivity serve only as an adjunct to molecular tests because of the potential for false-negative results. © 2015 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Improvement of an enzyme immunosorbent assay for detecting antibodies against Dioctophyma renale.

PubMed

Pedrassani, Daniela; do Nascimento, Adjair Antonio; André, Marcos Rogério; Machado, Rosangela Zacarias

2015-09-15

An available enzyme-linked immunosorbent assay (ELISA) was studied for the detection of anti-Dioctophyma renale antibodies in the sera of dogs using, detection of parasite eggs in urine sediment as a reference test. ELISA uses a soluble antigenic preparation of esophagus of D. renale and the optimal dilutions of the antigen, serum and conjugate were determined by means of checker board titration, using positive (n=13) and negative (n=27) reference serum. The specificity and sensitivity of the ELISA were 93.8% and 92.3% respectively and the kappa index was good (0.76). These results suggest that ELISA described may prove to be an effective serological test for detecting dogs infected and exposed to this parasite mainly dogs that are not eliminating parasite eggs through their urine. Copyright © 2015 Elsevier B.V. All rights reserved.

Gold nanoparticles paper as a SERS bio-diagnostic platform.

PubMed

Ngo, Ying Hui; Then, Whui Lyn; Shen, Wei; Garnier, Gil

2013-11-01

Bioactive papers are usually challenged by four major limitations: sensitivity, selectivity, simplicity and strength (4S). Gold nanoparticles (AuNPs) treated paper has previously been demonstrated as a Surface Enhanced Raman Scattering (SERS) active substrate, capable of addressing the 4S issues. In this study, AuNPs on paper substrate were functionalized by a series of biomolecules to develop a generic SERS platform for antibody-antigen detection. The functionalization steps were performed by taking advantage of the high affinity association between Streptomyces avidinii-derived protein, streptavidin, and biotin. Streptavidin was firstly bound onto the AuNPs treated paper using biotinylated-thiol. Subsequently, desired biotinylated-antibody was bound onto the streptavidin. SERS spectra of each functionalization step were obtained to ensure specific adsorption of the bio-molecules. The binding interaction of the antibody with its specific antigen was detected using SERS. Shifts of Raman band associated with α-helix and β-sheet structures indicated structural modification of the antibody upon interaction with its antigen. Predominant tryptophan and tyrosine residue bands were also detected, confirming the presence of antigen. Reproducible spectral features were quantified as AuNP papers were subjected to different concentrations of antigen; the spectra intensity increased as a function of the antigen concentration. The retention of AuNPs on paper remained constant after all the consecutive washing and functionalization steps. The feasibility of AuNPs paper as a low-cost and generic SERS platform for bio-diagnostic applications was demonstrated. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

[Clinical value of combined determination of serum and pleural effusion level of CEA,CYFRA21-1, TPS in the diagnosis of lung cancer].

PubMed

Huang, Fang; Wang, Xiao-li; Yang, Lian; Yin, Bin-xia; Geng, Yan; Li, Ting-ting

2008-04-01

To study the clinical value of combined determination of carcinoembryonic antigen (CEA), cytokeratin-19-fragments (CYFRA21-1) and tissue polypeptide specific antigen (TPS) levels in both serum and pleural effusion in the diagnosis of lung cancer. The levels of CEA, CYFRA21-1 and TPS in serum and pleural effusion were measured in 78 patients with lung cancer and 45 patients with benign lung disease by using electrochemiluminescence and ELISA methods respectively. The levels of CEA, CYFRA21-1 and TPS in pleural effusion of patients with lung cancer were much higher than those with benign lung disease (P<0.01). The levels of CEA and TPS in serum of patients with lung cancer were much higher than those with benign lung disease (P<0.05, P<0.01). It was also found that the levels of these tumor markers in pleural effusion were higher and more sensitive than those in sera, especially TPS. TPS showed the highest sensitivity in single tumor marker detection, and TPS+CYFRA21-1+ CEA showed the highest sensitivity and accuracy in combined tumor marker detection for diagnosis of lung cancer in pleural effusion. The combined detection of CEA, CYFRA21-1 and TPS in pleural effusion showed higher sensitivity, better accuracy and higher clinical value than those in serum for diagnosis of lung cancer.

Rapid and broad detection of H5 hemagglutinin by an immunochromatographic kit using novel monoclonal antibody against highly pathogenic avian influenza virus belonging to the genetic clade 2.3.4.4.

PubMed

Nguyen, Lam Thanh; Nakaishi, Kazunari; Motojima, Keiko; Ohkawara, Ayako; Minato, Erina; Maruyama, Junki; Hiono, Takahiro; Matsuno, Keita; Okamatsu, Masatoshi; Kimura, Takashi; Takada, Ayato; Kida, Hiroshi; Sakoda, Yoshihiro

2017-01-01

Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds.

The Mutation-Associated Neoantigen Functional Expansion of Specific T cells (MANAFEST) assay: a sensitive platform for monitoring antitumor immunity.

PubMed

Danilova, Ludmila; Anagnostou, Valsamo; Caushi, Justina X; Sidhom, John-William; Guo, Haidan; Chan, Hok Yee; Suri, Prerna; Tam, Ada J; Zhang, Jiajia; El Asmar, Margueritta; Marrone, Kristen A; Naidoo, Jarushka; Brahmer, Julie R; Forde, Patrick M; Baras, Alexander S; Cope, Leslie; Velculescu, Victor E; Pardoll, Drew; Housseau, Franck; Smith, Kellie N

2018-06-12

Mutation-associated neoantigens (MANAs) are a target of antitumor T-cell immunity. Sensitive, simple, and standardized assays are needed to assess the repertoire of functional MANA-specific T cells in oncology. Assays analyzing in vitro cytokine production such as ELISpot and intracellular cytokine staining (ICS) have been useful but have limited sensitivity in assessing tumor-specific T-cell responses and do not analyze antigen-specific T-cell repertoires. The FEST (Functional Expansion of Specific T cells) assay described herein integrates TCR sequencing of short-term, peptide-stimulated cultures with a bioinformatic platform to identify antigen-specific clonotypic amplifications. This assay can be adapted for all types of antigens, including mutation associated neoantigens (MANAs) via tumor exome-guided prediction of MANAs. Following in vitro identification by the MANAFEST assay, the MANA-specific CDR3 sequence can be used as a molecular barcode to detect and monitor the dynamics of these clonotypes in blood, tumor, and normal tissue of patients receiving immunotherapy. MANAFEST is compatible with high-throughput routine clinical and lab practices. Copyright ©2018, American Association for Cancer Research.

Simultaneous multicolor detection system of the single-molecular microbial antigen by total internal reflection fluorescence microscopy with fluorescent nanocrystal quantum dots

NASA Astrophysics Data System (ADS)

Hoshino, Akiyoshi; Fujioka, Kouki; Yamamoto, Mayu; Manabe, Noriyoshi; Yasuhara, Masato; Suzuki, Kazuo; Yamamoto, Kenji

2005-11-01

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystals QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

Nylon bead enzyme-linked immunosorbent assay for detection of sub-picogram quantities of Brucella antigens.

PubMed Central

Perera, V Y; Creasy, M T; Winter, A J

1983-01-01

An indirect sandwich enzyme-linked immunosorbent assay, using antibody covalently coupled to nylon beads, has been adapted for the detection of Brucella antigens. Optimum conditions were achieved by incubation of 1 ml of reaction mixture with a single bead, and by minimizing nonspecific interactions through the use of beads coated with purified bovine antibodies, preabsorption of third layer rabbit antibodies with normal bovine serum, and treatment of beads with normal goat serum before addition of the goat anti-rabbit enzyme conjugate. Beta-galactosidase was selected for use with clinical samples primarily because of low levels of endogenous enzyme in bovine leukocytes. Use of a fluorogenic substrate enhanced sensitivity 20-fold. Under these conditions, 100 fg of solubilized crude lipopolysaccharide or 8 to 10 Brucella cells was detectable in a fixed volume of 1 ml. A system was also devised for concentrating antigen which permitted ready detection of 2 pg of lipopolysaccharide in a volume of 50 ml (40 fg/ml). Attempts to detect lipopolysaccharide in the presence of concentrated serum or plasma were unsuccessful, but 10 brucellae added to a suspension of leukocytes from 100 ml of normal bovine blood were easily measured. PMID:6415094

Semiquantitative Dot Blot with the GRA8 antigen to differentiate the stages of toxoplasmosis infection.

PubMed

Costa, Juan Gabriel; Vilariño, María Julia

2018-06-01

In this work we present a novel methodology to differentiate the phases of toxoplasmosis infection: the "semiquantitative Dot Blot". It is a simple technique that does not require expensive equipment, does not involve a long technique development, and can be used in a low-complexity laboratory. In this study, two recombinant sequences of Toxoplasma gondii GRA8 antigen were used, and specific IgG antibodies were detected in selected patient samples. This method makes it possible to obtain a score for each serum and define whether the patient is in the acute or chronic phase of the infection. The sensitivity and specificity results varied depending on the antigenic sequence used. With GRA8A, 62.1% and 72.7% were obtained, while with GRA8B, 82.8% and 72.1% were obtained, respectively. Although the sensitivity and specificity values were not close to 100%, they were similar to those reported with the same antigens in ELISA. Therefore, this quantitative technique would be a good alternative to ELISA. Copyright © 2018 Elsevier B.V. All rights reserved.

Tissue tropism of highly pathogenic avian influenza virus subtype H5N1 in naturally infected mute swans (Cygnus Olor ), domestic geese (Aser Anser var. domestica), pekin ducks (Anas platyrhynchos) and mulard ducks ( Cairina moschata x anas platyrhynchos).

PubMed

Szeredi, Levente; Dán, Adám; Pálmai, Nimród; Ursu, Krisztina; Bálint, Adám; Szeleczky, Zsófia; Ivanics, Eva; Erdélyi, Károly; Rigó, Dóra; Tekes, Lajos; Glávits, Róbert

2010-03-01

The 2006 epidemic due to highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Hungary caused the most severe losses in waterfowl which were, according to the literature at the time, supposed to be the most resistant to this pathogen. The presence of pathological lesions and the amount of viral antigen were quantified by gross pathology, histopathology and immunohistochemistry (IHC) in the organs of four waterfowl species [mute swans (n = 10), domestic geese (n = 6), mulard ducks (n = 6) and Pekin ducks (n = 5)] collected during the epidemic. H5N1 subtype HPAIV was isolated from all birds examined. Quantitative real-time reverse transcriptase-polymerase chain reaction (qRRT-PCR) was also applied on a subset of samples [domestic geese (n = 3), mulard (n = 4) and Pekin duck (n = 4)] in order to compare its sensitivity with IHC. Viral antigen was detected by IHC in all cases. However, the overall presence of viral antigen in tissue samples was quite variable: virus antigen was present in 56/81 (69%) swan, 22/38 (58%) goose, 28/46 (61%) mulard duck and 5/43 (12%) Pekin duck tissue samples. HPAIV subtype H5N1 was detected by qRRT-PCR in all birds examined, in 19/19 (100%) goose, 7/28 (25%) mulard duck and 12/28 (43%) Pekin duck tissue samples. As compared to qRRTPCR, the IHC was less sensitive in geese and Pekin ducks but more sensitive in mulard ducks. The IHC was consistently positive above 4.31 log10 copies/reaction but it gave very variable results below that level. Neurotropism of the isolated virus strains was demonstrated by finding the largest amount of viral antigen and the highest average RNA load in the brain in all four waterfowl species examined.

Deglycosylation of Toxocara excretory-secretory antigens improves the specificity of the serodiagnosis for human toxocariasis.

PubMed

Roldán, W H; Elefant, G R; Ferreira, A W

2015-11-01

Serodiagnosis of human toxocariasis is difficult in tropical areas where other helminthiasis are endemic. Many studies have shown that glycans from helminths may be the responsible for cross-reactions in the immunoassays. In this study, we have evaluated the deglycosylation of the Toxocara canis excretory-secretory (TES) antigens for the detection of IgG antibodies using a panel of 228 serum samples (58 patients with toxocariasis, 75 patients with other helminth infections and 95 healthy individuals) by ELISA and Western blot assays. Our results showed that the deglycosylation of TES antigens resulted in a single fraction of 26 kDa (dTES) and was able to detect IgG antibodies with a sensitivity and specificity of 100% in both above-mentioned assays. The rate of cross-reactions, observed in ELISA with TES (13·3%), was significantly reduced (5·3%) when the dTES antigens were used. Likewise, the cross-reactivity observed with the fractions of 32, 55 and 70 kDa of the TES antigens was totally eliminated when the dTES were used in the Western blot. All these results showed that the deglycosylation of the TES antigens really improves the specificity of the serodiagnosis of human toxocariasis in endemic areas for helminth infections. © 2015 John Wiley & Sons Ltd.

Validation of an enzyme-linked immunosorbent assay that detects Histoplasma capsulatum antigenuria in Colombian patients with AIDS for diagnosis and follow-up during therapy.

PubMed

Caceres, Diego H; Scheel, Christina M; Tobón, Angela M; Ahlquist Cleveland, Angela; Restrepo, Angela; Brandt, Mary E; Chiller, Tom; Gómez, Beatriz L

2014-09-01

We validated an antigen capture enzyme-linked immunosorbent assay (ELISA) in Colombian persons with AIDS and proven histoplasmosis and evaluated the correlation between antigenuria and clinical improvement during follow-up. The sensitivity of the Histoplasma capsulatum ELISA was 86%, and the overall specificity was 94%. The antigen test successfully monitored the response to therapy. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Performance of pfHRP2 versus pLDH antigen rapid diagnostic tests for the detection of Plasmodium falciparum: a systematic review and meta-analysis.

PubMed

Li, Bo; Sun, Zhiqiang; Li, Xiaohan; Li, Xiaoxi; Wang, Han; Chen, Weijiao; Chen, Peng; Qiao, Mengran; Mao, Yuanli

2017-04-01

There have been many inconsistent reports about the performance of histidine-rich protein 2 (HRP2) and lactate dehydrogenase (LDH) antigens as rapid diagnostic tests (RDTs) for the diagnosis of past Plasmodium falciparum infections. This meta-analysis was performed to determine the performance of pfHRP2 versus pLDH antigen RDTs in the detection of P. falciparum . After a systematic review of related studies, Meta-DiSc 1.4 software was used to calculate the pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). Forest plots and summary receiver operating characteristic curve (SROC) analysis were used to summarize the overall test performance. Fourteen studies which met the inclusion criteria were included in the meta-analysis. The summary performances for pfHRP2- and pLDH-based tests in the diagnosis of P. falciparum infections were as follows: pooled sensitivity, 96.3% (95.8-96.7%) vs. 82.6% (81.7-83.5%); specificity, 86.1% (85.3-86.8%) vs. 95.9% (95.4-96.3%); diagnostic odds ratio (DOR), 243.31 (97.679-606.08) vs. 230.59 (114.98-462.42); and area under ROCs, 0.9822 versus 0.9849 (all p < 0.001). The two RDTs performed satisfactorily for the diagnosis of P. falciparum , but the pLDH tests had higher specificity, whereas the pfHRP2 tests had better sensitivity. The pfHRP2 tests had slightly greater accuracy compared to the pLDH tests. A combination of both antigens might be a more reliable approach for the diagnosis of malaria.

Hybridoma cell agglutination as a novel test to detect circulating antigen of Schistosoma japonicum.

PubMed

Li, Yong-Long; Liu, Wenqi; Ruppel, Andreas

2003-01-01

We developed a serodiagnostic test which is based on the agglutination of hybridoma cells. In the presence of specific antigen, agglutination of the fixed and stained cells occurs and can be visualized in analogy to traditional erythrocyte agglutination. The procedures were developed with a murine cell line producing a monoclonal antibody against a schistosome gut protein and sera of patients and mice infected with Schistosoma japonicum. This test is capable of detecting circulating antigen during pre-patency in mice infected with 50 cercariae. Its sensitivity was high with acute schistosomiasis japonica (97%, n = 32) and moderate with chronic cases (75%, n = 57). No positive reactions were obtained with healthy persons (n = 78) or patients infected with other parasites (Chlonorchis sinensis, n = 20; Paragonimus westermani, n = 20; Plasmodium vivax, n = 10) or suffering from lupus erythomatodus (n = 5) or mononucleosis (n = 10).

[Imaging of surface cell antigens on the tumor sections of lymph nodes using fluorescence quantum dots].

PubMed

Rafalovskaia-Orlovskaia, E P; Gorgidze, L A; Gladkikh, A A; Tauger, S M; Vorob'ev, I A

2012-01-01

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.

High-contrast grating resonators for label-free detection of disease biomarkers

PubMed Central

Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

2016-01-01

A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI. PMID:27265624

High-contrast grating resonators for label-free detection of disease biomarkers

NASA Astrophysics Data System (ADS)

Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

2016-06-01

A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI.

Antigen retrieval, blocking, detection and visualisation systems in immunohistochemistry: a review and practical evaluation of tyramide and rolling circle amplification systems.

PubMed

Warford, Anthony; Akbar, Hameed; Riberio, Deise

2014-11-01

To achieve specificity and sensitivity using immunohistochemistry it is necessary to combine the application of validated primary antibodies with optimised pre-treatment, detection and visualisation steps. The influence of these surrounding procedures is reviewed. A practical evaluation of tyramide signal amplification and rolling circle amplification detection methods is provided in which formalin fixed paraffin embedded sections of adenocarcinomas of breast, colon and lung together with squamous metaplasia of lung were immunostained with CD20 and CK19 primary antibodies. The results indicate that the detection systems are of comparable sensitivity and specificity. Copyright © 2014 Elsevier Inc. All rights reserved.

Which is the best method to trace group A streptococci in sore throat patients: culture or GAS antigen test?

PubMed

Lindbaek, Morten; Høiby, Ernst Arne; Lermark, Gro; Steinsholt, Inger Marie; Hjortdahl, Per

2004-12-01

To compare an antigen detection test (GAS antigen test) with the results from combinations of two various bacteriological test media in general practice patients with sore throat. Furthermore to assess the diagnostic properties of the chosen GAS antigen test and to compare semi-quantitative results of this test with the bacterial load found in the throat culture. Two Norwegian general practices in Stokke and Kongsberg communities. 306 patients with sore throat lasting less than 7 days; 244 were adults, 62 were children under 10 years old, mean age 23.9 years (SD 15.0), 40% were men. Results from GAS antigen test, and distribution of bacteriological findings in throat cultures, compared with the results of our GAS antigen test; semi-quantitative results of the GAS antigen test compared with the bacterial load by culture. In the primary culture 110 patients harboured group A streptococci (GAS) infection, while the second culture identified another 17, giving a total of 127 patients. Some 33 patients harboured large-colony groups C and G. The GAS antigen test used had a sensitivity of 97% and specificity of 95% regarding GAS when compared with the two cultures. We found a significant correlation between the bacterial loads by culture and the semi-quantitative results of the GAS antigen test. By using a second, different set of bacteriological media, we identified an additional 17 patients with GAS infections. This raises the question of validity of frequently used reference standards in studies related to streptococcal infections. Compared with the combined results of the two throat cultures, the GAS antigen test used showed high sensitivity and specificity. Semi-quantitative evaluations of the rapid immunological test may also be of clinical value.

Application of a SERS-based lateral flow immunoassay strip for the rapid and sensitive detection of staphylococcal enterotoxin B

NASA Astrophysics Data System (ADS)

Hwang, Joonki; Lee, Sangyeop; Choo, Jaebum

2016-06-01

A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as determined with the SERS-based LFA strip, was estimated to be 0.001 ng mL-1. This value is approximately three orders of magnitude more sensitive than that achieved with the corresponding ELISA-based method. The proposed SERS-based LFA strip sensor shows significant potential for the rapid and sensitive detection of target markers in a simplified manner.A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as determined with the SERS-based LFA strip, was estimated to be 0.001 ng mL-1. This value is approximately three orders of magnitude more sensitive than that achieved with the corresponding ELISA-based method. The proposed SERS-based LFA strip sensor shows significant potential for the rapid and sensitive detection of target markers in a simplified manner. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07243c

Design of Indigenous ELISA Using Tachyzoites from the RH Strain of Toxoplasma gondii and Comparison with Commercial Kits in Ahvaz, Southwest of Iran, 2015.

PubMed

Mohammadpour, Niloofar; Saki, Jasem; Rafiei, Abdollah; Khodadadi, Ali; Tavalla, Mehdi; Cheraghian, Bahman

2016-10-01

Toxoplasma gondii is one of the most common causes of latent infections in humans worldwide. Detecting anti- Toxoplasma antibodies in serum using serological tests is a common method to diagnose toxoplasmosis. In the present study, an indigenous ELISA kit was prepared using tachyzoites from the RH strain of T. gondii , and its sensitivity and specificity were compared with those of commercial kits. To produce antigens, 0.02 mL of locally isolated T. gondii RH strain parasites along with 10 9 tachyzoites were injected into the peritoneal cavities of 50 laboratory mice (BALB/C). Parasites were collected after 4 days. After filtering and washing, the concentration of protein in sonicated tachyzoites was calculated using the Lowry protein assay. The dilution of antigen, serum and alkaline phosphatase conjugate was assessed in designing an indigenous ELISA method; then ELISA was performed based on these dilutions, and its sensitivity was determined using 200 serum samples. In addition, the specificity of the assay was evaluated using 40 serum samples from patients with tuberculosis, leukemia or hydatid cyst. Indigenous ELISA was used to examine 100 serum samples containing anti- T. gondii IgG, with a sensitivity of 98% (commercial kits: 100%). Another 100 serum samples containing anti- T. gondii IgM were also tested, with a sensitivity of 99% (commercial kits: 100%). When 40 serum samples from patients with leukemia, hydatid cyst or tuberculosis were examined using anti- T. gondii IgG, the specificity was 100%, identical to commercial kits. However, the specificity of a similar test with anti- T. gondii IgM was just 28.6% for serum samples from leukemia patients, 21.4% for hydatid cyst and 16.7% for tuberculosis. We found that purified locally isolated soluble crude antigens of the RH strain of T. gondii from the peritoneal cavity of mice may be one of the most promising antigens for detection of human toxoplasmosis in routine screening.

Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

PubMed

Gray, Sean A; Weigel, Kris M; Ali, Ibne K M; Lakey, Annie A; Capalungan, Jeremy; Domingo, Gonzalo J; Cangelosi, Gerard A

2012-01-01

The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

Detection of prostate specific antigen (PSA) in human saliva using an ultra-sensitive nanocomposite of graphene nanoplatelets with diblock-co-polymers and Au electrodes.

PubMed

Khan, M S; Dighe, K; Wang, Z; Srivastava, I; Daza, E; Schwartz-Dual, A S; Ghannam, J; Misra, S K; Pan, D

2018-02-26

Prostate-specific antigen (PSA) is a commonly used biomarker for the detection of prostate cancer (PCa) and there are numerous data available for its invasive detection in the serum and whole blood. In this work, an electrochemical sensing method was devised to detect traces of PSA in human saliva using a hybrid nanocomposite of graphene nanoplatelets with diblock co-polymers and Au electrodes (GRP-PS 67 -b-PAA 27 -Au). The pure graphitic composition on filter paper provides significantly high electrical and thermal conductivity while PS 67 -b-PAA 27 makes an amphiphilic bridge between GRP units. The sensor utilizes the binding of an anti-PSA antibody with an antigen-PSA to act as a resistor in a circuit providing an impedance change that in turn allows for the detection and quantification of PSA in saliva samples. A miniaturized electrical impedance analyzer was interfaced with a sensor chip and the data were recorded in real-time using a Bluetooth-enabled module. This fully integrated and optimized sensing device exhibited a wide PSA range of detection from 0.1 pg mL -1 to 100 ng mL -1 (R 2 = 0.963) with a lower limit of detection of 40 fg mL -1 . The performance of the biosensor chip was validated with an enzyme-linked immunosorbent assay technique with a regression coefficient as high as 0.940. The advantages of the newly developed saliva-PSA electrical biosensor over previously reported serum-PSA electrochemical biosensors include a faster response time (3-5 min) to achieve a stable electrical signal for PSA detection, high selectivity, improved sensitivity, no additional requirement of a redox electrolyte for electron exchange and excellent shelf life. The presented sensor is aimed for clinical commercialization to detect PSA in human saliva.

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

PubMed

Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

2016-01-01

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

IMMUNOGLOBULIN ISOANTIGENS (ALLOTYPES) IN THE MOUSE

PubMed Central

Herzenberg, Leonard A.; Warner, Noel L.; Herzenberg, Leonore A.

1965-01-01

Eight antigens of 7S γ2-immunoglobulins controlled by alleles at a single locus Ig-1, have been identified in mice. This locus has previously been shown to determine antigenic specificities on the F fragments of 7S γ2a-globulins. The reactions of these antigens with various isoantisera have shown that the antigens all cross react with one another. New methods for the analysis of antigenic specificities of soluble proteins are presented in detail. A sensitive method for detecting in the order of 0.01 µg of these isoantigens has been developed, based on the quantitative inhibition of precipitation of I125-labeled antigen. Cross-reactions of the antigens were analysed in inhibition assays and the data is compatible with the existence of a minimum of eight antigenic specificities. Each of the antigens is composed of different combinations of these specificities, with only one antigen having a specificity not present in any other. Sixty-eight mouse strains have been tested with specific isoantisera, and on the basis of the results, have been placed into the eight allele groups. Evidence for close genetic linkage of the Ig-1 locus and 11 chromosome markers has been sought and not found. PMID:14270242

Diagnosis of hypersensitivity pneumonitis by measurement of antibodies against environmental antigens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dewair, M.

1989-01-01

Hypersensitivity pneumonitis (HP), an immunologically mediated chronic pulmonary disease, is the result of an inflammatory response of the lung initiated by the inhalation of environmental organic dusts. These organic dusts usually contain substances (antigens) capable of eliciting immune responses in humans. The symptoms of HP generally present as recurrent flu-like episodes which makes it difficult to establish the proper diagnosis. However, detection in patients' sera of high-titer antibodies against the environmental antigens could be of great help in identifying those materials causing the disease and which must be avoided. A highly specific and sensitive serodiagnostic test, a radioimmuno assay (RIA),more » was developed for measurement of antibodies against antigens relevant to Farmer's Lung Disease (FLD), a type of HP affecting farmers.« less

Recombinant gp19 as a potential antigen for detecting anti-Ehrlichia canis antibodies in dog sera.

PubMed

Oliveira, Rômulo Silva de; Cunha, Rodrigo Casquero; Moraes-Filho, Jonas; Gonçales, Relber Aguiar; Lara, Ana Paula de Souza Stori de; Avila, Luciana Farias da Costa de; Labruna, Marcelo Bahia; Leite, Fábio Pereira Leivas

2015-01-01

The canine monocytic ehrlichiosis, caused by Ehrlichia canis, is endemic in several regions of Brazil. Some serological diagnostic techniques using immunodominant proteins of E. canis as antigens are available, but their specificities and sensitivities are questionable. Based on this, the objective of this study was to test the antigenic potential of the recombinant gp19 protein (rGP19) for subsequent use in diagnostic tests. The rGP19 expressed in the Escherichia coli strain BL21 (DE3) C41 was recognized in the sera from experimentally infected dogs using ELISA and Western blotting. Thus, it was possible to obtain a promising antigen with the ability to differentiate between E. canis-positive and -negative animals, even 1 week after infection.

Determination by enzyme-linked immunosorbent assay of immunoglobulin G (IgG), IgM, and IgA to Brucella melitensis major outer membrane proteins and whole-cell heat-killed antigens in sera of patients with brucellosis.

PubMed Central

Araj, G F; Kaufmann, A F

1989-01-01

An enzyme-linked immunosorbent assay was used to compare Brucella melitensis major outer membrane proteins (MOMP) and whole-cell heat-killed antigens (HK) in measuring antibrucella immunoglobulin G (IgG), IgM, and IgA in sera of brucellosis patients and controls. Antibodies to MOMP were generally similar to those against HK, and the correlation coefficients between the two antigens and IgG, IgM, and IgA in patients varied between 0.73 and 0.94. Both antigens are comparably suitable in detecting antibrucella immunoglobulin isotypes for the serologic diagnosis of patients with brucellosis, with high (greater than or equal to 95%) sensitivity and specificity. PMID:2768476

Eliciting an antibody response against a recombinant TSH containing fusion protein.

PubMed

Mard-Soltani, Maysam; Rasaee, Mohamad Javad; Sheikhi, AbdolKarim; Hedayati, Mehdi

2017-01-01

Designing novel antigens to rise specific antibodies for Thyroid Stimulating Hormone (TSH) detection is of great significance. A novel fusion protein consisting of the C termini sequence of TSH beta subunit and a fusion sequence was designed and produced for rabbit immunization. Thereafter, the produced antibodies were purified and characterized for TSH detection. Our results indicate that the produced antibody is capable of sensitive and specific detection of TSH with low cross reactivity. This study underscores the applicability of designed fusion protein for specific and sensitive polyclonal antibody production and the importance of selecting an amenable region of the TSH for immunization.

Antigen based detection of cystic echinococcosis in buffaloes using ELISA and Dot-EIA.

PubMed

Sangaran, A; Bino Sundar, S T; Latha, Bhaskaran Ravi

2017-03-01

Cystic echinococcosis is caused by the larval stage of the dog tapeworm, Echinococcus granulosus . The disease is recognized as one of the world's major zoonoses affecting human beings and domestic animals apart from its economic and public health importance. Development of the cysts in the intermediate host such as buffaloes occurs in the lungs, liver and other organs. In this study, detection of circulating antigen in the diagnosis of cystic echinococcosis in buffaloes was done using enzyme linked immunosorbent assay and Dot-Enzyme immunoassay (Dot-EIA). The sensitivity and specificity were determined as 89 and 92 % respectively, whereas those of Dot-EIA were determined as 94 and 96 %.

[Early diagnosis of streptococcal pharyngitis in paediatric practice: Validity of a rapid antigen detection test].

PubMed

Flores Mateo, Gemma; Conejero, Jaume; Grenzner Martinel, Elisabet; Baba, Zeki; Dicono, Susana; Echasabal, Mildrey; Gonzalo Santos, Concepción; Aliaga, Arantxa; Barredo, María; Ruiz, Luis; Carrau, Montserrat

2010-07-01

To determine the validity of the rapid antigen test for the diagnoses of acute pharyngitis caused by group A beta-haemolytic Streptococcus (GABHS) compared with culture. Observational study of a consecutive sample of paediatric patients. Two primary care centres (PCC) from the metropolitan area of Barcelona. Children aged 1-14 years with sore throat of no more than 5 days duration were chosen at PCC. Oropharyngeal samples were collected from tonsillar bed and posterior pharynx. A rapid diagnostic test was performed, as well as a throat culture. A total of 211 patients were studied. The overall prevalence of pharyngitis due to Streptococcus was 34.1%. Compared with the throat culture, the sensitivity of the rapid test was 90.3% (95% CI: 81.0-96.0), the specificity was 78.4% (95% CI: 70.6-84.9). The percentage of false negatives was 9.7% and the false positives was 21.6%. Spectrum bias was present, inasmuch as the rapid test sensitivity increased with Centor scores. The diagnostic value of a rapid antigen test for the diagnosis of streptococcal pharyngitis in paediatric patients at PCC is high. However, the percentage of false positives and negatives is too high, and also the sensitivity is too low in patients with fewer symptoms to support the use of rapid antigenic test without culture confirmation and bacterial sensitivity test. 2009 Elsevier España, S.L. All rights reserved.

Identification of anti-SF3B1 autoantibody as a diagnostic marker in patients with hepatocellular carcinoma.

PubMed

Hwang, Hai-Min; Heo, Chang-Kyu; Lee, Hye Jung; Kwak, Sang-Seob; Lim, Won-Hee; Yoo, Jong-Shin; Yu, Dae-Yuel; Lim, Kook Jin; Kim, Jeong-Yoon; Cho, Eun-Wie

2018-06-28

Tumor-associated (TA) autoantibodies, which are generated by the immune system upon the recognition of abnormal TA antigens, are promising biomarkers for the early detection of tumors. In order to detect autoantibody biomarkers effectively, antibody-specific epitopes in the diagnostic test should maintain the specific conformations that are as close as possible to those presenting in the body. However, when using patients' serum as a source of TA autoantibodies the characterization of the autoantibody-specific epitope is not easy due to the limited amount of patient-derived serum. To overcome these limits, we constructed a B cell hybridoma pool derived from a hepatocellular carcinoma (HCC) model HBx-transgenic mouse and characterized autoantibodies derived from them as tumor biomarkers. Their target antigens were identified by mass spectrometry and the correlations with HCC were examined. With the assumption that TA autoantibodies generated in the tumor mouse model are induced in human cancer patients, the enzyme-linked immunosorbent assays (ELISA) based on the characteristics of mouse TA autoantibodies were developed for the detection of autoantibody biomarkers in human serum. To mimic natural antigenic structures, the specific epitopes against autoantibodies were screened from the phage display cyclic random heptapeptide library, and the streptavidin antigens fused with the specific epitopes were used as coating antigens. In this study, one of HCC-associated autoantibodies derived from HBx-transgenic mouse, XC24, was characterized. Its target antigen was identified as splicing factor 3b subunit 1 (SF3B1) and the high expression of SF3B1 was confirmed in HCC tissues. The specific peptide epitopes against XC24 were selected and, among them, XC24p11 cyclic peptide (-CDATPPRLC-) was used as an epitope of anti-SF3B1 autoantibody ELISA. With this epitope, we could effectively distinguish between serum samples from HCC patients (n = 102) and healthy subjects (n = 85) with 73.53% sensitivity and 91.76% specificity (AUC = 0.8731). Moreover, the simultaneous detection of anti-XC24p11 epitope autoantibody and AFP enhanced the efficiency of HCC diagnosis with 87.25% sensitivity and 90.59% specificity (AUC = 0.9081). ELISA using XC24p11 peptide epitope that reacts against anti-SF3B1 autoantibody can be used as a novel test to enhance the diagnostic efficiency of HCC.

Surface plasmon-enhanced fluorescence on Au nanohole array for prostate-specific antigen detection

PubMed Central

Zhang, Qingwen; Wu, Lin; Wong, Ten It; Zhang, Jinling; Liu, Xiaohu; Zhou, Xiaodong; Bai, Ping; Liedberg, Bo; Wang, Yi

2017-01-01

Localized surface plasmon (LSP) has been widely applied for the enhancement of fluorescence emission for biosensing owing to its potential for strong field enhancement. However, due to its small penetration depth, LSP offers limited fluorescence enhancement over a whole sensor chip and, therefore, insufficient sensitivity for the detection of biomolecules, especially large molecules. We demonstrate the simultaneous excitation of LSP and propagating surface plasmon (PSP) on an Au nanohole array under Kretschmann configuration for the detection of prostate-specific antigen with a sandwich immunoassay. The proposed method combines the advantages of high field enhancement by LSP and large surface area probed by PSP field. The simulated results indicated that a maximum enhancement of electric field intensity up to 1,600 times can be achieved under the simultaneous excitation of LSP and PSP modes. The sandwich assay of PSA carried out on gold nanohole array substrate showed a limit of detection of 140 fM supporting coexcitation of LSP and PSP modes. The limit of detection was approximately sevenfold lower than that when only LSP was resonantly excited on the same substrate. The results of this study demonstrate high fluorescence enhancement through the coexcitation of LSP and PSP modes and pave a way for its implementation as a highly sensitive bioassay. PMID:28392689

Rapid and ultrasensitive flexible palladium nano-thin film biosensing electrode development for cancer antigen HER2 detection

NASA Astrophysics Data System (ADS)

Huang, Yun-Tzu; Chang, Chia-Yu; Chen, Wei; Su, Chien-Hao; Hsu, Guo-Cheng; Chang, Chia-Ching

HER2 (human epidermal growth factor receptor 2) is one of the significant surface antigens of breast cancer Trace amount of HER2 protein in human serum is highly correlated to the tumor progression in breast cancers especially in the cases of recurrence. Therefore, HER2 detection of human serum is significant for early detection of cancer recurrence. Conventional HER2 detection approaches may not be sensitive enough or contain highly false positive rate or time consuming for accurate detection. Therefore, a rapid, highly sensitive and specific sensing is highly desired. By using HER2 specific binding peptide functionalized palladium thin film electrochemical electrode the HER2 protein concentration can be determined at sub-nanogram level by electrochemical impedance spectroscopy (EIS) within 10 mins. The Pd nano-film is sputtered on the flexible plastics substrate and reduces the cost of this electrode. Due to the low cost of the electrode, it is designed as a disposable biosensing probe which may reduce the concern of human sample contamination. The self-management after breast cancer operation may be feasible in the near future. Keywords: Electrochemical impedance spectroscopy(EIS), breast cancer, biosensor Corresponding author: ccchang01@faculty.nctu.edu.tw; Cheeshin Technology Co. Collaboration.

On the importance of controlling film architecture in detecting prostate specific antigen

NASA Astrophysics Data System (ADS)

Graça, Juliana Santos; Miyazaki, Celina Massumi; Shimizu, Flavio Makoto; Volpati, Diogo; Mejía-Salazar, J. R.; Oliveira, Osvaldo N., Jr.; Ferreira, Marystela

2018-03-01

Immunosensors made with nanostructured films are promising for detecting cancer biomarkers, even at early stages of the disease, but this requires control of film architecture to preserve the biological activity of immobilized antibodies. In this study, we used electrochemical impedance spectroscopy (EIS) to detect Prostate Specific Antigen (PSA) with immunosensors produced with layer-by-layer (LbL) films containing anti-PSA antibodies in two distinct film architectures. The antibodies were either adsorbed from solutions in which they were free, or from solutions where they were incorporated into liposomes of dipalmitoyl phosphatidyl glycerol (DPPG). Incorporation into DPPG liposomes was confirmed with surface plasmon resonance experiments, while the importance of electrostatic interactions on the electrical response was highlighted using the Finite Difference Time-Domain Method (FDTD). The sensitivity of both architectures was sufficient to detect the threshold value to diagnose prostate cancer (ca. 4 ng mL-1). In contrast to expectation, the sensor with the antibodies incorporated into DPPG liposomes had lower sensitivity, though the range of concentrations amenable to detection increased, according to the fitting of the EIS data using the Langmuir-Freundlich adsorption model. The performance of the two film architectures was compared qualitatively by plotting the data with a multidimensional projection technique, which constitutes a generic approach for optimizing immunosensors and other types of sensors.

Immunodiagnostic monoclonal antibody-based sandwich ELISA of fasciolosis by detection of Fasciola gigantica circulating fatty acid binding protein.

PubMed

Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

2016-09-01

Up to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinant Fasciola gigantica fatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected with F. gigantica. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL-1, and no cross-reaction with other parasite antigens was observed. This assay could detect F. gigantica infection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection.

PubMed

Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

2016-06-01

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

HLA-typing analysis following allogeneic bone grafting for sinus lifting.

PubMed

Piaia, Marcelo; Bub, Carolina Bonet; Succi, Guilherme de Menezes; Torres, Margareth; Costa, Thiago Henrique; Pinheiro, Fabricio Costa; Napimoga, Marcelo Henrique

2017-03-01

According to the Brazilian Association of Organ Transplants, in 2015, 19,408 bone transplants were performed in Brazil, over 90% by Dental Surgeons. The surgical technique itself has a respectable number of reports regarding its clinical efficacy, as measured by long-term survival of dental implants in grafted areas. Uncertainty remains, however, as to whether fresh frozen grafts from human bone donors remain immunologically innocuous in the body of the host. Six male with no previous medical history of note, including systemic diseases, surgery or blood transfusion were selected. These patients underwent reconstructive procedures (sinus lifting) using fresh frozen human bone from a tissue bank. All patients had venous blood samples collected prior to surgery and 6 months after the procedure. Anti-HLA analysis for the detection of HLA (human leukocyte antigen) antibodies was performed using methods such as the LABScreen PRA Class I and Class II, LABScreen Single Antigen Class I and Class II, Luminex Platform. Reactive individuals to the screening tests (LABScreen PRA) were further investigated to determine the specificity of the antibodies detected (LABScreen Single Antigen) with a cutoff value of median fluorescence intensity ≥500. As a result, it was observed that two patients (33%) were positive in screening tests, one presenting with anti-HLA Class I and II sensitization and the other with anti-HLA class II. The specificity analysis showed that the patients sensitized to HLA class II presented 4 specificities, 3 of which immunologically relevant. In the second individual, 23 specificities were identified, 6 of which immunologically important for HLA class I and 4 specificities for HLA class II, 3 of these were immunologically important. All specificities detected had average fluorescence. These findings are suggestive that sinus-lifting procedures with allogeneic bone can induce immunological sensitization.

A sandwich-type optical immunosensor based on the alkaline phosphatase enzyme for Salmonella thypimurium detection

NASA Astrophysics Data System (ADS)

Widyastuti, E.; Puspitasari Schonherr, M. F.; Masruroh, A.; Anggraeni, R. A.; Nisak, Y. K.; Mursidah, S.

2018-03-01

Salmonella is pathogenic bacteria that caused foodborne diseases which being called Salmonellosis. Prevalence of Salmonellosis that being caused by Salmonella thypimurium in Indonesia is quite high. However, detection of Salmonella bacteria in food still limited, complicated, and required a lot time. Sensitive optical assay for Salmonella thypimurium paper based detection has been developed by integrating sandwich assay between antibody-antigen complex and alkaline phosphatase enzyme that produce visible bluish-purple colour with presence of NBT-BCIP substrate. The results showed that Limit of Quantitation of detection is 105 CFU mL-1 with detection time 15 minutes. Linearity test between Colour intensity that produced from Salmonella concentration presence on samples showed that detection has good linearity. Selectivity test exhibited excellent sensitivity with good discrimination against Escherichia coli.

Comparative evaluation of commercially available point-of-care heartworm antigen tests using well-characterized canine plasma samples.

PubMed

Starkey, Lindsay A; Bowles, Joy V; Payton, Mark E; Blagburn, Byron L

2017-11-09

Dirofilaria immitis is a worldwide parasite that is endemic in many parts of the United States. There are many commercial assays available for the detection of D. immitis antigen, one of which was modified and has reentered the market. Our objective was to compare the recently reintroduced Witness® Heartworm (HW) Antigen test Kit (Zoetis, Florham Park, NJ) and the SNAP® Heartworm RT (IDEXX Laboratories, Inc., Westbrook, ME) to the well-based ELISA DiroChek® Heartworm Antigen Test Kit (Zoetis, Florham Park, NJ). Canine plasma samples were either received at the Auburn Diagnostic Parasitology Laboratory from veterinarians submitting samples for additional heartworm testing (n = 100) from 2008 to 2016 or purchased from purpose-bred beagles (n = 50, presumed negative) in 2016. Samples were categorized as "positive," "borderline" or "negative" using our established spectrophotometric cutoff value with the DiroChek® assay when a sample was initially received and processed. Three commercially available heartworm antigen tests (DiroChek®, Witness® HW, and SNAP® RT) were utilized for simultaneous testing of the 150 samples in random order as per their package insert with the addition of spectrophotometric optical density (OD) readings of the DiroChek® assay. Any samples yielding discordant test results between assays were further evaluated by heat treatment of plasma and retesting. Chi-square tests for the equality of proportions were utilized for statistical analyses. Concordant results occurred in 140/150 (93.3%) samples. Discrepant results occurred in 10/150 samples tested (6.6%): 9/10 occurring in the borderline heartworm (HW) category and 1/10 occurring in the negative HW category. The sensitivity and specificity of each test compared to the DiroChek® read by spectrophotometer was similar to what has been reported previously (Witness®: sensitivity 97.0% [94.1-99.4%], specificity 96.4% [95.5-100.0%]; SNAP® RT: sensitivity 90.9% [78.0-100.0%], specificity 98.8% [96.0-100.0%]). There were significant differences detected when comparing the sensitivities of the SNAP® RT and the Witness® HW to the DiroChek® among the 150 total samples (p = 0.003) and the 50 "borderline" samples (p = 0.001). In this study, the sensitivity of the Witness® HW was higher than the sensitivity of the SNAP® RT when compared with the DiroChek® test results prior to heat treatment of samples.

Top-down nanofabrication of silicon nanoribbon field effect transistor (Si-NR FET) for carcinoembryonic antigen detection.

PubMed

Bao, Zengtao; Sun, Jialin; Zhao, Xiaoqian; Li, Zengyao; Cui, Songkui; Meng, Qingyang; Zhang, Ye; Wang, Tong; Jiang, Yanfeng

2017-01-01

Sensitive and quantitative detection of tumor markers is highly required in the clinic for cancer diagnosis and consequent treatment. A field-effect transistor-based (FET-based) nanobiosensor emerges with characteristics of being label-free, real-time, having high sensitivity, and providing direct electrical readout for detection of biomarkers. In this paper, a top-down approach is proposed and implemented to fulfill a novel silicon nano-ribbon FET, which acts as biomarker sensor for future clinical application. Compared with the bottom-up approach, a top-down fabrication approach can confine width and length of the silicon FET precisely to control its electrical properties. The silicon nanoribbon (Si-NR) transistor is fabricated on a Silicon-on-Insulator (SOI) substrate by a top-down approach with complementary metal oxide semiconductor (CMOS)-compatible technology. After the preparation, the surface of Si-NR is functionalized with 3-aminopropyltriethoxysilane (APTES). Glutaraldehyde is utilized to bind the amino terminals of APTES and antibody on the surface. Finally, a microfluidic channel is integrated on the top of the device, acting as a flowing channel for the carcinoembryonic antigen (CEA) solution. The Si-NR FET is 120 nm in width and 25 nm in height, with ambipolar electrical characteristics. A logarithmic relationship between the changing ratio of the current and the CEA concentration is measured in the range of 0.1-100 ng/mL. The sensitivity of detection is measured as 10 pg/mL. The top-down fabricated biochip shows feasibility in direct detecting of CEA with the benefits of real-time, low cost, and high sensitivity as a promising biosensor for tumor early diagnosis.

Pitfalls in Serological Diagnosis of Cryptococcus gattii Infections.

PubMed

Tintelnot, Kathrin; Hagen, Ferry; Han, Chang Ok; Seibold, Michael; Rickerts, Volker; Boekhout, Teun

2015-11-01

The detection of cryptococcal antigen by latex agglutination tests (LATs), enzyme-linked immunoassays (ELISA), or lateral flow assay (LFA) is an important tool for diagnosis of a Cryptococcus infection. Cerebrospinal fluid and/or serum samples of 10 patients with cryptococcosis due to Cryptococcus gattii or a hybrid of Cryptococcus neoformans and C. gattii were examined by three LATs (the IMMY Latex-Crypto(®) test, the Pastorex(TM) Crypto Plus, and the Remel Cryptococcus Antigen Test Kit) and the LFA made by Immuno-Mycologics. LATs based on monoclonal antibodies (mAbs) like the Pastorex(TM) Crypto Plus or the Remel Cryptococcus Antigen Test Kit turned out to have an insufficient sensitivity to detect four out of 10 C. gattii infections, including one infection by a hybrid between C. gattii and C. neoformans. Reflecting the ongoing expansion of C. gattii in geographical zones outside of tropical and subtropical areas like Mediterranean countries, Vancouver Island (British Columbia, Canada) and the Pacific Northwest region (USA), these findings are alarming because of the risk of delayed diagnosis of infections caused by C. gattii. Therefore, the preliminary serological screening for cryptococcal antigen in the case of a suspected Cryptococcus infection should be performed by using an assay with a broad range specificity and sensitivity for C. neoformans and C. gattii, including their hybrids. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Using the AD12-ICT rapid-format test to detect Wuchereria bancrofti circulating antigens in comparison to Og4C3-ELISA and nucleopore membrane filtration and microscopy techniques.

PubMed

El-Moamly, Amal Abdul-Rasheed; El-Sweify, Mohamed Aly; Hafez, Mohamad Abdul

2012-09-01

Lymphatic filariasis (LF) continues to be a major source of permanent disability and an impediment to socio-economic development in 73 countries where more than 1 billion people are at risk and over 120 millions are infected. The global drive to eliminate LF necessitates an increasing demand for valid, reliable and rapid diagnostic tests. This study aimed to assess the performance of the AD12 rapid format immunochromatographic test (ICT) to detect Wuchereria bancrofti circulating antigens, against the combined gold standard: TropBio Og4C3-ELISA (enzyme-linked immunosorbent assay) which detects circulating filarial antigen (CFA) and the nucleopore membrane filtration and microscopic examination. This prospective case-control study involved 647 asymptomatic migrant workers from filariasis-endemic countries. Of these specimens, 32 were positive for microfilaremia using the membrane filtration and microscopy, 142 positive by ELISA (of which 32 had microfilaremia), and 128 positive by the ICT (of which 31 had microfilaremia). The performance of the ICT was calculated against 32 true-positive and 90 true-negative cases. For the detection of CFA, the ICT had a sensitivity of 97% (95% confidence interval [CI] 91-103), specificity 100% (95% CI 100-100), Positive Predictive Value (PPV) 100% (95% CI 100-100), Negative Predictive Value (NPV) 99% (95% CI 97-101); and the total accuracy of the test was 99% (95% CI 98-101). The agreement between ICT and ELISA in detecting W. bancrofti antigens was excellent (kappa = 0.934; p = 0.000). In conclusion, the AD12-ICT test for the detection of W. bancrofti-CFA was sensitive and specific and comparable to the performance of ELISA. The ICT would be a useful additional test to facilitate the proposed strategies for control and elimination of LF. Because it is rapid, simple to perform, and does not require the use of special equipment, the ICT may be most appropriate in screening programs and in monitoring the possible risk of introducing the disease to the non-endemic countries.

Polyhydroquinone-graphene composite as new redox species for sensitive electrochemical detection of cytokeratins antigen 21-1

NASA Astrophysics Data System (ADS)

Wang, Huiqiang; Rong, Qinfeng; Ma, Zhanfang

2016-07-01

Polyhydroquinone-graphene composite as a new redox species was synthesized simply by a microwave-assisted one-pot method through oxidative polymerization of hydroquinone by graphene oxide, which exhibited excellent electrochemical redox activity at 0.124 V and can remarkably promote electron transfer. The as-prepared composite was used as immunosensing substrate in a label-free electrochemical immunosensor for the detection of cytokeratins antigen 21-1, a kind of biomarker of lung cancer. The proposed immunosensor showed wide liner range from 10 pg mL-1 to 200 ng mL-1 with a detection limit 2.3 pg mL-1, and displayed a good stability and selectivity. In addition, this method has been used for the analysis of human serum sample, and the detection results showed good consistence with those of ELISA. The present substrate can be easily extended to other polymer-based nanocomposites.

Label-free protein assay based on a nanomechanical cantilever array

NASA Astrophysics Data System (ADS)

Arntz, Y.; Seelig, J. D.; Lang, H. P.; Zhang, J.; Hunziker, P.; Ramseyer, J. P.; Meyer, E.; Hegner, M.; Gerber, Ch

2003-01-01

We demonstrate continuous label-free detection of two cardiac biomarker proteins (creatin kinase and myoglobin) using an array of microfabricated cantilevers functionalized with covalently anchored anti-creatin kinase and anti-myoglobin antibodies. This method allows biomarker proteins to be detected via measurement of surface stress generated by antigen-antibody molecular recognition. Reference cantilevers are used to eliminate thermal drifts, undesired chemical reactions and turbulences from injections of liquids by calculating differential deflection signals with respect to sensor cantilevers. The sensitivity achieved for myoglobin detection is below 20 µg ml-1. Both myoglobin and creatin kinase could be detected independently using cantilevers functionalized with the corresponding antibodies, in unspecific protein background. This approach permits the use of up to seven different antigen-antibody reactions simultaneously, including an additional thermomechanical and chemical in situ reference. Applications lie in the field of early and rapid diagnosis of acute myocardial infarction.

Epstein-Barr virus glycoprotein gH/gL antibodies complement IgA-viral capsid antigen for diagnosis of nasopharyngeal carcinoma

PubMed Central

Tang, Lin-Quan; Zhang, Hua; Li, Yan; Liu, Wan-Li; Zhong, Qian; Zeng, Mu-Sheng; Huang, Xiao-Ming

2016-01-01

To determine whether measuring antibodies against Epstein-Barr virus (EBV) glycoprotein gH/gL in serum could improve diagnostic accuracy in nasopharyngeal carcinoma (NPC) cases, gH/gL expressed in a recombinant baculovirus system was used in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in two independent cohorts. Binary logistic regression analyses were performed using results from a training cohort (n = 406) to establish diagnostic mathematical models, which were validated in a second independent cohort (n = 279). Levels of serum gH/gL antibodies were higher in NPC patients than in healthy controls (p < 0.001). In the training cohort, the IgA-gH/gL ELISA had a sensitivity of 83.7%, specificity of 82.3% and area under the curve (AUC) of 0.893 (95% CI, 0.862-0.924) for NPC diagnosis. Furthermore, gH/gL maintained diagnostic capacity in IgA-VCA negative NPC patients (sensitivity = 78.1%, specificity = 82.3%, AUC = 0.879 [95% CI, 0.820 - 0.937]). Combining gH/gL and viral capsid antigen (VCA) detection improved diagnostic capacity as compared to individual tests alone in both the training cohort (sensitivity = 88.5%, specificity = 97%, AUC = 0.98 [95% CI, 0.97 - 0.991]), and validation cohort (sensitivity = 91.2%, specificity = 96.5%, AUC = 0.97 [95% CI, 0.951-0.988]). These findings suggest that EBV gH/gL detection complements VCA detection in the diagnosis of NPC and aids in the identification of patients with VCA-negative NPC. PMID:27093005

Development of ELISA using recombinant antigens for specific detection of mouse parvovirus infection.

PubMed

Kunita, Satoshi; Chaya, Miyuki; Hagiwara, Kozue; Ishida, Tomoko; Takakura, Akira; Sugimoto, Tatsuya; Iseki, Hiroyoshi; Fuke, Kumiko; Sugiyama, Fumihiro; Yagami, Ken-ichi

2006-04-01

Nucleotide sequences of mouse parvovirus (MPV) isolate, named MPV/UT, and mouse minute virus (MMV) were analyzed and used for expressing recombinant proteins in E. coli. ELISA tests using recombinant major capsid protein (rVP2) and recombinant major non-structural protein (rNS1) as antigens were developed and their performance in serologic detection of rodent parvovirus infection was assessed. MPV-rVP2 and MMV-rVP2 ELISAs reacted specifically with anti-MPV and anti-MMV mouse sera, respectively. MMV-rNS1 antigen had a wide reaction range with antisera to rodent parvoviruses including MPV, MMV, Kilham rat virus (KRV) and H-1 virus. All mice oronasally infected with MPV were seropositive at 4 weeks post-infection in screening by ELISAs using MPV-rVP2 and MMV-rNS1 antigens, but were negative by conventional ELISA using whole MMV antigen. A contact transmission experiment revealed that transmission of MPV occurred up to 4 weeks post-infection, and all cage mates were seropositive in screening with MPV-rVP2 and MMV-rNS1 ELISAs. These results indicate that MPV-rVP2 and MMV-rVP2 are specific ELISA antigens which distinguish between MPV and MVM infection, while MMV-rNS1 antigen can be used in generic ELISA for a variety of rodent parvoviruses. The higher sensitivity of MPV-rVP2 ELISA than conventional ELISA for detecting seroconversion to MPV in oronasally infected mice as well as in cage mates suggests the usefulness of MPV-rVP2 ELISA in quarantine and microbiological monitoring of MPV infection in laboratory mice.

Detection of CMV pneumonitis after lung transplantation using PCR of DNA from bronchoalveolar lavage cells.

PubMed

Bewig, B; Haacke, T C; Tiroke, A; Bastian, A; Böttcher, H; Hirt, S W; Rautenberg, P; Haverich, A

2000-01-01

Cytomegalovirus (CMV) is known as a common pathogen causing infections after lung transplantation. Rapid diagnosis of CMV infection is important for the initiation of a specific treatment. Evaluation of methods for the rapid diagnosis of CMV pneumonitis. The detection rates of CMV DNA in bronchoalveolar lavage (BAL) and bronchial brushes by polymerase chain reaction (PCR), of viral antigens (p52 and IE1) in BAL and of pp65 antigen in peripheral blood leukocytes were compared to the clinical status after lung transplantation. In 28 patients, 105 BAL, 96 blood samples and 14 brushes were analyzed. In 6 patients, a total of eight episodes of CMV pneumonitis occurred. Five of the 6 with positive CMV antigens in BAL (p52 or IE1) showed signs of CMV pneumonitis. All episodes of CMV pneumonitis were detected by the PCR of BAL cells. Fourteen samples positive for CMV pp65 antigen in blood were negative in BAL PCR. In these cases, no clinical signs of pulmonary CMV infection occurred. Overall sensitivity, specificity, and positive and negative predictive values for the detection of CMV pneumonitis by PCR of BAL cells were 100, 98.9, 88.9 and 100%, respectively. In brush samples, PCR did not provide additional information to the results of the PCR of BAL cells. PCR of DNA from BAL cells is suitable for reliable and rapid detection of CMV pneumonitis. Copyright 2000 S. Karger AG, Basel.

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26.

PubMed

Anderson, John P; Rascoe, Lisa N; Levert, Keith; Chastain, Holly M; Reed, Matthew S; Rivera, Hilda N; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E; Hotez, Peter J; Wilkins, Patricia P; Pohl, Jan; Handali, Sukwan

2015-01-01

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26

PubMed Central

Anderson, John P.; Rascoe, Lisa N.; Levert, Keith; Chastain, Holly M.; Reed, Matthew S.; Rivera, Hilda N.; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E.; Hotez, Peter J.; Wilkins, Patricia P.; Pohl, Jan; Handali, Sukwan

2015-01-01

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag. PMID:26485145

Herpes simplex keratitis in South India: clinico-virological correlation.

PubMed

Pramod, N P; Rajendran, P; Kannan, K A; Thyagarajan, S P

1999-01-01

A retrospective cross-section study to analyze the prevalence of herpes simplex virus-induced keratitis (HSK) among 3,000 patients attending a corneal clinic in South India between 1995 and 1997, and to evaluate laboratory techniques for detecting HSK. The clinico-virological correlation was studied using herpes simplex virus (HSV) isolation on the Vero cell line, HSV-specific antigen detection by indirect immunofluorescence (IF) microscopy, and serum anti-HSV IgG quantitation, IgM estimation, and tear secretory IgA (sIgA) detection by ELISA. HSK had a prevalence of 7.8% (234 patients) in this study. A virological correlation could be obtained in 44.4% of the cases that had epithelial manifestations and in 14.8% of the cases that had only stromal disease. In 161 cases where both culture and IF microscopy were used, IF detected 27 cases (26.8%) more than cell culture. The difference in sensitivity between cell culture and IF was found to be statistically significant (McNemar's test, P < .05). An elevation in IgG titer was seen in 17 (30.4%) cases. IgM was detected in only 2 cases of the 62 (3.2%) analyzed. Of the 138 cases analyzed, sIgA was positive in 28 (20.3%) cases. A proved diagnosis could be made in 58% of cases when the specimen was collected during the first week after disease onset, and in only 5% when the time interval increased to 4 weeks. HSV antigen detection by indirect IF is a rapid and sensitive diagnostic tool for HSK. Tear secretory IgA (sIgA) is a specific marker for acute herpetic keratitis, and the detection of HSV-specific tear sIgA is a valuable adjunct to virus isolation and antigen detection in the laboratory diagnosis of HSK. For a successful diagnosis, the specimen should be collected as soon as possible after HSK onset.

Serodiagnosis of Toxocariasis by ELISA Using Crude Antigen of Toxocara canis Larvae

PubMed Central

Jin, Yan; Shen, Chenghua; Huh, Sun; Sohn, Woon-Mok; Choi, Min-Ho

2013-01-01

Toxocariasis is a worldwide zoonosis caused by larvae of ascarid nematodes of dogs or cats, Toxocara canis or T. cati. Diagnosis of human toxocariasis currently relies on serology that uses T. canis excretory-secretory antigen to detect specific IgG antibodies by ELISA. We investigated the serodiagnostic efficacy of ELISA using crude antigen of T. canis larvae (TCLA). Serum specimens of 64 clinically confirmed toxocariasis, 115 healthy controls, and 119 other tissue-invading helminthiases were screened by ELISA using TCLA. The ELISA using TCLA showed 92.2% (59/64 patient samples) sensitivity and 86.6% (103/119) specificity. Its positive diagnostic predictivity was 78.7% and negative predictivity was 97.8%. No serum of healthy controls reacted but that of anisakiasis (45.5%), gnathostomiasis (19.2%), clonorchiasis (15.8%), sparganosis (11.1%), and cysticercosis (6.3%) cross-reacted. Immunoblot analysis on TCLA recognized antigenic proteins of 28- and 30-kDa bands in their dominant protein quantity and strong blotting reactivity. The present results indicate that the ELISA using our TCLA antigen is acceptable by the sensitivity and specificity for serodiagnosis of human toxocariasis. ELISA with TCLA is recommended to make differential diagnosis for patients with any sign of organ infiltration and eosinophilia. PMID:24039286

Identification of threshold prostate specific antigen levels to optimize the detection of clinically significant prostate cancer by magnetic resonance imaging/ultrasound fusion guided biopsy.

PubMed

Shakir, Nabeel A; George, Arvin K; Siddiqui, M Minhaj; Rothwax, Jason T; Rais-Bahrami, Soroush; Stamatakis, Lambros; Su, Daniel; Okoro, Chinonyerem; Raskolnikov, Dima; Walton-Diaz, Annerleim; Simon, Richard; Turkbey, Baris; Choyke, Peter L; Merino, Maria J; Wood, Bradford J; Pinto, Peter A

2014-12-01

Prostate specific antigen sensitivity increases with lower threshold values but with a corresponding decrease in specificity. Magnetic resonance imaging/ultrasound targeted biopsy detects prostate cancer more efficiently and of higher grade than standard 12-core transrectal ultrasound biopsy but the optimal population for its use is not well defined. We evaluated the performance of magnetic resonance imaging/ultrasound targeted biopsy vs 12-core biopsy across a prostate specific antigen continuum. We reviewed the records of all patients enrolled in a prospective trial who underwent 12-core transrectal ultrasound and magnetic resonance imaging/ultrasound targeted biopsies from August 2007 through February 2014. Patients were stratified by each of 4 prostate specific antigen cutoffs. The greatest Gleason score using either biopsy method was compared in and across groups as well as across the population prostate specific antigen range. Clinically significant prostate cancer was defined as Gleason 7 (4 + 3) or greater. Univariate and multivariate analyses were performed. A total of 1,003 targeted and 12-core transrectal ultrasound biopsies were performed, of which 564 diagnosed prostate cancer for a 56.2% detection rate. Targeted biopsy led to significantly more upgrading to clinically significant disease compared to 12-core biopsy. This trend increased more with increasing prostate specific antigen, specifically in patients with prostate specific antigen 4 to 10 and greater than 10 ng/ml. Prostate specific antigen 5.2 ng/ml or greater captured 90% of upgrading by targeted biopsy, corresponding to 64% of patients who underwent multiparametric magnetic resonance imaging and subsequent fusion biopsy. Conversely a greater proportion of clinically insignificant disease was detected by 12-core vs targeted biopsy overall. These differences persisted when controlling for potential confounders on multivariate analysis. Prostate cancer upgrading with targeted biopsy increases with an increasing prostate specific antigen cutoff. Above a prostate specific antigen threshold of 5.2 ng/ml most upgrading to clinically significant disease was achieved by targeted biopsy. In our population this corresponded to potentially sparing biopsy in 36% of patients who underwent multiparametric magnetic resonance imaging. Below this value 12-core biopsy detected more clinically insignificant cancer. Thus, the diagnostic usefulness of targeted biopsy is optimized in patients with prostate specific antigen 5.2 ng/ml or greater. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Comparative Study of rK39 Leishmania Antigen for Serodiagnosis of Visceral Leishmaniasis: Systematic Review with Meta-Analysis

PubMed Central

Maia, Zuinara; Lírio, Monique; Mistro, Sóstenes; Mendes, Carlos Maurício Cardeal; Mehta, Sanjay R.; Badaro, Roberto

2012-01-01

Background The rK39 recombinant protein is derived from a specific antigen produced by the Leishmania donovani complex, and has been used in the last two decades for the serodiagnosis of visceral leishmaniasis. We present here a systematic review and meta-analysis of studies evaluating serologic assays to diagnose visceral leishmaniasis to determine the accuracy of rK39 antigen in comparison to the use of other antigen preparations. Methodology/Principal Findings A systematic review with meta-analysis of the literature was performed to compare the rK39 strip-test and ELISA formats against serological tests using promastigote antigens derived from whole or soluble parasites for Direct Aglutination Test (DAT), Indirect Immunofluorescence test (IFAT) and ELISA with a promastigote antigen preparation (p-ELISA). Gold standard diagnosis was defined by the demonstration of amastigotes on hematological specimens. A database search was performed on Medline, Lilacs, Scopus, Isi Web of Science, and Cochrane Library. Quality of data was assessed using the QUADAS questionnaire. A search of the electronic databases found 352 papers of which only 14 fulfilled the selection criteria. Three evaluated the rK39 ELISA, while 13 evaluated the rK39 immunochromatographic strip test. The summarized sensitivity for the rK39-ELISA was 92% followed by IFAT 88% and p-ELISA 87%. The summarized specificity for the three diagnostic tests was 81%, 90%, and 77%. Studies comparing the rK39 strip test with DAT found a similar sensitivity of 94%, although the DAT had a slightly higher specificity. The rK39 strip test was more sensitive and specific than the IFAT and p-ELISA. We did not detect any difference in the sensitivity and specificity between strips produced by different manufacturers. Conclusions The rK39 protein used either in a strip test or in an ELISA, and the DAT are the best choices for implementation of rapid, easy and efficient test for serodiagnosis of VL. PMID:22303488

Comparative study of rK39 Leishmania antigen for serodiagnosis of visceral leishmaniasis: systematic review with meta-analysis.

PubMed

Maia, Zuinara; Lírio, Monique; Mistro, Sóstenes; Mendes, Carlos Maurício Cardeal; Mehta, Sanjay R; Badaro, Roberto

2012-01-01

The rK39 recombinant protein is derived from a specific antigen produced by the Leishmania donovani complex, and has been used in the last two decades for the serodiagnosis of visceral leishmaniasis. We present here a systematic review and meta-analysis of studies evaluating serologic assays to diagnose visceral leishmaniasis to determine the accuracy of rK39 antigen in comparison to the use of other antigen preparations. A systematic review with meta-analysis of the literature was performed to compare the rK39 strip-test and ELISA formats against serological tests using promastigote antigens derived from whole or soluble parasites for Direct Aglutination Test (DAT), Indirect Immunofluorescence test (IFAT) and ELISA with a promastigote antigen preparation (p-ELISA). Gold standard diagnosis was defined by the demonstration of amastigotes on hematological specimens. A database search was performed on Medline, Lilacs, Scopus, Isi Web of Science, and Cochrane Library. Quality of data was assessed using the QUADAS questionnaire. A search of the electronic databases found 352 papers of which only 14 fulfilled the selection criteria. Three evaluated the rK39 ELISA, while 13 evaluated the rK39 immunochromatographic strip test. The summarized sensitivity for the rK39-ELISA was 92% followed by IFAT 88% and p-ELISA 87%. The summarized specificity for the three diagnostic tests was 81%, 90%, and 77%. Studies comparing the rK39 strip test with DAT found a similar sensitivity of 94%, although the DAT had a slightly higher specificity. The rK39 strip test was more sensitive and specific than the IFAT and p-ELISA. We did not detect any difference in the sensitivity and specificity between strips produced by different manufacturers. The rK39 protein used either in a strip test or in an ELISA, and the DAT are the best choices for implementation of rapid, easy and efficient test for serodiagnosis of VL.

Country-wide assessment of the genetic polymorphism in Plasmodium falciparum and Plasmodium vivax antigens detected with rapid diagnostic tests for malaria

PubMed Central

Mariette, Natacha; Barnadas, Céline; Bouchier, Christiane; Tichit, Magali; Ménard, Didier

2008-01-01

Background Rapid diagnostic tests (RDTs) are becoming increasingly indispensable in malaria management, as a means of increasing the accuracy of diagnosis. The WHO has issued recommendations, but the selection of the most suitable RDT remains difficult for users in endemic countries. The genetic variability of the antigens detected with RDTs has been little studied, but may affect the sensitivity of RDTs. This factor has been studied by comparisons between countries at continental level, but little information is available concerning antigen variability within a given country. Methods A country-wide assessment of polymorphism of the PfHRP2, PfHRP3, pLDH and aldolase antigens was carried out in 260 Plasmodium falciparum and 127 Plasmodium vivax isolates, by sequencing the genes encoding these antigens in parasites originating from the various epidemiological strata for malaria in Madagascar. Results Higher levels of polymorphism were observed for the pfhrp2 and pfhrp3 genes than for the P. falciparum and P. vivax aldolase and pldh genes. Pfhrp2 sequence analysis predicted that 9% of Malagasy isolates would not be detected at parasite densities ≤ 250 parasites/μl (ranging from 6% in the north to 14% in the south), although RDTs based on PfHRP2 detection are now recommended in Madagascar. Conclusion These findings highlight the importance of training of health workers and the end users of RDTs in the provision of information about the possibility of false-negative results for patients with clinical symptoms of malaria, particularly in the south of Madagascar. PMID:18957099

Quantitative and multiplexed detection for blood typing based on quantum dot-magnetic bead assay.

PubMed

Xu, Ting; Zhang, Qiang; Fan, Ya-Han; Li, Ru-Qing; Lu, Hua; Zhao, Shu-Ming; Jiang, Tian-Lun

2017-01-01

Accurate and reliable blood grouping is essential for safe blood transfusion. However, conventional methods are qualitative and use only single-antigen detection. We overcame these limitations by developing a simple, quantitative, and multiplexed detection method for blood grouping using quantum dots (QDs) and magnetic beads. In the QD fluorescence assay (QFA), blood group A and B antigens were quantified using QD labeling and magnetic beads, and the blood groups were identified according to the R value (the value was calculated with the fluorescence intensity from dual QD labeling) of A and B antigens. The optimized performance of QFA was established by blood typing 791 clinical samples. Quantitative and multiplexed detection for blood group antigens can be completed within 35 min with more than 10 5 red blood cells. When conditions are optimized, the assay performance is satisfactory for weak samples. The coefficients of variation between and within days were less than 10% and the reproducibility was good. The ABO blood groups of 791 clinical samples were identified by QFA, and the accuracy obtained was 100% compared with the tube test. Receiver-operating characteristic curves revealed that the QFA has high sensitivity and specificity toward clinical samples, and the cutoff points of the R value of A and B antigens were 1.483 and 1.576, respectively. In this study, we reported a novel quantitative and multiplexed method for the identification of ABO blood groups and presented an effective alternative for quantitative blood typing. This method can be used as an effective tool to improve blood typing and further guarantee clinical transfusion safety.

Modulation of thymus-leukemia antigens on mouse leukemia cells induced by IgG, but not IgM, antibody.

PubMed

Stackpole, C W

1980-04-01

Exposure of mouse leukemia cells bearing thymus-leukemia (TL) surface antigens to whole TL alloantiserum has previously been shown to desensitize the cells to subsequent lysis by guinea pig complement (C) and fresh antiserum (antigenic modulation) and to correlate with the ability of cells to escape immune destruction in mice immunized against TL antigens. Tested in vitro, IgG of TL.1,2,3,5 antiserum modulated RADA1 leukemia cells (TL.1,2,3,5) completely within 2 hours at 37 degrees C when fully sensitizing amounts were used, with normal mouse serum as a source of C3. Similar results were obtained with IgG1, IgG2a, and IgG2b fractions of TL antiserum. An IgG2a monoclonal TL.3 antibody also completely modulated TL.3 antigens and partially modulated all antigens detected with TL.1,2,3,5 antiserum. IgM anti-TL.1,2,3,5 failed to modulate RADA1 cells even after 6 hours in vitro when fully sensitizing amounts of antibody were used. An IgM monoclonal TL antibody also failed to induce modulation. Modulation did occur on cells incubated with fully sensitizing amounts of IgG and IgM TL.1,2,3,5 antibody simultaneously, and nearly all cell-bound immunoglobulins were IgG. In mice passively immunized with IgG TL antibody, RADA1 cells modulated completely within 24 hours, whereas no modulation occurred during 4 days in mice immunized with IgM antibody. However, in both instances, tumor cells grew actively, which indicated that tumor escape did not depend on achievement of a modulated state.

ANTIGENIC MODULATION

PubMed Central

Old, Lloyd J.; Stockert, Elisabeth; Boyse, Edward A.; Kim, Jae Ho

1968-01-01

Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro. An ascites leukemia, phenotype TL.1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon. Over a wide range of TL antibody concentrations modulation at 37°C was detectable within 10 min and was complete within approximately 1 hr. The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test. The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0°C. Thus modulation apparently is an active cellular process. Antigens TL. 1,2, and 3 are all modulated by anti-TL.1,3 serum and by anti-TL.3 serum. This modulation affects all three TL components together, even when antibody to one or two of them is lacking. aAnti-TL.2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies. The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro. Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region. Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions. PMID:5636556

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen

PubMed Central

BANNAI, Hiroshi; NEMOTO, Manabu; TSUJIMURA, Koji; YAMANAKA, Takashi; MAEDA, Ken; KONDO, Takashi

2015-01-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA. PMID:26424485

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen.

PubMed

Bannai, Hiroshi; Nemoto, Manabu; Tsujimura, Koji; Yamanaka, Takashi; Maeda, Ken; Kondo, Takashi

2016-02-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.

Plasmonic welded single walled carbon nanotubes on monolayer graphene for sensing target protein

NASA Astrophysics Data System (ADS)

Kim, Jangheon; Kim, Gi Gyu; Kim, Soohyun; Jung, Wonsuk

2016-05-01

We developed plasmonic welded single walled carbon nanotubes (SWCNTs) on monolayer graphene as a biosensor to detect target antigen molecules, fc fusion protein without any treatment to generate binder groups for linker and antibody. This plasmonic welding induces atomic networks between SWCNTs as junctions containing carboxylic groups and improves the electrical sensitivity of a SWCNTs and the graphene membrane to detect target protein. We investigated generation of the atomic networks between SWCNTs by field-emission scanning electron microscopy and atomic force microscopy after plasmonic welding process. We compared the intensity ratios of D to G peaks from the Raman spectra and electrical sheet resistance of welded SWCNTs with the results of normal SWCNTs, which decreased from 0.115 to 0.086 and from 10.5 to 4.12, respectively. Additionally, we measured the drain current via source/drain voltage after binding of the antigen to the antibody molecules. This electrical sensitivity of the welded SWCNTs was 1.55 times larger than normal SWCNTs.

Fabrication of fiber-optic localized surface plasmon resonance sensor and its application to detect antibody-antigen reaction of interferon-gamma

NASA Astrophysics Data System (ADS)

Jeong, Hyeon-Ho; Erdene, Norov; Lee, Seung-Ki; Jeong, Dae-Hong; Park, Jae-Hyoung

2011-12-01

A fiber-optic localized surface plasmon (FO LSPR) sensor was fabricated by gold nanoparticles (Au NPs) immobilized on the end-face of an optical fiber. When Au NPs were formed on the end-face of an optical fiber by chemical reaction, Au NPs aggregation occurred and the Au NPs were immobilized in various forms such as monomers, dimers, trimers, etc. The component ratio of the Au NPs on the end-face of the fabricated FO LSPR sensor was slightly changed whenever the sensors were fabricated in the same condition. Including this phenomenon, the FO LSPR sensor was fabricated with high sensitivity by controlling the density of Au NPs. Also, the fabricated sensors were measured for the resonance intensity for the different optical systems and analyzed for the effect on sensitivity. Finally, for application as a biosensor, the sensor was used for detecting the antibody-antigen reaction of interferon-gamma.

Detection of Entamoeba histolytica by Recombinase Polymerase Amplification

PubMed Central

Nair, Gayatri; Rebolledo, Mauricio; White, A. Clinton; Crannell, Zachary; Richards-Kortum, R. Rebecca; Pinilla, A. Elizabeth; Ramírez, Juan David; López, M. Consuelo; Castellanos-Gonzalez, Alejandro

2015-01-01

Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. PMID:26123960

Evaluation of a surface plasmon resonance imaging-based multiplex O-antigen serogrouping for Escherichia coli using eleven major serotypes of Shiga -toxin-producing E. coli.

PubMed

Nakano, Satoshi; Nagao, Miki; Yamasaki, Tomomi; Morimura, Hiroyuki; Hama, Natsuki; Iijima, Yoshio; Shinomiya, Hiroto; Tanaka, Michio; Yamamoto, Masaki; Matsumura, Yasufumi; Miyake, Shiro; Ichiyama, Satoshi

2018-06-01

The early detection of Shiga toxin-producing Escherichia coli (STEC) is important for early diagnosis and preventing the spread of STEC. Although the confirmatory test for STEC should be based on the detection of Shiga toxin using molecular analysis, isolation permits additional characterization of STEC using a variety of methods, including O:H serotyping. The conventional slide agglutination O-antigen serogrouping used in many clinical laboratories is laborious and time-consuming. Surface plasmon resonance (SPR)-based immunosensors are commonly used to investigate a large variety of bio-interactions such as antibody/antigen, peptide/antibody, DNA/DNA, and antibody/bacteria interactions. SPR imaging (SPRi) is characterized by multiplexing capabilities for rapidly screening (approximately 100 to several hundred sensorgrams in parallel) molecules. SPRi-based O-antigen serogrouping method for STEC was recently developed by detecting the interactions between O-antigen-specific antibodies and bacterial cells themselves. The aim of this study was to evaluate its performance for E. coli serogrouping using clinical STEC isolates by comparing the results of slide agglutination tests. We tested a total of 188 isolates, including O26, O45, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The overall sensitivity of SPRi-based O-antigen serogrouping was 98.9%. Only two O157 isolates were misidentified as nontypeable and O121. The detection limits of all serotypes were distributed between 1.1 × 10 6 and 17.6 × 10 6  CFU/ml. Pulsed-field gel electrophoresis (PFGE) revealed the heterogeneity of the examined isolates. In conclusion, SPRi is a useful method for the O-antigen serogrouping of STEC isolates, but the further evaluation of non-O157 minor serogroups is needed. Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Multiplex serology of paraneoplastic antineuronal antibodies.

PubMed

Maat, Peter; Brouwer, Eric; Hulsenboom, Esther; VanDuijn, Martijn; Schreurs, Marco W J; Hooijkaas, Herbert; Smitt, Peter A E Sillevis

2013-05-31

Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diagnosing a neurological syndrome as paraneoplastic (70% is not yet known to have cancer) and in directing the search for the underlying neoplasm. At present six onconeural antibodies are considered 'well characterized' and recognize the antigens HuD, CDR62 (Yo), amphiphysin, CRMP-5 (CV2), NOVA-1 (Ri), and Ma2. The gold standard of detection is the characteristic immunohistochemical staining pattern on brain tissue sections combined with confirmation by immunoblotting using recombinant purified proteins. Since all six onconeural antibodies are usually analyzed simultaneously and objective cut-off values for these analyses are warranted, we developed a multiplex assay based on Luminex technology. Reaction of serial dilutions of six onconeural standard sera with microsphere-bound antigens showed lower limits of detection than with Western blotting. Using the six standard sera at a dilution of 1:200, the average within-run coefficient of variation (CV) was 4% (range 1.9-7.3%). The average between-run within-day CV was 5.1% (range 2.9-6.7%) while the average between-day CV was 8.1% (range 2.8-11.6%). The shelf-life of the antigen coupled microspheres was at least two months. The sensitivity of the multiplex assay ranged from 83% (Ri) to 100% (Yo, amphiphysin, CV2) and the specificity from 96% (CV2) to 100% (Ri). In conclusion, Luminex-based multiplex serology is highly reproducible with high sensitivity and specificity for the detection of onconeural antibodies. Conventional immunoblotting for diagnosis of onconeural antibodies in the setting of a routine laboratory may be replaced by this novel, robust technology. Copyright © 2013 Elsevier B.V. All rights reserved.

Selective testing strategies for diagnosing group A streptococcal infection in children with pharyngitis: a systematic review and prospective multicentre external validation study

PubMed Central

Cohen, Jérémie F.; Cohen, Robert; Levy, Corinne; Thollot, Franck; Benani, Mohamed; Bidet, Philippe; Chalumeau, Martin

2015-01-01

Background: Several clinical prediction rules for diagnosing group A streptococcal infection in children with pharyngitis are available. We aimed to compare the diagnostic accuracy of rules-based selective testing strategies in a prospective cohort of children with pharyngitis. Methods: We identified clinical prediction rules through a systematic search of MEDLINE and Embase (1975–2014), which we then validated in a prospective cohort involving French children who presented with pharyngitis during a 1-year period (2010–2011). We diagnosed infection with group A streptococcus using two throat swabs: one obtained for a rapid antigen detection test (StreptAtest, Dectrapharm) and one obtained for culture (reference standard). We validated rules-based selective testing strategies as follows: low risk of group A streptococcal infection, no further testing or antibiotic therapy needed; intermediate risk of infection, rapid antigen detection for all patients and antibiotic therapy for those with a positive test result; and high risk of infection, empiric antibiotic treatment. Results: We identified 8 clinical prediction rules, 6 of which could be prospectively validated. Sensitivity and specificity of rules-based selective testing strategies ranged from 66% (95% confidence interval [CI] 61–72) to 94% (95% CI 92–97) and from 40% (95% CI 35–45) to 88% (95% CI 85–91), respectively. Use of rapid antigen detection testing following the clinical prediction rule ranged from 24% (95% CI 21–27) to 86% (95% CI 84–89). None of the rules-based selective testing strategies achieved our diagnostic accuracy target (sensitivity and specificity > 85%). Interpretation: Rules-based selective testing strategies did not show sufficient diagnostic accuracy in this study population. The relevance of clinical prediction rules for determining which children with pharyngitis should undergo a rapid antigen detection test remains questionable. PMID:25487666

Magnetic bead-quantum dot assay for detection of a biomarker for traumatic brain injury

NASA Astrophysics Data System (ADS)

Kim, Chloe; Searson, Peter C.

2015-10-01

Current diagnostic methods for traumatic brain injury (TBI), which accounts for 15% of all emergency room visits, are limited to neuroimaging modalities. The challenges of accurate diagnosis and monitoring of TBI have created the need for a simple and sensitive blood test to detect brain-specific biomarkers. Here we report on an assay for detection of S100B, a putative biomarker for TBI, using antibody-conjugated magnetic beads for capture of the protein, and antibody-conjugated quantum dots for optical detection. From Western Blot, we show efficient antigen capture and concentration by the magnetic beads. Using magnetic bead capture and quantum dot detection in serum samples, we show a wide detection range and detection limit below the clinical cut-off level.Current diagnostic methods for traumatic brain injury (TBI), which accounts for 15% of all emergency room visits, are limited to neuroimaging modalities. The challenges of accurate diagnosis and monitoring of TBI have created the need for a simple and sensitive blood test to detect brain-specific biomarkers. Here we report on an assay for detection of S100B, a putative biomarker for TBI, using antibody-conjugated magnetic beads for capture of the protein, and antibody-conjugated quantum dots for optical detection. From Western Blot, we show efficient antigen capture and concentration by the magnetic beads. Using magnetic bead capture and quantum dot detection in serum samples, we show a wide detection range and detection limit below the clinical cut-off level. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05608j

Functionalized vertical GaN micro pillar arrays with high signal-to-background ratio for detection and analysis of proteins secreted from breast tumor cells.

PubMed

Choi, Mun-Ki; Kim, Gil-Sung; Jeong, Jin-Tak; Lim, Jung-Taek; Lee, Won-Yong; Umar, Ahmad; Lee, Sang-Kwon

2017-11-02

The detection of cancer biomarkers has recently attracted significant attention as a means of determining the correct course of treatment with targeted therapeutics. However, because the concentration of these biomarkers in blood is usually relatively low, highly sensitive biosensors for fluorescence imaging and precise detection are needed. In this study, we have successfully developed vertical GaN micropillar (MP) based biosensors for fluorescence sensing and quantitative measurement of CA15-3 antigens. The highly ordered vertical GaN MP arrays result in the successful immobilization of CA15-3 antigens on each feature of the arrays, thereby allowing the detection of an individual fluorescence signal from the top surface of the arrays owing to the high regularity of fluorophore-tagged MP spots and relatively low background signal. Therefore, our fluorescence-labeled and CA15-3 functionalized vertical GaN-MP-based biosensor is suitable for the selective quantitative analysis of secreted CA15-3 antigens from MCF-7 cell lines, and helps in the early diagnosis and prognosis of serious diseases as well as the monitoring of the therapeutic response of breast cancer patients.

Evaluation of a New and Rapid Serologic Test for Detecting Brucellosis: Brucella Coombs Gel Test.

PubMed

Hanci, Hayrunisa; Igan, Hakan; Uyanik, Muhammet Hamidullah

2017-01-01

Many serological tests have been used for the diagnosis of human brucellosis. A new serological method is identified as Brucella Coombs gel test based on the principle of centrifugation gel system similar to the gel system used in blood group determination. In this system, if Brucella antibodies were present in the serum, antigen and antibody would remain as a pink complex on the gel. Otherwise, the pink Brucella antigens would precipitate at the bottom of the gel card system. In this study, we aimed to compare the Brucella Coombs gel test, a new, rapid screen and titration method for detection of non-agglutinating IgG with the Brucella Coombs test. For this study, a total of 88 serum samples were obtained from 45 healthy persons and 43 individuals who had clinical signs and symptoms of brucellosis. For each specimen, Rose Bengal test, standard agglutination test, Coombs test and Brucella Coombs gel test were carried out. Sensitivity and specificity of Brucella Coombs gel test were found as 100.0 and 82.2%, respectively. Brucella Coombs gel test can be used as a screening test with high sensitivity. By the help of pink Brucella antigen precipitation, the tests' evaluation is simple and objective. In addition, determination of Brucella antibody by rapid titration offers another important advantage.

An ultra-sensitive impedimetric immunosensor for detection of the serum oncomarker CA-125 in ovarian cancer patients

NASA Astrophysics Data System (ADS)

Johari-Ahar, M.; Rashidi, M. R.; Barar, J.; Aghaie, M.; Mohammadnejad, D.; Ramazani, A.; Karami, P.; Coukos, G.; Omidi, Y.

2015-02-01

Effective treatment of ovarian cancer depends upon the early detection of the malignancy. Here, we report on the development of a new nanostructured immunosensor for early detection of cancer antigen 125 (CA-125). A gold electrode was modified with mercaptopropionic acid (MPA), and then consecutively conjugated with silica coated gold nanoparticles (AuNP@SiO2), CdSe quantum dots (QDs) and anti-CA-125 monoclonal antibody (mAb). The engineered MPA|AuNP@SiO2|QD|mAb immunosensor was characterised using transmission electron microscopy (TEM), atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Successive conjugation of AuNP@SiO2, CdSe QD and anti-CA-125 mAb onto the gold electrode resulted in sensitive detection of CA-125 with a limit of detection (LOD) of 0.0016 U mL-1 and a linear detection range (LDR) of 0-0.1 U mL-1. Based on the high sensitivity and specificity of the immunosensor, we propose this highly stable and reproducible biosensor for the early detection of CA-125.Effective treatment of ovarian cancer depends upon the early detection of the malignancy. Here, we report on the development of a new nanostructured immunosensor for early detection of cancer antigen 125 (CA-125). A gold electrode was modified with mercaptopropionic acid (MPA), and then consecutively conjugated with silica coated gold nanoparticles (AuNP@SiO2), CdSe quantum dots (QDs) and anti-CA-125 monoclonal antibody (mAb). The engineered MPA|AuNP@SiO2|QD|mAb immunosensor was characterised using transmission electron microscopy (TEM), atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Successive conjugation of AuNP@SiO2, CdSe QD and anti-CA-125 mAb onto the gold electrode resulted in sensitive detection of CA-125 with a limit of detection (LOD) of 0.0016 U mL-1 and a linear detection range (LDR) of 0-0.1 U mL-1. Based on the high sensitivity and specificity of the immunosensor, we propose this highly stable and reproducible biosensor for the early detection of CA-125. Electronic supplementary information (ESI) available: Additional materials including Figures and discussion as described in the text. See DOI: 10.1039/c4nr06687a

Highly sensitive and robust peroxidase-like activity of Au-Pt core/shell nanorod-antigen conjugates for measles virus diagnosis.

PubMed

Long, Lin; Liu, Jianbo; Lu, Kaishun; Zhang, Tao; Xie, Yunqing; Ji, Yinglu; Wu, Xiaochun

2018-05-02

As a promising candidate for artificial enzymes, catalytically active nanomaterials show several advantages over natural enzymes, such as controlled synthesis at low cost, tunability of catalytic activities, and high stability under stringent conditions. Rod-shaped Au-Pt core/shell nanoparticles (Au@Pt NRs), prepared by Au nanorod-mediated growth, exhibit peroxidase-like activities and could serve as an inexpensive replacement for horseradish peroxidase, with potential applications in various bio-detections. The determination of measles virus is accomplished by a capture-enzyme-linked immunosorbent assay (ELISA) using Au@Pt NR-antigen conjugates. Based on the enhanced catalytic properties of this nanozyme probe, a linear response was observed up to 10 ng/mL measles IgM antibodies in human serum, which is 1000 times more sensitive than commercial ELISA. Hence, these findings provide positive proof of concept for the potential of Au@Pt NR-antigen conjugates in the development of colorimetric biosensors that are simple, robust, and cost-effective.

Detection of Clonorchis sinensis circulating antigen in sera from Chinese patients by immunomagnetic bead ELISA based on IgY.

PubMed

Nie, Ge; Wang, Ting; Lu, Shengjun; Liu, Wenqi; Li, Yonglong; Lei, Jiahui

2014-01-01

Clonorchiasis, caused by Clonorchis sinensis, is widely distributed in Southeast Asia including China. Clonorchiasis is included in control programs of neglected tropical diseases by World Health Organization (WHO) because it is one of the major health problems in most endemic areas. Diagnosis of clonorchiasis plays a key role in the control programs. However, so far, there is no satisfactory method for clonorchiasis because of low sensitivity, poor practicality and high false positivity of available diagnostic tools. We developed an immunomagnetic bead enzyme-linked immunosorbent assay (ELISA) based on IgY (egg yolk immunoglobulin) against cysteine proteinase of C. sinensis for detection of circulating antigen in serum samples of patients infected with C. sinensis. The polyclonal IgY, coated with magnetic beads, was used as a capture antibody and a monoclonal IgG labeled with horseradish peroxidase as a detection antibody in the IgY-based immunomagnetic bead ELISA system (IgY-IMB-ELISA). The results showed that the sensitivity of IgY-IMB-ELISA was 93.3% (14 of 15) in cases of heavy infection (5000 to 9999 eggs per gram feces, i.e, EPG 5000-9999), 86.7% (13 of 15) in cases of moderate infection (EPG 1000-4999) and 75.0% (9 of 12) in cases of light infection (EPG <1000) of clonorchiasis. Together 36 of total 42 (85.7%) serum samples of human clonorchiasis gave a positive reaction. There was a significant correlation between ELISA optical density and egg counts (EPG) with a correlation coefficient of 0.83 in total 42 patients. There were no positive results in patients with trichinosis (n = 10) or cysticercosis (n = 10). Cross-reactivity was 6.7% (2 of 30) with schistosomiasis japonica and 10.0% (3 of 30) with paragonimiasis, respectively. No positive reaction was found in 20 healthy persons. Our findings suggest that IgY-IMB-ELISA appears to be a sensitive and specific assay for detection of circulating antigen in human clonorchiasis.

Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage.

PubMed

Talarico, Sarah; Safaeian, Mahboobeh; Gonzalez, Paula; Hildesheim, Allan; Herrero, Rolando; Porras, Carolina; Cortes, Bernal; Larson, Ann; Fang, Ferric C; Salama, Nina R

2016-08-01

Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning. Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital. The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool. The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen. © 2015 John Wiley & Sons Ltd.

An auto-biotinylated bifunctional protein nanowire for ultra-sensitive molecular biosensing.

PubMed

Men, Dong; Zhang, Zhi-Ping; Guo, Yong-Chao; Zhu, Duan-Hao; Bi, Li-Jun; Deng, Jiao-Yu; Cui, Zong-Qiang; Wei, Hong-Ping; Zhang, Xian-En

2010-12-15

In order to obtain an ultra-sensitive molecular biosensor, we designed an auto-biotinylated bifunctional protein nanowire (bFPNw) based on the self-assembly of a yeast amyloid protein, Sup35, to which protein G and a biotin acceptor peptide (BAP) were genetically fused. These auto-biotinylated bFPNws can transfer hundreds of commercially available diagnostic enzymes to an antigen-antibody complex via the biotin-avidin system, greatly enhancing the sensitivity of immune-biosensing. Compared to our previously reported seeding-induced bFPNws (Men et al., 2009), these auto-biotinylated bFPNws gave greater signal amplification, reduced non-specific binding and improved stability. The auto-biotinylated self-assembled bFPNw molecular biosensors were applied to detect Yersinia pestis (Y. pestis) F1 antigen and showed a 2000- to 4000-fold increase in sensitivity compared to traditional immunoassays, demonstrating the potential use of these self-assembling protein nanowires in biosensing. Copyright © 2010 Elsevier B.V. All rights reserved.

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis.

PubMed

Makam, Shivakiran S; Majumder, Saugata; Kingston, Joseph J; Urs, Radhika M; Tuteja, Urmil; Sripathi, Murali H; Batra, Harsh V

2013-12-01

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml⁻¹ of bacterial cells and spores, respectively. IPCR was found to be 2-3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.

Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay

PubMed Central

Ahn, Ki Chang; Ranganathan, Anupama; Bever, Candace S.; Hwang, Sung Hee; Holland, Erika B.; Morisseau, Kevin; Pessah, Isaac N.; Hammock, Bruce D.; Gee, Shirley J.

2016-01-01

A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclosan (TCS; 2,4,4’-trichloro-2’-hydroxydiphenyl ether) was developed. Novel immunizing haptens were synthesized by derivatizing at the 4-Cl position of the TCS molecule. Compounds derived from substitutions at 4’-Cl and that replaced the 2’–OH with a –Cl were designed as unique coating antigen haptens. Polyclonal rabbit antisera were screened against the coating antigen library in order to identify combinations of immunoreagents resulting in the most sensitive assays. The most sensitive assay identified was one utilizing antiserum #1155 and a heterologous competitive hapten where the 2’–OH group was substituted with a Cl. An IC50 value and the detection range for TCS in assay buffer were 1.19 and 0.21–6.71 μg/L, respectively. The assay was selective for TCS, providing low cross-reactivity (< 5%) to the major metabolites of TCS and to brominated diphenyl ether-47. A second assay utilizing a competitive hapten containing Br instead of Cl substitutions was broadly selective for both brominated and chlorinated diphenylethers. Using the most sensitive assay combination, TCS concentrations were measured in water samples following dilution. Biosolid samples were analyzed following dilution of a simple solvent extract. The immunoassay results were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid and convenient tool to screen for human and environmental exposure. PMID:26937944

Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay.

PubMed

Ahn, Ki Chang; Ranganathan, Anupama; Bever, Candace S; Hwang, Sung Hee; Holland, Erika B; Morisseau, Kevin; Pessah, Isaac N; Hammock, Bruce D; Gee, Shirley J

2016-04-05

A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) was developed. Novel immunizing haptens were synthesized by derivatizing at the 4-Cl position of the TCS molecule. Compounds derived from substitutions at 4'-Cl and that replaced the 2'-OH with a Cl atom were designed as unique coating antigen haptens. Polyclonal rabbit antisera were screened against the coating antigen library to identify combinations of immunoreagents resulting in the most sensitive assays. The most sensitive assay identified was one utilizing antiserum no. 1155 and a heterologous competitive hapten, where the 2'-OH group was substituted with a Cl atom. An IC50 value and the detection range for TCS in assay buffer were 1.19 and 0.21-6.71 μg/L, respectively. The assay was selective for TCS, providing low cross-reactivity (<5%) to the major metabolites of TCS and to brominated diphenyl ether-47. A second assay utilizing a competitive hapten containing Br instead of Cl substitutions was broadly selective for both brominated and chlorinated diphenylethers. Using the most sensitive assay combination, we measured TCS concentrations in water samples following dilution. Biosolid samples were analyzed following the dilution of a simple solvent extract. The immunoassay results were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid and convenient tool to screen for human and environmental exposure.

Development of an Antigen Capture Enzyme-Linked Immunosorbent Assay for Virus Detection Based on Porcine Epidemic Diarrhea Virus Monoclonal Antibodies

PubMed Central

Wang, Zanyu; Jiyuan, Yin; Su, Chen; Xinyuan, Qiao

2015-01-01

Abstract Porcine epidemic diarrhea virus (PEDV), a coronavirus, can cause acute diarrhea and dehydration in pigs. In the current study, two positive monoclonal cell lines (5D7 and 3H4) specific for PEDV were established, and the immunoreactivity of the monoclonal antibodies was confirmed by immunofluorescence and dot-immunobinding assays. A method, termed antigen capture enzyme-linked immunosorbent assay (AC-ELISA), which used the monoclonal antibody 5D7 as the detecting antibody and rabbit antiserum of PEDV protein S as the capture antibody, was developed. Compared with the reverse transcription polymerase chain reaction method of detecting PEDV in fecal samples, AC-ELISA showed similar sensitivity and specificity. These results suggested that AC-ELISA would be useful for the diagnosis and epidemiological studies of PEDV. PMID:25658793

Multianalyte detection using a capillary-based flow immunosensor.

PubMed

Narang, U; Gauger, P R; Kusterbeck, A W; Ligler, F S

1998-01-01

A highly sensitive, dual-analyte detection system using capillary-based immunosensors has been designed for explosive detection. This model system consists of two capillaries, one coated with antibodies specific for 2,4,6-trinitrotoluene (TNT) and the other specific for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) combined into a single device. The fused silica capillaries are prepared by coating anti-TNT and anti-RDX antibodies onto the silanized inner walls using a hetero-bifunctional crosslinker. After immobilization, the antibodies are saturated with a suitable fluorophorelabeled antigen. A "T" connector is used to continuously flow the buffer solution through the individual capillaries. To perform the assay, an aliquot of TNT or RDX or a mixture of the two analytes is injected into the continuous flow stream. In each capillary, the target analyte displaces the fluorophore-labeled antigen from the binding pocket of the antibody. The labeled antigen displaced from either capillary is detected downstream using two portable spectrofluorometers. The limits of detection for TNT and RDX in the multi-analyte formate are 44 fmol (100 microliters of 0.1 ng/ml TNT solution) and 224 fmol (100 microliters of 0.5 ng/ml RDX solution), respectively. The entire assay for both analytes can be performed in less than 3 min.

The performance characteristics of prostate-specific antigen and prostate-specific antigen density in Chinese men.

PubMed

Teoh, Jeremy Yc; Yuen, Steffi Kk; Tsu, James Hl; Wong, Charles Kw; Ho, Brian Sh; Ng, Ada Tl; Ma, Wai-Kit; Ho, Kwan-Lun; Yiu, Ming-Kwong

2017-01-01

We investigated the performance characteristics of prostate-specific antigen (PSA) and PSA density (PSAD) in Chinese men. All Chinese men who underwent transrectal ultrasound-guided prostate biopsy (TRUS-PB) from year 2000 to 2013 were included. The receiver operating characteristic (ROC) curves for both PSA and PSAD were analyzed. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) at different cut-off levels were calculated. A total of 2606 Chinese men were included. For the ROC, the area under curve was 0.770 for PSA (P < 0.001) and 0.823 for PSAD (P < 0.001). PSA of 4.5 ng ml-1 had sensitivity of 94.4%, specificity of 14.1%, PPV of 29.5%, and NPV of 86.9%; PSAD of 0.12 ng ml-1 cc-1 had sensitivity of 94.5%, specificity of 26.6%, PPV of 32.8%, and NPV of 92.7%. On multivariate logistic regression analyses, PSA cut-off at 4.5 ng ml-1 (OR 1.61, 95% CI 1.05-2.45, P= 0.029) and PSAD cut-off at 0.12 ng ml-1 cc-1 (OR 6.22, 95% CI 4.20-9.22, P< 0.001) were significant predictors for prostate cancer detection on TRUS-PB. In conclusion, the performances of PSA and PSAD at different cut-off levels in Chinese men were very different from those in Caucasians. PSA of 4.5 ng ml-1 and PSAD of 0.12 ng ml-1 cc-1 had near 95% sensitivity and were significant predictors of prostate cancer detection in Chinese men.

Comparative study between cytotoxicity and flowcytometry crossmatches before and after renal transplantation.

PubMed

Abdel Rahman, Afaf S; Fahim, Nehal M A; El Sayed, Abeer A; El Hady, Soha A R; Ahmad, Yasser S

2005-01-01

Renal transplantation, in most countries, is based on human leukocyte antigen (HLA) matching of the donor kidney with the recipient. Traditional human leukocyte antigen matching is based on defining human leukocyte antigen specificities by antibodies utilizing cytotoxicity crossmatch techniques. Newer techniques have emerged, which challenge the accuracy of serological typing and crossmatching. We compared the results of the standard complement-dependent cytotoxicity crossmatch (CDCXM) with the anti-human globulin augmented cytotoxicity (AHG-CDC), and Flowcytometry crossmatch (FCXM) for the detection of anti-HLA antibodies in 150 pre-transplant patients. The development of post-transplantation sensitization was screened utilizing these three techniques within two weeks post-operative and correlated with rejection episodes. Comparison between the results of CDCXM and AHG-CDC in 150 recipients, revealed no significant correlation (P>0.05). When comparing these results with that of FCXM in 50 recipients a significant correlation was shown (P<0.05). Relative to CDCXM, the sensitivity of AHG-CDC was 100%, specificity 97.4%, positive predictive value 92.3%, and negative predictive value 100%. On the other hand, the sensitivity of FCXM was 100%, specificity 76.3%, positive predictive value 57.1%, and negative predictive value 100%. According to the results of CDCXM, AHG-CDC, and FCXM, no difference was detected between pre- and posttransplant anti-HLA sensitization within two weeks after the operation. Patients with negative cytotoxicity crossmatch (CDCXM and AHG-CDC) and positive FCXM may have an increased risk of early graft loss and may represent a relative contraindication to transplantation. Given the important theoretical advantages of FCXM over the CDC XM, further testing of the clinical relevance is warranted.

Immuno-PCR: Achievements and Perspectives.

PubMed

Ryazantsev, D Y; Voronina, D V; Zavriev, S K

2016-12-01

The immuno-PCR (iPCR) method combines advantages of enzyme-linked immunosorbent assay and polymerase chain reaction, which is used in iPCR as a method of "visualization" of antigen-antibody interaction. The use of iPCR provides classical PCR sensitivity to objects traditionally detected by ELISA. This method could be very sensitive and allow for detection of quantities of femtograms/ml order. However, iPCR is still not widely used. The aim of this review is to highlight the special features of the iPCR method and to show the main aspects of its development and application in recent years.

Repeated human leukocyte antigen mismatches in lung re-transplantation.

PubMed

Sommer, Wiebke; Hallensleben, Michael; Ius, Fabio; Kühn, Christian; Tudorache, Igor; Avsar, Murat; Salman, Jawad; Siemeni, Thierry; Greer, Mark; Gottlieb, Jens; Boethig, Dietmar; Blasczyk, Rainer; Haverich, Axel; Warnecke, Gregor

2017-02-01

The role of HLA-sensitization in the absence of detectable DSA in lung re-transplantation is unclear. Antigens of the second donor matching the HLA typing of the first donor are considered 'unacceptable', by some tissue typing laboratories, especially in kidney re-transplantation. Thus, we performed a retrospective analysis of all lung re-transplantations focussing on the impact of HLA-homologies between the first and the second donor ('unacceptable' antigens; repeated HLA mismatch) on patient and graft survival. A total of 132 lung re-transplantations were performed at our centre between 1985 and 2014, of which 120 with complete HLA data were analysed. 55.8% of the recipients received re-transplants with repeated HLA mismatched antigens whereas 43.2% of the re-transplants were transplanted without repeated HLA mismatched antigens. Postoperative survival showed no difference between re-transplant procedures with or without repeated HLA mismatches (p=0.99). While neither homologies on the HLA-A, -B, -C, or -DR locus, nor the addition of several locus homologies (p=0.72) had an impact on survival, unexpectedly, repeated HLA mismatching on the HLA-DQ locus was correlated with better survival. Re-transplantations with repeated HLA mismatches did not result in more development of CLAD as compared to recipients without repeated HLA mismatches (p=0.99). Neither the number of repeated HLA mismatched antigens (p=0.52) nor the HLA locus (HLA-A(p=0.34), HLA-B(p=0.97), HLA-C (p=0.80), HLA-DR(p=0.49) and HLA-DQ(p=0.07)) had an impact on the development of CLAD after re-transplantation. Transplantation with repeated HLA mismatches due to sensitization by a previous transplantation in the absence of detectable HLA-antibodies does not have a negative impact on patient or graft survival. Copyright © 2016 Elsevier B.V. All rights reserved.

Mitochondrial DNA Targets Increase Sensitivity of Malaria Detection Using Loop-Mediated Isothermal Amplification ▿

PubMed Central

Polley, Spencer D.; Mori, Yasuyoshi; Watson, Julie; Perkins, Mark D.; González, Iveth J.; Notomi, Tsugunori; Chiodini, Peter L.; Sutherland, Colin J.

2010-01-01

Loop-mediated isothermal amplification (LAMP) of DNA offers the ability to detect very small quantities of pathogen DNA following minimal tissue sample processing and is thus an attractive methodology for point-of-care diagnostics. Previous attempts to diagnose malaria by the use of blood samples and LAMP have targeted the parasite small-subunit rRNA gene, with a resultant sensitivity for Plasmodium falciparum of around 100 parasites per μl. Here we describe the use of mitochondrial targets for LAMP-based detection of any Plasmodium genus parasite and of P. falciparum specifically. These new targets allow routine amplification from samples containing as few as five parasites per μl of blood. Amplification is complete within 30 to 40 min and is assessed by real-time turbidimetry, thereby offering rapid diagnosis with greater sensitivity than is achieved by the most skilled microscopist or antigen detection using lateral flow immunoassays. PMID:20554824

[Sensitivity of three inmunocromathographic tests in faeces samples for Campylobacter and Salmonella detection in comparison to culture].

PubMed

Liébana-Martos, Ma del Carmen; Gutierrez, José; Riazzo, Cristina; Navarro, José Ma

2014-06-01

Introduction: Campylobacter sp. and Salmonella enterica are two of the main organisms causing gastroenteritis in our environment. Immunochromatographic tests for antigen detection performed directly on stool samples for its simplicity and rapid results may make them useful diagnostic elements in the context of primary care. During October 2012 we selected all feces in which enteropathogenic bacteria are isolated from those received for stool culture in the laboratory of Microbiology of the University Hospital Virgen de las Nieves of Granada. After standard management of faeces samples and isolation of any enteropathogen, the commercial kits: Campy Leti, Ridaquick Campylobacterscreen and Salmonella Leti were tested for simultaneous research of Campylobacter and Salmonella antigens. Sensitivity and specificity were determined. Two hundred and thirty five stool samples were received in which 8 Salmonella enterica (7 B serogroup and 1 D serogroup), 7 Campylobacter jejuni, 4 Aeromonas hydrophila and 1 Yersinia enterocolitica were isolated. Campy Leti, Ridaquick Campylobacterscreen and Salmonella Leti presented a sensitivity of 100%, 100% and 75%, respectively. Specificities corresponded to 46%, 69% and 100%, respectively. Immunocromatographic tests can be useful for a first screening of enteropathogen in primary care.

A comparison of two antigen-detection ELISA for detecting infection of Dirofilaria immitis in dogs.

PubMed

Euclid, J M; Copeman, D B

1997-09-01

A survey on 87 dogs necropsied in the Townsville region revealed 34 (39%) were infected with Dirofilaria immitis. Infected dogs had an average of 6.1 adult worms in the heart and associated blood vessels. The VetRED assay detected 23 of the 34 infected dogs (sensitivity 65%) and the Og4C3 ELISA detected 27 (sensitivity 80%). Sensitivity of the VetRED and Og4C3 ELISA increased to 88 and 94% respectively in dogs with three or more worms. Both tests detected correctly all uninfected dogs. Despite the higher accuracy of the Og4C3 ELISA, compared to the VetRED assay, it is unlikely to be used widely as a field test for heartworm unless it can be modified from its present plate ELISA format which takes 4 hours, into one which is more rapid and convenient. However, as a reference ELISA, it may well be worthwhile in situations which require considerable accuracy for detecting D. immitis infection.

ZnO thin film transistor immunosensor with high sensitivity and selectivity

NASA Astrophysics Data System (ADS)

Reyes, Pavel Ivanoff; Ku, Chieh-Jen; Duan, Ziqing; Lu, Yicheng; Solanki, Aniruddh; Lee, Ki-Bum

2011-04-01

A zinc oxide thin film transistor-based immunosensor (ZnO-bioTFT) is presented. The back-gate TFT has an on-off ratio of 108 and a threshold voltage of 4.25 V. The ZnO channel surface is biofunctionalized with primary monoclonal antibodies that selectively bind with epidermal growth factor receptor (EGFR). Detection of the antibody-antigen reaction is achieved through channel carrier modulation via pseudo double-gating field effect caused by the biochemical reaction. The sensitivity of 10 fM detection of pure EGFR proteins is achieved. The ZnO-bioTFT immunosensor also enables selectively detecting 10 fM of EGFR in a 5 mg/ml goat serum solution containing various other proteins.

A biomolecular detection method based on charge pumping in a nanogap embedded field-effect-transistor biosensor

NASA Astrophysics Data System (ADS)

Kim, Sungho; Ahn, Jae-Hyuk; Park, Tae Jung; Lee, Sang Yup; Choi, Yang-Kyu

2009-06-01

A unique direct electrical detection method of biomolecules, charge pumping, was demonstrated using a nanogap embedded field-effect-transistor (FET). With aid of a charge pumping method, sensitivity can fall below the 1 ng/ml concentration regime in antigen-antibody binding of an avian influenza case. Biomolecules immobilized in the nanogap are mainly responsible for the acute changes of the interface trap density due to modulation of the energy level of the trap. This finding is supported by a numerical simulation. The proposed detection method for biomolecules using a nanogap embedded FET represents a foundation for a chip-based biosensor capable of high sensitivity.

Usage of Leptospira spp. local strains as antigens increases the sensitivity of the serodiagnosis of bovine leptospirosis.

PubMed

Pinto, Priscila S; Loureiro, Ana P; Penna, Bruno; Lilenbaum, Walter

2015-09-01

Leptospirosis is a zoonotic disease that occurs worldwide, particularly in tropical countries. In livestock the agent is responsible for reproductive problems such as infertility and abortion. Serogroup Sejroe, particularly serovar Hardjo, prevails in cattle in several regions. The microscopic agglutination test (MAT) is the current method for diagnosing leptospirosis. It has been proposed that the inclusion of local strains could detect a larger set of seroreactive animals. In that context, the aim of the present study was to evaluate if the usage of local strains as antigens increases the sensitivity of the serodiagnosis of bovine leptospirosis. Blood and urine samples were collected from 314 bovines from several herds randomly selected in a slaughterhouse in Rio de Janeiro, Brazil. Serological diagnosis was made with MAT using a 21 reference-strains panel (MAT21). Additionally, 12 local strains (MAT33) were included as antigens. PCR was performed with the urine samples and it was positive on 71 out of 222 samples (31.9%). MAT21 identified as seroreactive 173 (55.1%) out of the 314 animals studied, with Sejroe the most common (38.1%). In MAT33, 204 (65.0%) animals were seroreactive with a significant increase on seroreactivity (9.9%). In conclusion, MAT presented with a significant increase of sensitivity when local strains were used as antigens. Among the local strains, 2013_U152 (KP263062) (serogroup Shermani) and 2013_U280 (KP263069) (serogroup Grippotyphosa) showed to be more antigenic. Copyright © 2015. Published by Elsevier B.V.

Utilization of microparticles in next-generation assays for microflow cytometers.

PubMed

Kim, Jason S; Ligler, Frances S

2010-11-01

Micron-sized particles have primarily been used in microfabricated flow cytometers for calibration purposes and proof-of-concept experiments. With increasing frequency, microparticles are serving as a platform for assays measured in these small analytical devices. Light scattering has been used to measure the agglomeration of antibody-coated particles in the presence of an antigen. Impedance detection is another technology being integrated into microflow cytometers for microparticle-based assays. Fluorescence is the most popular detection method in flow cytometry, enabling highly sensitive multiplexed assays. Finally, magnetic particles have also been used to measure antigen levels using a magnetophoretic micro-device. We review the progress of microparticle-based assays in microflow cytometry in terms of the advantages and limitations of each approach.

Systematic review: Tumor-associated antigen autoantibodies and ovarian cancer early detection.

PubMed

Fortner, Renée Turzanski; Damms-Machado, Antje; Kaaks, Rudolf

2017-11-01

Tumor-associated autoantibodies (AAbs), produced as an immune response to tumor-associated antigens (TAAs), are a novel pathway of early detection markers. We conducted a systematic review on AAbs and ovarian cancer to summarize the diagnostic performance of individual AAbs and AAb panels. A total of 29 studies including 85 AAbs were included; 27 of the studies were conducted in prevalent cases and cancer-free controls and 2 investigations included pre-diagnosis samples. The majority of studies were hypothesis-driven, evaluating AAbs to target TAAs; 10 studies used screening approaches such as serological expression cloning (SEREX) and nucleic acid-programmable protein arrays (NAPPA). The highest sensitivities for individual AAbs were reported for RhoGDI-AAbs (89.5%) and TUBA1C-AAbs (89%); however, specificity levels were relatively low (80% and 75%, respectively). High sensitivities at high specificities were reported for HOXA7-AAbs for detection of moderately differentiated ovarian tumors (66.7% sensitivity at 100% specificity) and IL8-AAbs in stage I-II ovarian cancer (65.5% sensitivity at 98% specificity). A panel of 11 AAbs (ICAM3, CTAG2, p53, STYXL1, PVR, POMC, NUDT11, TRIM39, UHMK1, KSR1, and NXF3) provided 45% sensitivity at 98% specificity for serous ovarian cancer, when at least 2 AAbs were above a threshold of 95% specificity. Twelve of the AAbs identified in this review were investigated in more than one study. Data on diagnostic discrimination by tumor histology and stage at diagnosis are sparse. Limited data suggest select AAb markers improve diagnostic discrimination when combined with markers such as CA125 and HE4. AAbs for ovarian cancer early detection is an emerging area, and large-scale, prospective investigations considering histology and stage are required for discovery and validation. However, data to date suggests panels of AAbs may eventually reach sufficient diagnostic discrimination to allow earlier detection of disease as a complement to existing markers and transvaginal ultrasound. Copyright © 2017. Published by Elsevier Inc.

Rapid Serodiagnosis of Active Pulmonary Mycobacterium tuberculosis by Analysis of Results from Multiple Antigen-Specific Tests

PubMed Central

Okuda, Yoshinari; Maekura, Ryoji; Hirotani, Atsusi; Kitada, Seigo; Yoshimura, Kenji; Hiraga, Touru; Yamamoto, Yuoko; Itou, Masami; Ogura, Takeshi; Ogihara, Toshio

2004-01-01

We have prospectively analyzed three antigens for serodiagnosis of tuberculosis (TB). These antigens were tuberculous glycolipid antigen, lypoarabinomannan polysaccharide antigen, and antigen 60 (A60), which was derived from purified protein derivatives. Of the 131 patients with active pulmonary TB, 57 were both smear and culture negative and 14 had chronic active pulmonary TB that remained smear positive for >12 months of chemotherapy. One hundred twenty healthy adults were controls. The percentages of patients positive in all three tests were 58.8% for smear-positive active pulmonary TB and 71.4% for chronic active pulmonary TB. When the results of the three serodiagnostic tests were evaluated in combination, the sensitivity increased to 91.5% in patients with active pulmonary TB and to 86.0% in smear- and culture-negative patients. The false-positive rate of the three-test combination was 12.5% in the healthy control groups. In conclusion, it was not possible to detect all of the antibodies against antigenic substances in the cell walls of the tuberculous bacilli in the sera of all TB patients by using available serodiagnostic tests. However, the combined use of tests with three separate antigens maximizes the effectiveness of serodiagnosis. PMID:15004065

Immuno-analysis of microparticles: probing at the limits of detection

PubMed Central

Latham, Sharissa L.; Tiberti, Natalia; Gokoolparsadh, Naveena; Holdaway, Karen; Olivier Couraud, Pierre; Grau, Georges E. R.; Combes, Valery

2015-01-01

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30–40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data. PMID:26553743

An ultrasensitive electrochemical immunosensor for the detection of prostate-specific antigen based on conductivity nanocomposite with halloysite nanotubes.

PubMed

Li, Yueyuan; Khan, Malik Saddam; Tian, Lihui; Liu, Li; Hu, Lihua; Fan, Dawei; Cao, Wei; Wei, Qin

2017-05-01

A sensitive label-free amperometric electrochemical immunosensor for detection of prostate-specific antigen (PSA) was proposed in this work. The nanocomposite of halloysite nanotubes with polypyrrole shell and palladium nanoparticles (HNTs@PPy-Pd) was used as a novel signal label. The HNTs with adequate hydroxyl groups are economically available raw materials. PPy, as an electrically conducting polymer material, can be absorbed to the surface of HNTs by in situ oxidative polymerization of the pyrrole monomer and form a shell on the HNTs. The shell of PPy could not only improve the conductivity of the nanocomposite but also absorb large amounts of Pd nanoparticles (NPs). The Pd NPs with high electrocatalytic activity toward the reduction of H 2 O 2 and the HNTs@PPy-Pd nanocomposite as the analytical signal label could improve the sensitivity of the immunosensor. Under optimal conditions, the immunosensor showed a low detection limit (0.03 pg/mL) and a wide linear range (0.0001 to 25 ng/mL) of PSA. Moreover, its merits such as good selectivity, acceptable reproducibility, and stability indicate that the fabricated immunosensor has a promising application potential in clinical diagnosis. Graphical Abstract A new label-free amperometric electrochemical immunosensor based on HNTs@PPy-Pd nanocomposite for quantitative detection of PSA.

Electrophoretically deposited reduced graphene oxide platform for food toxin detection

NASA Astrophysics Data System (ADS)

Srivastava, Saurabh; Kumar, Vinod; Ali, Md Azahar; Solanki, Pratima R.; Srivastava, Anchal; Sumana, Gajjala; Saxena, Preeti Suman; Joshi, Amish G.; Malhotra, B. D.

2013-03-01

Reduced graphene oxide (RGO) due to its excellent electrochemical properties and large surface area, has recently aroused much interest for electrochemical biosensing application. Here, the chemically active RGO has been synthesized and deposited onto an indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. This novel platform has been utilized for covalent attachment of the monoclonal antibodies of aflatoxin B1 (anti-AFB1) for food toxin (AFB1) detection. The electron microscopy, X-ray diffraction, and UV-visible studies reveal successful synthesis of reduced graphene oxide while the XPS and FTIR studies suggest its carboxylic functionalized nature. The electrochemical sensing results of the anti-AFB1/RGO/ITO based immunoelectrode obtained as a function of aflatoxin concentration show high sensitivity (68 μA ng-1 mL cm-2) and improved detection limit (0.12 ng mL-1). The association constant (ka) for antigen-antibody interaction obtained as 5 × 10-4 ng mL-1 indicates high affinity of antibodies toward the antigen (AFB1).Reduced graphene oxide (RGO) due to its excellent electrochemical properties and large surface area, has recently aroused much interest for electrochemical biosensing application. Here, the chemically active RGO has been synthesized and deposited onto an indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. This novel platform has been utilized for covalent attachment of the monoclonal antibodies of aflatoxin B1 (anti-AFB1) for food toxin (AFB1) detection. The electron microscopy, X-ray diffraction, and UV-visible studies reveal successful synthesis of reduced graphene oxide while the XPS and FTIR studies suggest its carboxylic functionalized nature. The electrochemical sensing results of the anti-AFB1/RGO/ITO based immunoelectrode obtained as a function of aflatoxin concentration show high sensitivity (68 μA ng-1 mL cm-2) and improved detection limit (0.12 ng mL-1). The association constant (ka) for antigen-antibody interaction obtained as 5 × 10-4 ng mL-1 indicates high affinity of antibodies toward the antigen (AFB1). Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr32242d

Human oesophagus: a convenient antigenic substrate for the determination of anti-endomysium antibodies in the serological diagnosis of coeliac disease.

PubMed

Uibo, O; Lambrechts, A; Mascart-Lemone, F

1995-01-01

Immunoglobulin (Ig) A-class anti-endomysium antibodies are superior to other current antibody tests for detecting coeliac disease. We aimed to evaluate the suitability of human oesophagus for the determination of anti-endomysium antibodies. The specificity of monkey and human oesophageal tissue as antigenic substrate were compared using indirect immunofluorescence analysis. Overall, 159 individuals were studied: 56 patients with biopsy-proven coeliac disease (39 with active disease) and 103 controls. The patients' IgA-class anti-endomysium antibodies were compared using unfixed cryostat sections of human and monkey oesophagus. Indirect immunofluorescence analysis was performed with an initial serum sample dilution of 1:5, and if positive, the highest dilution yielding a positive reaction was reported. The anti-endomysium antibody test was positive in 38 out of 39 patients with active coeliac disease using monkey oesophagus (sensitivity 97%) and in all 39 patients with active coeliac disease using human oesophagus (sensitivity 100%). Ten out of 17 coeliac patients on a gluten-free diet had positive anti-endomysium antibodies using monkey oesophagus and 12 using human oesophagus as the antigenic substrate. This test was negative in all 103 controls using both substrates. Our study shows that human oesophageal tissue can be used instead of monkey tissue for determining anti-endomysium antibodies. Human tissue is a more sensitive antigenic substrate than monkey oesophagus and can be used to determine low titres of antibodies. Improving the diagnostic sensitivity of the anti-endomysium antibody test would make an important contribution to screening for coeliac disease.

18F-DCFBC Prostate-Specific Membrane Antigen-Targeted PET/CT Imaging in Localized Prostate Cancer: Correlation With Multiparametric MRI and Histopathology.

PubMed

Turkbey, Baris; Mena, Esther; Lindenberg, Liza; Adler, Stephen; Bednarova, Sandra; Berman, Rose; Ton, Anita T; McKinney, Yolanda; Eclarinal, Philip; Hill, Craig; Afari, George; Bhattacharyya, Sibaprasad; Mease, Ronnie C; Merino, Maria J; Jacobs, Paula M; Wood, Bradford J; Pinto, Peter A; Pomper, Martin G; Choyke, Peter L

2017-10-01

To assess the ability of (N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-F-fluorobenzyl-L-cysteine) (F-DCFBC), a prostate-specific membrane antigen-targeted PET agent, to detect localized prostate cancer lesions in correlation with multiparametric MRI (mpMRI) and histopathology. This Health Insurance Portability and Accountability Act of 1996-compliant, prospective, institutional review board-approved study included 13 evaluable patients with localized prostate cancer (median age, 62.8 years [range, 51-74 years]; median prostate-specific antigen, 37.5 ng/dL [range, 3.26-216 ng/dL]). Patients underwent mpMRI and F-DCFBC PET/CT within a 3 months' window. Lesions seen on mpMRI were biopsied under transrectal ultrasound/MRI fusion-guided biopsy, or a radical prostatectomy was performed. F-DCFBC PET/CT and mpMRI were evaluated blinded and separately for tumor detection on a lesion basis. For PET image analysis, MRI and F-DCFBC PET images were fused by using software registration; imaging findings were correlated with histology, and uptake of F-DCFBC in tumors was compared with uptake in benign prostatic hyperplasia nodules and normal peripheral zone tissue using the 80% threshold SUVmax. A total of 25 tumor foci (mean size, 1.8 cm; median size, 1.5 cm; range, 0.6-4.7 cm) were histopathologically identified in 13 patients. Sensitivity rates of F-DCFBC PET/CT and mpMRI were 36% and 96%, respectively, for all tumors. For index lesions, the largest tumor with highest Gleason score, sensitivity rates of F-DCFBC PET/CT and mpMRI were 61.5% and 92%, respectively. The average SUVmax for primary prostate cancer was higher (5.8 ± 4.4) than that of benign prostatic hyperplasia nodules (2.1 ± 0.3) or that of normal prostate tissue (2.1 ± 0.4) at 1 hour postinjection (P = 0.0033). The majority of index prostate cancers are detected with F-DCFBC PET/CT, and this may be a prognostic indicator based on uptake and staging. However, for detecting prostate cancer with high sensitivity, it is important to combine prostate-specific membrane antigen PET/CT with mpMRI.

Sensitivity improvement of an electrical sensor achieved by control of biomolecules based on the negative dielectrophoretic force.

PubMed

Kim, Hye Jin; Kim, Jinsik; Yoo, Yong Kyoung; Lee, Jeong Hoon; Park, Jung Ho; Hwang, Kyo Seon

2016-11-15

Effective control of nano-scale biomolecules can enhance the sensitivity and limit of detection of an interdigitated microelectrode (IME) sensor. Manipulation of the biomolecules by dielectrophoresis (DEP), especially the negative DEP (nDEP) force, so that they are trapped between electrodes (sensing regions) was predicted to increase the binding efficiency of the antibody and target molecules, leading to a more effective reaction. To prove this concept, amyloid beta 42 (Aβ42) and prostate specific antigen (PSA) protein were respectively trapped between the sensing region owing to the nDEP force under 5V and 0.05V, which was verified with COMSOL simulation. Using the simulation value, the resistance change (ΔR/Rb) of the IME sensor from the specific antibody-antigen reaction of the two biomolecules and the change in fluorescence intensity were compared in the reference (pDEP) and nDEP conditions. The ΔR/Rb value improved by about 2-fold and 1.66-fold with nDEP compared to the reference condition with various protein concentrations, and these increases were confirmed with fluorescence imaging. Overall, nDEP enhanced the detection sensitivity for Aβ42 and PSA by 128% and 258%, respectively, and the limit of detection improved by up to 2-orders of magnitude. These results prove that DEP can improve the biosensor's performance. Copyright © 2016 Elsevier B.V. All rights reserved.

Utility of galactomannan antigen detection in bronchoalveolar lavage fluid in immunocompromised patients.

PubMed

Brownback, Kyle R; Pitts, Lucas R; Simpson, Steven Q

2013-09-01

Diagnosis of invasive pulmonary aspergillosis (IPA) is a challenging process in immunocompromised patients. Galactomannan (GM) antigen detection in bronchoalveolar lavage (BAL) fluid is a method to detect IPA with improved sensitivity over conventional studies. We sought to determine the diagnostic yield of BAL GM assay in a diverse population of immunocompromised patients. A retrospective review of 150 fiberoptic bronchoscopy (FOB) with BAL for newly diagnosed pulmonary infiltrate in immunocompromised patients was performed. Patient information, procedural details and laboratory studies were collected. BAL and serum samples were evaluated for GM using enzyme-linked immunoassay. Of 150 separate FOB with BAL, BAL GM was obtained in 143 samples. There were 31 positive BAL GM assays. In those 31 positive tests, 13 were confirmed as IPA, giving a positive predictive value of 41.9%. There was one false negative BAL GM. Of the 18 false positive BAL GM, 4 were receiving piperacillin-tazobactam and 11 were receiving an alternative beta-lactam antibiotic. BAL GM assay shows excellent sensitivity for diagnosing IPA. There was a significant number of false positive BAL GM assays and several of those patients were receiving beta-lactam antibiotics at the time of bronchoscopy. © 2013 Blackwell Verlag GmbH.

3D-Printed Supercapacitor-Powered Electrochemiluminescent Protein Immunoarray

PubMed Central

Kadimisetty, Karteek; Mosa, Islam M.; Malla, Spundana; Satterwhite-Warden, Jennifer E.; Kuhns, Tyler; Faria, Ronaldo C.; Lee, Norman H.; Rusling, James F.

2015-01-01

Herein we report a low cost, sensitive, supercapacitor-powered electrochemiluminescent (ECL) protein immunoarray fabricated by an inexpensive 3-dimensional (3D) printer. The immunosensor detects three cancer biomarker proteins in serum within 35 min. The 3D-printed device employs hand screen printed carbon sensors with gravity flow for sample/reagent delivery and washing. Prostate cancer biomarker proteins, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA) and platelet factor-4 (PF-4) in serum were captured on the antibody-coated carbon sensors followed by delivery of detection-antibody-coated Ru(bpy)32+ (RuBPY)-doped silica nanoparticles in a sandwich immunoassay. ECL light was initiated from RuBPY in the silica nanoparticles by electrochemical oxidation with tripropylamine (TPrA) co-reactant using supercapacitor power and ECL was captured with a CCD camera. The supercapacitor was rapidly photo-recharged between assays using an inexpensive solar cell. Detection limits were 300–500 fg mL−1 for the 3 proteins in undiluted calf serum. Assays of 6 prostate cancer patient serum samples gave good correlation with conventional single protein ELISAs. This technology could provide sensitive onsite cancer diagnostic tests in resource-limited settings with the need for only moderate-level training. PMID:26406460

Development of a Sensitive and Specific Antigen-Detection System for Strongyloides Stercoralis and Hookworm Infections

DTIC Science & Technology

1997-06-01

deficiency. Treatment usually includes blood transfusions and a three day course of mebendazole and iron substitution and/or single dose albendazole ...1983 Treatment of hookworm, ascariasis and trichuriasis with albendazole or mebendazole. Revista do Instituto de Medicina Tropical de Sao Paulo 25:294

[Establishment of A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and its preliminary application].

PubMed

Cai, Yu-Chun; Chen, Shao-Hong; Tian, Li-Guang; Chu, Yan-Hong; Lu, Yan; Chen, Mu-Xin; Ai, Lin; Zhou, Yang; Chen, Jia-Xu

2014-02-01

To establish A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and explore its application value in the field. The characteristics of A1E3 and B1C4 monoclonal antibodies were analyzed by SDS-PAGE and Western blotting. The SEA-based ELISA was used to evaluate the titers of A1E3 and B1C4. The orthogonal test was used to determine the best concentration of coating antibody B1C4 and optimal working concentration of A1E3-HRP. Under the optimal conditions, the serum samples of 20 acute schistosomiasis cases, 46 chronic schistosomiasis cases, and 20 control sera were tested to evaluate its detection sensitivity and specificity. Seventy-two antibody positive serum samples from Jiangling County of Hubei Province were detected and compared to a commercially available ELISA kit, to evaluate the detection effects of this method. The results of SDS-PAGE demonstrated that the purified A1E3 and B1C4 contained a clear heavy chain with molecular weight of 88,000 and 52,000 respectively and had the same light chain with molecular weight of 20,000; while Western blotting demonstrated that A1E3 and B1C4 could be recognized by SEA and serum samples of acute schistosomiasis cases. The SEA-based ELISA demonstrated the titers of B1C4 and A1E3 were 1:10(5) and 1:30,000, respectively. The serum samples from all the acute cases and 86.9% of the chronic cases showed a positive reaction. All of the control sera from healthy persons gave a negative response. The positive rates of the double monoclonal antibody ELISA and commercial ELISA for detecting the circulating antigen were 45.8% and 43.1% respectively, and there was no significant difference between the results of the two methods. A1E3 and B1C4 monoclonal antibody-based ELISA is established successfully. It exhibits a high sensitivity and specificity in detecting circulating antigen of Schistosoma japonicum.

Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool.

PubMed

Mesquita, Flávio da Silva; Oliveira, Danielle Bruna Leal de; Crema, Daniela; Pinez, Célia Miranda Nunes; Colmanetti, Thaís Cristina; Thomazelli, Luciano Matsumia; Gilio, Alfredo Elias; Vieira, Sandra Elisabeth; Martinez, Marina Baquerizo; Botosso, Viviane Fongaro; Durigon, Edison Luiz

The aim of this study was to evaluate the QuickVue ® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue ® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue ® RSV Test and viral load or specific strain. The QuickVue ® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. This study demonstrated that the QuickVue ® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Antibody profiling sensitivity through increased reporter antibody layering

DOEpatents

Apel, William A.; Thompson, Vicki S.

2013-02-26

A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

Antibody profiling sensitivity through increased reporter antibody layering

DOEpatents

Apel, William A.; Thompson, Vicki S.

2017-03-28

A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

Detection of Entamoeba histolytica by Recombinase Polymerase Amplification.

PubMed

Nair, Gayatri; Rebolledo, Mauricio; White, A Clinton; Crannell, Zachary; Richards-Kortum, R Rebecca; Pinilla, A Elizabeth; Ramírez, Juan David; López, M Consuelo; Castellanos-Gonzalez, Alejandro

2015-09-01

Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. © The American Society of Tropical Medicine and Hygiene.

Culturing and extraction of Coprococcus comes, absorption of serumagglutinins by soluble fractions and relation between agglutinins and antibodies in sera of patients with Crohn's disease.

PubMed

Hazenberg, M P; Pennock-Schröder, A M; van de Merwe, J P

1986-01-01

Agglutinating antibodies to Coprococcus comes and three other obligately anaerobic coccoid rods from the intestinal flora are used in the diagnosis of Crohn's disease. Further studies on the pathogenetic role as well as the development of more sensitive and specific methods for detecting antibodies require extraction of the antigen fractions. Culturing methods to obtain C. comes with optimal antigen presentation and isolation of soluble antigen fractions were therefore developed. Hot water extraction of whole cells and subsequent removal of proteins with trichloroacetic acid provided a fraction that absorbed serum agglutinins, was useful for an enzyme-linked immunosorbent assay and induced agglutinating antibodies in rats.

Effect of wavelength and dye selection on biosensor response

NASA Astrophysics Data System (ADS)

Ligler, Frances S.; Breslin, Kristen A.; Cao, Lynn K.; Anderson, George P.

1995-05-01

The availability of low cost laser diodes and new fluorescent dyes has made portable biosensors a reality. Previously, we have examined the variation in the fluorescent signal generated in an antigen-antibody reaction when the antigen is labeled with dyes exciting at different wavelengths. In this study, we looked at the effect of changing dyes and wavelengths on a sandwich immunoassay for the F1 antigen from Yersinia pestis, the etiologic agent of plaque. The F1 immunoassay has previously been demonstrated to work in serum, plasma, and even whole blood, when performed using a fiber optic biosensor. In this study, we demonstrated that changing to cyanine dyes enhanced the sensitivity of the detection without altering the immunochemistry of the assay.

The genetic origin of minor histocompatibility antigens.

PubMed

Roopenian, D C; Christianson, G J; Davis, A P; Zuberi, A R; Mobraaten, L E

1993-01-01

The purpose of this study was to elucidate the genetic origin of minor histocompatibility (H) antigens. Toward this end common inbred mouse strains, distinct subspecies, and species of the subgenus Mus were examined for expression of various minor H antigens. These antigens were encoded by the classical minor H loci H-3 and H-4 or by newly identified minor H antigens detected as a consequence of mutation. Both minor H antigens that stimulate MHC class I-restricted cytotoxic T cells (Tc) and antigens that stimulate MHC class II-restricted helper T cells (Th) were monitored. The results suggested that strains of distinct ancestry commonly express identical or cross-reactive antigens. Moreover, a correlation between the lack of expression of minor H antigens and ancestral heritage was observed. To address whether the antigens found on unrelated strains were allelic with the sensitizing minor H antigens or a consequence of antigen cross-reactivity, classical genetic segregation analysis was carried out. Even in distinct subspecies and species, the minor H antigens always mapped to the site of the appropriate minor H locus. Together the results suggest: 1) minor H antigen sequences are evolutionarily stable in that their pace of antigenic change is slow enough to predate subspeciation and speciation; 2) the minor H antigens originated in the inbred strains as a consequence of a rare polymorphism or loss mutation carried in a founder mouse stock that caused the mouse to perceive the wild-type protein as foreign; 3) there is a remarkable lack of antigenic cross-reactivity between the defined minor H antigens and other gene products.

PAPERS DEVOTED TO THE MEMORY OF ACADEMICIAN A M PROKHOROV: Immunosensor systems with the Langmuir-film-based fluorescence detection

NASA Astrophysics Data System (ADS)

Chudinova, G. K.; Nagovitsyn, I. A.; Karpov, R. E.; Savranskii, V. V.

2003-09-01

A method is developed for detecting protein antigens for fluorescent immunoassay using a model system based on the technique for preparation of Langmuir films. Fluorescein isothiocyanate and donor-acceptor energy-transfer pairs of markers (the Yb complex of tetraphenyl porphyrin — benzoyl trifluoroacetoneisothiocyanate and derivatives of tetra(carboxyphenyl) porphyrin — cyanine dye containing a five-membered polyene chain), which were nor studied earlier, were used as markers for detecting the binding of an antigen on the surface of Langmuir films of antibodies. Fluorescence was detected in the near-IR region (for the first pair) and in the visible spectral range (for the second pair). To reduce the nonspecific sorption of a protein (antigen), a method was proposed for the preparation of a nonpolar surface by applying an even number of layers of stearic acid as a substrate for the Langmuir — Blodgett film. A high sensitivity of model systems to a protein antigen in solution was achieved (~10-11 M), the assay time being 6 — 8 min. The model system with the first donor — acceptor pair was tested in analysis of the blood plasma. The fluorescence of the Dy3+, Tm3+, and Yb3+ complexes of tetraphenyl porphyrin sensitised by diketonate complexes of lanthanides was studied for the first time and the enhancement of the IR fluorescence of these complexes in a Langmuir film was demonstrated.

Laboratory Diagnosis of Echinococcus spp. in Human Patients and Infected Animals.

PubMed

Siles-Lucas, M; Casulli, A; Conraths, F J; Müller, N

2017-01-01

Among the species composing the genus Echinococcus, four species are of human clinical interest. The most prevalent species are Echinococcus granulosus and Echinococcus multilocularis, followed by Echinococcus vogeli and Echinococcus oligarthrus. The first two species cause cystic echinococcosis (CE) and alveolar echinococcosis (AE) respectively. Both diseases have a complex clinical management, in which laboratory diagnosis could be an adjunctive to the imaging techniques. To date, several approaches have been described for the laboratory diagnosis and followup of CE and AE, including antibody, antigen and cytokine detection. All of these approaches are far from being optimal as adjunctive diagnosis particularly for CE, since they do not reach enough sensitivity and/or specificity. A combination of several methods (e.g., antibody and antigen detection) or of several (recombinant) antigens could improve the performance of the adjunctive laboratory methods, although the complexity of echinococcosis and heterogeneity of clinical cases make necessary a deep understanding of the host-parasite relationships and the parasite phenotype at different developmental stages to reach the best diagnostic tool and to make it accepted in clinical practice. Standardization approaches and a deep understanding of the performance of each of the available antigens in the diagnosis of echinococcosis for the different clinical pictures are also needed. The detection of the parasite in definitive hosts is also reviewed in this chapter. Finally, the different methods for the detection of parasite DNA in different analytes and matrices are also reviewed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Diagnostic performance of influenza viruses and RSV rapid antigen detection tests in children in tertiary care.

PubMed

Moesker, F M; van Kampen, J J A; Aron, G; Schutten, M; van de Vijver, D A M C; Koopmans, M P G; Osterhaus, A D M E; Fraaij, P L A

2016-06-01

Rapid antigen detection tests (RADTs) are increasingly used to detect influenza viruses and respiratory syncytial virus (RSV). However, their sensitivity and specificity are a matter of debate, challenging their clinical usefulness. Comparing diagnostic performances of BinaxNow Influenza AB(®) (BNI) and BinaxNow RSV(®) (BNR), to those of real-time reverse transcriptase PCR (RT-PCR), virus isolation and direct immunofluorescence (D-IF) in paediatric patients. Between November 2005 and September 2013, 521 nasal washings from symptomatic children (age <5 years) attending our tertiary care centre were tested, with a combination of the respective assays using RT-PCR as gold standard. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of BNI were 69% (confidence interval [CI] [51-83]), 96% [94-97], 55% [39-70] and 98% [96-99] respectively. Of eleven false-negative samples, RT-PCR Ct-values were higher than all RT-PCR positive test results (27 vs 22, p=0.012). Of twenty false-positive samples, none were culture positive and two tested positive in D-IF. Sensitivity, specificity, PPV and NPV for BNR were 79% [73-85], 98% [96-99], 97% [93-99] and 88% [84-91]. Of the 42 false-negative samples the median Ct-value was higher than that of all RT-PCR positive samples (31 vs 23, p<0.0001). Five false-positive samples were detected. Three of these tested positive for RSV in virus isolation and D-IF. RADTs have a high specificity with BNR being superior to BNI. However, their relative low sensitivity limits their usefulness for clinical decision making in a tertiary care paediatric hospital. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Rapid and Accurate Diagnosis of Acute Pyogenic Meningitis Due to Streptococcus Pneumoniae, Haemophilus influenzae Type b and Neisseria meningitidis Using A Multiplex PCR Assay.

PubMed

Seth, Rajeev; Murthy, Peela Sree Ramchandra; Sistla, Sujatha; Subramanian, Mahadevan; Tamilarasu, Kadhiravan

2017-09-01

Acute bacterial meningitis is one of the major causes of morbidity and mortality in children and geriatric population, especially in developing countries. Methods of identification are standard culture and other phenotypic tests in many resource poor settings. To use molecular methods for the improvement of aetiological diagnosis of acute pyogenic meningitis in patients. CSF samples of 125 patients were included for the study. Gram staining and culture were performed according to standard procedures. Antigen was detected using commercial latex agglutination test kit. Multiplex PCR was performed using previously published primers and protocols. Fischer's exact test was used for finding association between presence of the disease and clinical/biochemical parameters, considering two tailed p<0.05 as statistically significant. Sensitivity, specificity, positive and negative predictive values were calculated using Graphpad QuicCalc software. A total of 39 cases (31.2%) were confirmed to be of acute pyogenic meningitis based on biochemical methods. Only 10/39 was positive for the three organisms tested. Multiplex PCR was able to detect one additional isolate each of Streptococcus pneumoniae and Haemophilus influenzae type b. When compared with multiplex PCR as the gold standard, culture and latex agglutination tests had same sensitivity (80%), specificity (100%), PPV (100%) and NPV (97.8%), whereas Gram stain had poor sensitivity (40%) and good specificity (95.6%). Detection rates were higher in multiplex PCR for the two organisms Streptococcus pneumoniae and Haemophilus influenzae type b. Multiplex PCR was more sensitive than culture or antigen detection, and employing this assay can significantly increase the speed and accuracy of identification of the pathogen.

1-D grating based SPR biosensor for the detection of lung cancer biomarkers using Vroman effect

NASA Astrophysics Data System (ADS)

Teotia, Pradeep Kumar; Kaler, R. S.

2018-01-01

Grating based surface plasmon resonance waveguide biosensor have been reported for the detection of lung cancer biomarkers using Vroman effect. The proposed grating based multilayered biosensor is designed with high detection accuracy for Epidermal growth factor receptor (EGFR) and also analysed to show high detection accuracy with acceptable sensitivity for both cancer biomarkers. The introduction of periodic grating with multilayer metals generates a good resonance that make it possible for early detection of cancerous cells. Using finite difference time domain method, it is observed wavelength of biosensor get red-shifted on variations of the refractive index due to the presence of both the cancerous bio-markers. The reported detection accuracy and sensitivity of proposed biosensor is quite acceptable for both lung cancer biomarkers i.e. Carcinoembryonic antigen (CEA) and Epidermal growth factor receptor (EGFR) which further offer us label free early detection of lung cancer using these biomarkers.

Impact of nucleic acid testing relative to antigen/antibody combination immunoassay on the detection of acute HIV infection.

PubMed

De Souza, Mark S; Phanuphak, Nittaya; Pinyakorn, Suteeraporn; Trichavaroj, Rapee; Pattanachaiwit, Supanit; Chomchey, Nitiya; Fletcher, James L; Kroon, Eugene D; Michael, Nelson L; Phanuphak, Praphan; Kim, Jerome H; Ananworanich, Jintanat

2015-04-24

To assess the addition of HIV nucleic acid testing (NAT) to fourth-generation (4thG) HIV antigen/antibody combination immunoassay in improving detection of acute HIV infection (AHI). Participants attending a major voluntary counseling and testing site in Thailand were screened for AHI using 4thG HIV antigen/antibody immunoassay and sequential less sensitive HIV antibody immunoassay. Samples nonreactive by 4thG antigen/antibody immunoassay were further screened using pooled NAT to identify additional AHI. HIV infection status was verified following enrollment into an AHI study with follow-up visits and additional diagnostic tests. Among 74 334 clients screened for HIV infection, HIV prevalence was 10.9% and the overall incidence of AHI (N = 112) was 2.2 per 100 person-years. The inclusion of pooled NAT in the testing algorithm increased the number of acutely infected patients detected, from 81 to 112 (38%), relative to 4thG HIV antigen/antibody immunoassay. Follow-up testing within 5 days of screening marginally improved the 4thG immunoassay detection rate (26%). The median CD4 T-cell count at the enrollment visit was 353 cells/μl and HIV plasma viral load was 598 289 copies/ml. The incorporation of pooled NAT into the HIV testing algorithm in high-risk populations may be beneficial in the long term. The addition of pooled NAT testing resulted in an increase in screening costs of 22% to identify AHI: from $8.33 per screened patient to $10.16. Risk factors of the testing population should be considered prior to NAT implementation given the additional testing complexity and costs.

Diagnostic strategies to reveal covert infections with intestinal helminths in dogs.

PubMed

Adolph, Chris; Barnett, Sharon; Beall, Melissa; Drake, Jason; Elsemore, David; Thomas, Jennifer; Little, Susan

2017-11-30

Intestinal helminths are common in dogs in the United States, particularly non-treated dogs in animal shelters, but surveys by fecal flotation may underestimate their prevalence. To determine the prevalence of intestinal helminths and evaluate the ability of fecal flotation and detection of nematode antigen to identify those infections, contents of the entire gastrointestinal tract of 97 adult (>1year) dogs previously identified for humane euthanasia at two animal control shelters in northeastern Oklahoma, USA, were screened. All helminths recovered were washed in saline and fixed prior to enumeration and identification to genus and species. Fecal samples from each dog were examined by passive sodium nitrate (SG 1.33) and centrifugal sugar solution (SG 1.25) flotation. Fecal antigen detection assays were used to confirm the presence of nematode antigen in frozen fecal samples from 92 dogs. Necropsy examination revealed Ancylostoma caninum in 45/97 (46.4%), Toxocara canis in 11/97 (11.3%), Trichuris vulpis in 38/97 (39.2%), Dipylidium caninum in 48/97 (49.5%), and Taenia sp. in 7/97 (7.2%) dogs. Passive fecal flotation identified 38/45 (84.4%) A. caninum, 6/11 (54.5%) T. canis, 26/38 (68.4%) T. vulpis, 2/48 (4.2%) D. caninum, and 1/7 (14.3%) Taenia sp. infections, while centrifugal flotation combined with antigen detection assays identified A. caninum in 97.7% (43/44), T. canis in 77.8% (7/9), and T. vulpis in 83.3% (30/36) of infected dogs based on necropsy recovery of nematodes. Taken together, these data indicate that detection of nematode antigen is a useful adjunct to microscopic examination of fecal samples for parasite eggs, and that this approach can improve diagnostic sensitivity for intestinal nematode infections in dogs. Copyright © 2017 Elsevier B.V. All rights reserved.

Allergen concentration in natural rubber latex.

PubMed

Yeang, H-Y; Hamilton, R G; Bernstein, D I; Arif, S A M; Chow, K-S; Loke, Y-H; Raulf-Heimsoth, M; Wagner, S; Breiteneder, H; Biagini, R E

2006-08-01

Hevea brasiliensis latex serum is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis as it contains the full complement of latex allergens. This study quantifies the concentrations of the significant allergens in latex serum and examines its suitability as an antigen source in latex allergy diagnosis and immunotherapy. The serum phase was extracted from centrifuged latex that was repeatedly freeze-thawed or glycerinated. Quantitation of latex allergens was performed by two-site immunoenzymetric assays. The abundance of RNA transcripts of the latex allergens was estimated from the number of their clones in an Expressed Sequence Tags library. The latex allergens, Hev b 1, 2, 3, 4, 5, 6, 7 and 13, were detected in freeze-thawed and glycerinated latex serum at levels ranging from 75 (Hev b 6) to 0.06 nmol/mg total proteins (Hev b 4). Hev b 6 content in the latex was up to a thousand times higher than the other seven latex allergens, depending on source and/or preparation procedure. Allergen concentration was reflected in the abundance of mRNA transcripts. When used as the antigen, latex serum may bias the outcome of latex allergy diagnostic tests towards sensitization to Hev b 6. Tests that make use of latex serum may fail to detect latex-specific IgE reactivity in subjects who are sensitized only to allergens that are present at low concentrations. Latex allergy diagnostics and immunotherapy that use whole latex serum as the antigen source may not be optimal because of the marked imbalance of its constituent allergens.

Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India.

PubMed

Deneke, Yosef; Sabarinath, T; Gogia, Neha; Lalsiamthara, Jonathan; Viswas, K N; Chaudhuri, Pallab

2014-08-01

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genus Leptospira, causing febrile infection characterized by multi-organ failure in humans and animals. Leptospiral Ig-like protein B (LigB) is a surface-expressed antigen that mediates host cell invasion or attachment. In this study, N-terminal conserved region of LigB protein (46 kDa) was evaluated for its diagnostic potential to detect anti-leptospiral antibodies in the sera of various animal species. Dot blot analysis revealed immunoreactivity of Leptospira-positive sera of cattle, buffalo, dog, sheep and goat to purified LigB protein. We have analyzed 1126 bovine serum samples, collected from Northern and Eastern part of India, by microscopic agglutination test (MAT) and recombinant LigB (rLigB) based ELISA and latex agglutination test (LAT). The sensitivity of rLigB based ELISA for 554 MAT positive sera was 96.9% and the specificity with 572 MAT negative sera was 91.08% whereas LAT showed sensitivity and specificity of 93.68% and 92.31%, respectively. Kappa values of 0.879 and 0.860 for recombinant antigen based ELISA and LAT indicate excellent agreement with the gold standard serological test, MAT, for the detection of anti-leptospiral antibodies in sera. Further, LAT based on rLigB antigen is a simple and rapid test, suitable for serodiagnosis of leptospirosis under field conditions, owing to its portability and longer shelf life. Copyright © 2014 Elsevier Ltd. All rights reserved.

Invited Review Article: Review of centrifugal microfluidic and bio-optical disks

PubMed Central

Nolte, David D.

2009-01-01

Spinning biodisks have advantages that make them attractive for specialized biochip applications. The two main classes of spinning biodisks are microfluidic disks and bio-optical compact disks (BioCD). Microfluidic biodisks take advantage of noninertial pumping for lab-on-a-chip devices using noninertial valves and switches under centrifugal and Coriolis forces to distribute fluids about the disks. BioCDs use spinning-disk interferometry, under the condition of common-path phase quadrature, to perform interferometric label-free detection of molecular recognition and binding. The optical detection of bound molecules on a disk is facilitated by rapid spinning that enables high-speed repetitive sampling to eliminate 1∕f noise through common-mode rejection of intensity fluctuations and extensive signal averaging. Multiple quadrature classes have been developed, such as microdiffraction, in-line, phase contrast, and holographic adaptive optics. Thin molecular films are detected through the surface dipole density with a surface height sensitivity for the detection of protein spots that is approximately 1 pm. This sensitivity easily resolves a submonolayer of solid-support immobilized antibodies and their antigen targets. Fluorescence and light scattering provide additional optical detection techniques on spinning disks. Immunoassays have been applied to haptoglobin using protein A∕G immobilization of antibodies and to prostate specific antigen. Small protein spots enable scalability to many spots per disk for high-throughput and highly multiplexed immonoassays. PMID:19895047

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

PubMed

Takahashi, Hitoshi; Nagata, Shiho; Odagiri, Takato; Kageyama, Tsutomu

2018-04-15

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections. Copyright © 2018 Elsevier Inc. All rights reserved.

Detection of hepatitis B virus core antigen by phage display mediated TaqMan real-time immuno-PCR.

PubMed

Monjezi, Razieh; Tan, Sheau Wei; Tey, Beng Ti; Sieo, Chin Chin; Tan, Wen Siang

2013-01-01

The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10ng of HBcAg with 10(8)pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples. Copyright © 2012 Elsevier B.V. All rights reserved.

[Antigens (CEA and CA 19-9) in diagnosis and prognosis colorectal cancer].

PubMed

Grotowski, Maciej

2002-01-01

carcinoembryonic antigen (CEA) was first described more than three decades ago, when its presence was demonstrated in fetal gut tissue and in tumors from gastrointestinal tract. Subsequently, CEA was detected in the circulation of patients and recognized as a serum marker for colorectal cancer. This tumor marker has not been advocated as a screening test for colorectal cancer, however a preoperative CEA serum level is useful for diagnosis and prognosis of recurrence and survival in colorectal cancer patients. The levels of CEA increased with increasing tumor stage. Expression of carbohydrate antigen (CA 19-9) has been described in various malignancies and also in colorectal cancer. This antigen also has not been advocated as a screening test for colorectal cancer. The levels of CA 19-9 increased in advanced stages of colorectal cancer. Despite its lower sensitivity than CEA in early stages of colorectal cancer, the combination of both antigens can provided more information than CEA alone for prognosis of recurrence and survival in those patients.

Comparison of Non-Culture-Based Methods for Detection of Systemic Fungal Infections, with an Emphasis on Invasive Candida Infections

PubMed Central

White, P. Lewis; Archer, Alice E.; Barnes, Rosemary A.

2005-01-01

The accepted limitations associated with classic culture techniques for the diagnosis of invasive fungal infections have lead to the emergence of many non-culture-based methods. With superior sensitivities and quicker turnaround times, non-culture-based methods may aid the diagnosis of invasive fungal infections. In this review of the diagnostic service, we assessed the performances of two antigen detection techniques (enzyme-linked immunosorbent assay [ELISA] and latex agglutination) with a molecular method for the detection of invasive Candida infection and invasive aspergillosis. The specificities for all three assays were high (≥97%), although the Candida PCR method had enhanced sensitivity over both ELISA and latex agglutination with values of 95%, 75%, and 25%, respectively. However, calculating significant sensitivity values for the Aspergillus detection methods was not feasible due to a low number of proven/probable cases. Despite enhanced sensitivity, the PCR method failed to detect nucleic acid in a probable case of invasive Candida infection that was detected by ELISA. In conclusion, both PCR and ELISA techniques should be used in unison to aid the detection of invasive fungal infections. PMID:15872239

Detection and localization of specific antigens in the reproductive tracts of cycling, pregnant, and ovariectomized hamsters.

PubMed

Fox, L L; Shivers, C A

1975-06-01

A systematic search was made for components specific to the female reproductive tract in golden hamsters. Antisera produced in rabbits against saline homogenates of hamster uteri (collected on the night of estrus) cross-reacted extensively with extracts of 12 other tissues in agar gel double-diffusion assays. Absorption of the antisera with small intestine, lung, and liver rendered the immune sera specific for uterine and oviductal antigens (within the limits of the sensitivity of the precipitin assays). Immunoelectrophoretic analysis resolved 12 uterine antigens, many of which were similar to components in several other tissues. Absorbed antisera specific for reproductive tract antigens formed one postalbumin arc with uterine and oviductal extracts in immunoelectrophoretic studies. No reactions were detected between specific antisera and five other organ extracts or plasma. An indirect immunofluorescent antibody technique was used to detect changes in the distribution of specific antigens in reproductive tracts of cycling, pregnant, and ovariectomized hamsters. The gamma-globulin fraction of anti-uterus sera (absorbed with small intestine, lung, and liver), shown to be specific for reproductive tract tissues in precipitin tests, was used to localize antigens. Appropriate controls indicated that the fluorescence observed was due to antigen-antibody interactions. During the cycle, specific antigens were usually confined to the ampullary lamina propria, except during estrus, when they were prominent in the lamina propria and luminal epithelium of the ampula. Specific antigens were never abundant in the isthmus of nonpregnant hamsters. On day 1 postcoitum, the components were found throughout the ampullary and isthmic regions. By day 2 postcoitum, ampullary antigens were usually confined to the lamina propria. The specific components were not prominent in the oviduct on day 3 postcoitum, but were conspicuous in both ampulla and isthmus on day 4. Specific antigens in the uterus were confined to endometrial glands in nonpregnant animals during proestrus, estrus, and (occasionally) metestrus. Diestrous uteri contained no specific antigens. During the first 2 days of pregnancy, antigens were not abundant and were usually confined to the glands and stroma. On days 3 and 4 of pregnancy the specific antigens were prominent in the endometrial glands and stroma and along the apical borders of some luminal epithelial cells. By day 5, these components were less conspicuous in all areas of the endometrium. Uteri of spayed animals receiving no hormones or estradiol alone lacked the specific antigens. However, progesterone (after estrogen priming) promoted the appearance of these components, and the distribution resembled that seen in uteri of 3- and 4-day pregnant animals.

A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis

PubMed Central

Cortina, María E.; Balzano, Rodrigo E.; Rey Serantes, Diego A.; Caillava, Ana J.; Elena, Sebastián; Ferreira, A. C.; Nicola, Ana M.; Ugalde, Juan E.

2016-01-01

Brucellosis is a highly zoonotic disease that affects animals and human beings. Brucella suis is the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide–protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected with B. suis biovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of “smooth” brucellosis in animals and humans. PMID:26984975

Rapid and broad detection of H5 hemagglutinin by an immunochromatographic kit using novel monoclonal antibody against highly pathogenic avian influenza virus belonging to the genetic clade 2.3.4.4

PubMed Central

Nguyen, Lam Thanh; Nakaishi, Kazunari; Motojima, Keiko; Ohkawara, Ayako; Minato, Erina; Maruyama, Junki; Hiono, Takahiro; Matsuno, Keita; Okamatsu, Masatoshi; Kimura, Takashi; Takada, Ayato; Kida, Hiroshi; Sakoda, Yoshihiro

2017-01-01

Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds. PMID:28787440

CMOS image sensor for detection of interferon gamma protein interaction as a point-of-care approach.

PubMed

Marimuthu, Mohana; Kandasamy, Karthikeyan; Ahn, Chang Geun; Sung, Gun Yong; Kim, Min-Gon; Kim, Sanghyo

2011-09-01

Complementary metal oxide semiconductor (CMOS)-based image sensors have received increased attention owing to the possibility of incorporating them into portable diagnostic devices. The present research examined the efficiency and sensitivity of a CMOS image sensor for the detection of antigen-antibody interactions involving interferon gamma protein without the aid of expensive instruments. The highest detection sensitivity of about 1 fg/ml primary antibody was achieved simply by a transmission mechanism. When photons are prevented from hitting the sensor surface, a reduction in digital output occurs in which the number of photons hitting the sensor surface is approximately proportional to the digital number. Nanoscale variation in substrate thickness after protein binding can be detected with high sensitivity by the CMOS image sensor. Therefore, this technique can be easily applied to smartphones or any clinical diagnostic devices for the detection of several biological entities, with high impact on the development of point-of-care applications.

Highly sensitive detection of target molecules using a new fluorescence-based bead assay

NASA Astrophysics Data System (ADS)

Scheffler, Silvia; Strauß, Denis; Sauer, Markus

2007-07-01

Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

Label-Free Electrical Immunosensor for Highly Sensitive and Specific Detection of Microcystin-LR in Water Samples.

PubMed

Tan, Feng; Saucedo, Nuvia Maria; Ramnani, Pankaj; Mulchandani, Ashok

2015-08-04

Microcystin-LR (MCLR) is one of the most commonly detected and toxic cyclic heptapeptide cyanotoxins released by cyanobacterial blooms in surface waters, for which sensitive and specific detection methods are necessary to carry out its recognition and quantification. Here, we present a single-walled carbon nanotube (SWCNTs)-based label-free chemiresistive immunosensor for highly sensitive and specific detection of MCLR in different source waters. MCLR was initially immobilized on SWCNTs modified interdigitated electrode, followed by incubation with monoclonal anti-MCLR antibody. The competitive binding of MCLR in sample solutions induced departure of the antibody from the antibody-antigen complexes formed on SWCNTs, resulting in change in the conductivity between source and drain of the sensor. The displacement assay greatly improved the sensitivity of the sensor compared with direct immunoassay on the same device. The immunosensor exhibited a wide linear response to log value of MCLR concentration ranging from 1 to 1000 ng/L, with a detection limit of 0.6 ng/L. This method showed good reproducibility, stability and recovery. The proposed method provides a powerful tool for rapid and sensitive monitoring of MCLR in environmental samples.

The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters.

PubMed

Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

2017-01-01

Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains.

The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters

PubMed Central

Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

2017-01-01

Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains. PMID:28224115

Production of Monoclonal Antibody Against Excretory-Secretory Antigen of Fasciola hepatica and Evaluation of Its Efficacy in the Diagnosis of Fascioliasis.

PubMed

Abdolahi Khabisi, Samaneh; Sarkari, Bahador; Moshfe, Abdolali; Jalali, Sedigheh

2017-02-01

Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately used for the diagnosis of fascioliasis.

Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii.

PubMed

Tomás, A L; Cardoso, F; Esteves, F; Matos, O

2016-11-08

Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis.

Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii

PubMed Central

Tomás, A. L.; Cardoso, F.; Esteves, F.; Matos, O.

2016-01-01

Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis. PMID:27824115

Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods.

PubMed

Raj, G Dhinakar; Rajanathan, T M C; Kumar, C Senthil; Ramathilagam, G; Hiremath, Geetha; Shaila, M S

2008-06-22

Peste des petits ruminants (PPR) is one of the most economically important diseases affecting sheep and goats in India. An immunofiltration-based test has been developed using either mono-specific serum/monoclonal antibodies (mAb) prepared against a recombinant truncated nucleocapsid protein of rinderpest virus (RPV) cross-reactive with PPR virus. This method consists of coating ocular swab eluate from suspected animals onto a nitrocellulose membrane housed in a plastic module, which is allowed to react with suitable dilutions of a mAb or a mono-specific polyclonal antibody. The antigen-antibody complex formed on the membrane is then detected by protein A-colloidal gold conjugate, which forms a pink colour. In the immunofiltration test, concordant results were obtained using either PPRV mAb or mono-specific serum. Another test, an antigen-competition ELISA which relies on the competition between plate-coated recombinant truncated 'N' protein of RPV and the PPRV 'N' protein present in ocular swab eluates (sample) for binding to the mono-specific antibody against N protein of RPV (in liquid phase) was developed. The cut-off value for this test was established using reverse transcription polymerase chain reaction (RT-PCR) positive and negative oculo-nasal swab samples. Linear correlation between percent inhibition (PI) values in antigen-competition ELISA and virus infectivity titres was 0.992. Comparison of the immunofiltration test with the antigen-competition ELISA yielded a sensitivity of 80% and specificity of 100%. These two tests can serve as a screening (immunofiltration) and confirmatory (antigen-competition ELISA) test, respectively, in the diagnosis of PPR in sheep or goats.

Ring-Resonator/Sol-Gel Interferometric Immunosensor

NASA Technical Reports Server (NTRS)

Bearman, Gregory; Cohen, David

2007-01-01

A proposed biosensing system would be based on a combination of (1) a sensing volume containing antibodies immobilized in a sol-gel matrix and (2) an optical interferometer having a ring resonator configuration. The antibodies would be specific to an antigen species that one seeks to detect. In the ring resonator of the proposed system, light would make multiple passes through the sensing volume, affording greater interaction length and, hence, greater antibody- detection sensitivity.

Should varicella-zoster virus culture be eliminated? A comparison of direct immunofluorescence antigen detection, culture, and PCR, with a historical review.

PubMed

Wilson, D A; Yen-Lieberman, B; Schindler, S; Asamoto, K; Schold, J D; Procop, G W

2012-12-01

A comparison of direct fluorescent-antibody assay (DFA), culture, and two PCR assays disclosed sensitivities of 87.8%, 46.3%, and 97.6% and 100%, respectively. We reviewed 1,150 results for clinical specimens submitted for DFA and culture and found that only 17 were culture positive/DFA negative. The incremental cost to detect these 17 positives was $3,078/specimen.

In vitro evaluation of five rapid antigen detection tests for group A beta-haemolytic streptococcal sore throat infections.

PubMed

Lasseter, Gemma M; McNulty, Cliodna A M; Richard Hobbs, F D; Mant, David; Little, Paul

2009-12-01

Using accurate and easy to use rapid antigen detection tests (RADTs) to identify group A beta-haemolytic Streptococci (GABHS) sore throat infections could reduce unnecessary antibiotic prescribing and antimicrobial resistance. Although there is no international consensus on the use of RADTs, these kits have been widely adopted in Finland, France and the USA. Yet in the UK, the Clinical Knowledge Summaries, that provide the main online guidance for GPs, discourage RADTs use, citing their poor sensitivity and inability to impact on prescribing decisions in acute sore throat infections. The purpose of this study was to evaluate the ease of use and in vitro accuracy (sensitivity and specificity) of the five most commonly used RADTs in Europe (OSOM Ultra, Quickvue Dipstick, Streptatest, Clearview Exact Strep A and IMI Test Pack). To ensure the RADTs were evaluated objectively, a standardized in vitro method using known concentrations of GABHS was used to remove the inherent biases associated with clinical studies. The IMI Test Pack was the easiest RADT to use overall. The ability to detect all positive GABHS (sensitivity) varied considerably between kits from 95% [95% confidence interval (CI): 88-98%], for the IMI Test Pack and OSOM, to 62% (95% CI: 51-72%) for Clearview, at the highest GABHS concentration. None of the RADTs gave any false-positive results with commensal flora-they were 100% specific. The IMI Test Pack is most suitable for use in primary care, as it had high sensitivity, high specificity and was easy to use.

Detection of lymphocystis disease virus (LCDV) in asymptomatic cultured gilt-head seabream (Sparus aurata, L.) using an immunoblot technique.

PubMed

Cano, I; Alonso, M C; Garcia-Rosado, E; Saint-Jean, S Rodriguez; Castro, D; Borrego, Juan J

2006-03-10

An immunoblot technique for the detection of lymphocystis disease virus (LCDV) in naturally infected gilt-head seabream (Sparus aurata, L.) has been developed. A specific antiserum against a 60 kDa viral protein has been proven to be an appropriate tool for LCDV diagnosis either from inoculated cell cultures or from fish tissues using the immunoblot assay. The sensitivity of this technique varied between 10(-1) and 10(2) TCID50. LCDV has also been detected in fish tissues from both, diseased and asymptomatic gilt-head seabream. For the asymptomatic fish detection, a viral amplification step in cell culture and a subsequent viral concentration using polyethylene glycol (PEG) (600 wt) are required. On the contrary, immunoblot allowed the detection of LCDV antigens directly from tissue homogenates of diseased fish. The method described in this study shows higher sensitivity than classical detection techniques based on cell culture inoculation.

Liver cancer immunoassay with magnetic nanoparticles and MgO-based magnetic tunnel junction sensors

NASA Astrophysics Data System (ADS)

Lei, Z. Q.; Li, L.; Li, G. J.; Leung, C. W.; Shi, J.; Wong, C. M.; Lo, K. C.; Chan, W. K.; Mak, C. S. K.; Chan, S. B.; Chan, N. M. M.; Leung, C. H.; Lai, P. T.; Pong, P. W. T.

2012-04-01

We have demonstrated the detection of alpha-fetoprotein (AFP) labeled with magnetic nanoparticles (MNPs) using MgO-based magnetic tunnel junction (MTJ) sensors. AFP is an important hepatic tumor biomarker and the detection of AFP has significant applications for clinical diagnostics and immunoassay for early-stage liver cancer indications. In this work, MgO-based MTJ sensors and 20-nm iron-oxide magnetic nanoparticles (MNPs) were used for detecting AFP antigens by a sandwich-assay configuration. The MTJ sensors with a sensing area of 4 × 2 μm2 possess tunneling magnetoresistance (TMR) of 122% and sensitivity of 0.95%/Oe at room temperature. The target AFP antigens of three concentrations were successfully detected, and the experimental data indicate that the resistance variations of the MTJ sensor increased with the AFP concentration ratios proportionally. These results demonstrate that MgO-based MTJ sensors together with MNPs are a promising biosensing platform for liver cancer immunoassay.

Canine parvovirus--a review of epidemiological and diagnostic aspects, with emphasis on type 2c.

PubMed

Decaro, Nicola; Buonavoglia, Canio

2012-02-24

Canine parvovirus type 2 (CPV-2) emerged in late 1970s causing severe epizootics in kennels and dog shelters worldwide. Soon after its emergence, CPV-2 underwent genetic evolution giving rise consecutively to two antigenic variants, CPV-2a and CPV-2b that replaced progressively the original type. In 2000, a new antigenic variant, CPV-2c, was detected in Italy and rapidly spread to several countries. In comparison to the original type CPV-2, the antigenic variants display increased pathogenicity in dogs and extended host range, being able to infect and cause disease in cats. Epidemiological survey indicate that the newest type CPV-2c is becoming prevalent in different geographic regions and is often associated to severe disease in adult dogs and also in dogs that have completed the vaccination protocols. However, the primary cause of failure of CPV vaccination is interference by maternally derived immunity. Diagnosis of CPV infection by traditional methods has been shown to be poorly sensitive, especially in the late stages of infections. New diagnostic approaches based on molecular methods have been developed for sensitive detection of CPV in clinical samples and rapid characterisation of the viral type. Continuous surveillance will help assess whether there is a real need to update currently available vaccines and diagnostic tests. Copyright © 2011 Elsevier B.V. All rights reserved.

Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay.

PubMed

Li, Ming; Kang, Jeon Woong; Sukumar, Saraswati; Dasari, Ramachandra Rao; Barman, Ishan

2015-07-01

Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognostication and monitoring of therapeutic response. Emerging data, however, suggest that single markers are inadequate in describing complex pathologic transformations. Architecting assays capable of parallel measurements of multiple biomarkers can help achieve the desired clinical sensitivity and specificity while conserving patient specimen and reducing turn-around time. Here we describe a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomolecular probes and spectroscopic imaging for multiplexed detection of disseminated breast cancer markers cancer antigen (CA) 15-3, CA 27-29 and cancer embryonic antigen (CEA). In the developed SERS assay, both the assay chip and surface-enhanced Raman spectroscopy (SERS) tags are functionalized with monoclonal antibodies against CA15-3, CA27-29 and CEA, respectively. Sequential addition of biomarkers and functionalized SERS tags onto the functionalized assay chip enable the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addition to offering extensive multiplexing capability, our method provides higher sensitivity than conventional immunoassays and demonstrates exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We envision that clinical translation of this assay may further enable asymptomatic surveillance of cancer survivors and speedy assessment of treatment benefit through a simple blood test.

Development and validation of an Enzyme Linked Immunosorbent Assay (ELISA) test for the diagnosis of toxoplasmosis in Sri Lanka.

PubMed

Iddawela, D; Ehambaram, K; Kumarasiri, P V; Wijesundera, S

2015-09-01

ELISA is the most widely used form of diagnosis for toxoplasmosis. Several commercial kits are currently used in Sri Lanka. However, these kits are not affordable in resource-limited settings. Objectives Aim of this study was to develop a cost effective in-house ELISA for the detection of Toxoplasma antibody and to estimate the diagnostic accuracy compared to a commercial kit. Vero cell lines were inoculated with tachyzoites and harvested after 2-6 days and sonicated to obtain somatic antigen. The antigen was used as coating material in ELISA to detect antibodies against T. gondii in patient sera. Hundred and three patients' sera were analysed by in-house ELISA and kit ELISA. Optical density (OD) values were analysed statistically. Toxoplasma IgG avidity test was used to determine the chronic and acute phase of infection. The optimum working dilutions for antigen was 0.846 μg/ml and for serum 1 in 100. The optimal cut-off values for the in-house ELISA within the range 0.85 to 0.98 at which the sensitivity was 95.3% and specificity was 98.3. The OD values of in-house ELISA were compared with OD values of kit ELISA and the results showed strong correlation between the two tests. The results of our study demonstrated that our in-house ELISA for detection of T. gondii antibody was as sensitive and specific as the commercial kit used in this study. Thus, the in-house ELISA is a useful, costeffective tool for diagnostic and screening purposes.

Application and evaluation of enzyme-linked immunosorbent assay and immunoblotting for detection of antibodies to Treponema hyodysenteriae in swine.

PubMed Central

Smith, S. C.; Barrett, L. M.; Muir, T.; Christopher, W. L.; Coloe, P. J.

1991-01-01

An enzyme-linked immunoassay (ELISA) has been developed to detect serum Immunoglobulin antibodies G and M to Treponema hyodysenteriae in vaccinated, experimentally infected and naturally infected swine. Naturally infected swine gave ELISA titres that were similar to experimentally infected swine, but were significantly less than the titres of vaccinated swine. When serum from naturally infected swine was used to probe nitrocellulose blots of sodium dodecyl sulphate-polyacrylamide gel electrophoresed whole cell proteins of T. hyodysenteriae, the immunoblotting patterns showed IgG antibodies were produced against many T. hyodysenteriae protein antigens and against lipopolysaccharide (LPS). The IgG antibodies directed against LPS were serotype-specific for that LPS and could be used to identify the serotype involved in the T. hyodysenteriae infection in that herd. IgM immunoblots also reacted with the many protein antigens but were less specific for LPS antigen, with a substantial degree of cross-reaction between the LPS of all serotypes. The data demonstrate that a microplate enzyme-linked immunosorbent assay, coupled with immunoblotting, is a very specific and sensitive test for detection of antibody to Treponema hyodysenteriae in swine. Images Fig. 3 Fig. 4 PMID:1936151

A simple and sensitive immunoassay for the determination of human chorionic gonadotropin by graphene-based chemiluminescence resonance energy transfer.

PubMed

Lei, Jiuqian; Jing, Tao; Zhou, Tingting; Zhou, Yusun; Wu, Wei; Mei, Surong; Zhou, Yikai

2014-04-15

In this study, we report a strategy of chemiluminescence resonance energy transfer (CRET) using graphene as an efficient long-range energy acceptor. Magnetic nanoparticles were also used in CRET for simple magnetic separation and immobilization of horseradish peroxidase (HRP)-labeled anti-HCG antibody. In the design of CRET system, the sandwich-type immunocomplex was formed between human chorionic gonadotropin (HCG, antigen) and two different antibodies bridged the magnetic nanoparticles and graphene (acceptors), which led to the occurrence of CRET from chemiluminescence light source to graphene. After optimizing the experimental conditions, the quenching of chemiluminescence signal depended linearly on the concentration of HCG in the range of 0.1 mIU mL(-1)-10 mIU mL(-1) and the detection limit was 0.06 mIU mL(-1). The proposed method was successfully applied for the determination of HCG levels in saliva and serum samples, and the results were in good agreement with the plate ELISA with colorimetric detection. It could also be developed for detection of other antigen-antibody immune complexes by using the corresponding antigens and respective antibodies. © 2013 Published by Elsevier B.V.

A silicon dioxide modified magnetic nanoparticles-labeled lateral flow strips for HBs antigen.

PubMed

Zhang, Xueqing; Jiang, Lin; Zhang, Chunlei; Li, Ding; Wang, Can; Gao, Feng; Cui, Daxiang

2011-12-01

Herein we reported a new type of silicon dioxide wrapped magnetic nanoparticles-labeled lateral flow strip for detection of HBs antigen in sera. The SiO2 wrapped Fe3O4 nanocomposites were prepared and characterized by HR-TEM, FTIR and magnetometer. As-prepared nanocomposites were used to label anti-HBV surface monoclonal antibody, the lateral flow strips were constructed, and 100 specimens of sera were collected and tested. Results showed that the prepared SiO2 wrapped Fe3O4 nanocomposites were shell/core structure, well dispersed, with the size of 25 nm in diameter, the thickness of the shell was about 3 nm, their magnetic saturation intensity was 44.3 meu g(-1). Clinical sera specimens test results showed that the prepared lateral flow strips were with the detection limitation of 5 pg/mL by naked eye observation, and 0.1 pg/mL by CCD reader or MAR Analyzer, specificity was 100%. In conclusion, one kind of silicon dioxide wrapped magnetic nanoparticles-labeled lateral flow strip for ultrasensitive detection of HBs antigen was successfully developed, its ease of use, sensitiveness and low-cost make it well-suited for population-based on-the-site hepatitis B screening.

[Membrane-filtration immunoassay: reagents, methods and the diagnostic and technical means for detection].

PubMed

Khramov, E N; Osin, N S; Pomelova, V G; Vikha, I V; Bychenkova, T A; Smirnova, V G; Grakina, G I; Kas'ianova, T A

1999-01-01

The comprehensive development of dot-EIA made at the State Research Institute of Biological Instrument-Making Industry has provided devices KIMF-02 and KIMF-03), a base of chemical reagents, immunoassays, test systems for detection of a wide range of causative agents of viral and bacterial infections, that of serodiagnosis of their related diseases. The KIMF-02 kit has undergone engineering and medical tests and recommended for the Ministry of Health of the Russian Federation to produce them in stock. The kit includes all required for analysis even in an ill-equipped laboratory, a set of attached agents ensures a valid visual recording of results. The developed procedures and test systems allow the immunoassay to be as sensitive as TIFA; however, they are laborious and much simpler in design. The simple and rapid procedures of dot-EIA are recommended for incorporation into the a package of laboratory methods for verification of the accumulation of virus-specific antigens in various biological substrata, environmental samples, for control of the activity of antigens and antibodies used in serological tests, for detection of specific antigens in the clinical samples, and for serodiagnosis of infections.

Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

PubMed

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-09-08

Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

Rapid, ultrasensitive detection of microorganisms based on interferometry and lab-on-a-chip nanotechnology

NASA Astrophysics Data System (ADS)

Ymeti, Aurel; Nederkoorn, Paul H. J.; Dudia, Alma; Subramaniam, Vinod; Kanger, Johannes S.

2009-05-01

Future viral outbreaks are a major threat to societal and economic development throughout the world. A rapid, sensitive, and easy-to-use test for viral infections is essential to prevent and to control such viral pandemics. Furthermore, a compact, portable device is potentially very useful in remote or developing regions without easy access to sophisticated laboratory facilities. We have developed a rapid, ultrasensitive sensor that could be used in a handheld device to detect various viruses and measure their concentration. The essential innovation in this technique is the combination of an integrated optical interferometric sensor with antibody-antigen recognition approaches to yield a very sensitive, very rapid test for virus detection. The sensor is able to spot the herpes virus at concentrations of just 850 particles per milliliter under physiological conditions. The sensitivity of the sensor approaches detection of a single virus particle, yielding a sensor of unprecedented sensitivity with wide applications for viral diagnostics. The sensor's detection principle can be extended to any biological target such as bacteria, cells and proteins and for which there are specific antibodies. The nature of the sensor enables multiplexed detection of several analytes at the same time.

Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

USGS Publications Warehouse

Pascho, Ronald J.; Chase, Dorothy M.; McKibben, Constance L.

1998-01-01

Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 × 109cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 × 104cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

A Coproantigen Diagnostic Test for Strongyloides Infection

PubMed Central

Sykes, Alex M.; McCarthy, James S.

2011-01-01

Accurate diagnosis of infection with the parasite Strongyloides stercoralis is hampered by the low concentration of larvae in stool, rendering parasitological diagnosis insensitive. Even if the more sensitive agar plate culture method is used repeated stool sampling is necessary to achieve satisfactory sensitivity. In this manuscript we describe the development of a coproantigen ELISA for diagnosis of infection. Polyclonal rabbit antiserum was raised against Strongyloides ratti excretory/secretory (E/S) antigen and utilized to develop an antigen capture ELISA. The assay enabled detection of subpatent rodent S. ratti and human S. stercoralis infection. No cross-reactivity was observed with purified E/S from Schistosoma japonicum, the hookworms Ancylostoma caninum, A. ceylanicum, nor with fecal samples collected from rodents harboring Trichuris muris or S. mansoni infection. Strongyloides coproantigens that appear stable when frozen as formalin-extracted fecal supernatants stored at −20°C remained positive up to 270 days of storage, whereas supernatants stored at 4°C tested negative. These results indicate that diagnosis of human strongyloidiasis by detection of coproantigen is an approach worthy of further development. PMID:21347447

Tissue polypeptide specific antigen (TPS) as a tumor marker in renal cell carcinoma.

PubMed

Chang, Chao-Hsiang; Wu, His-Chin; Yen, Ruoh-Fang; Kao, Albert; Lin, Cheng-Chieh; Lee, Cheng-Chun

2002-01-01

Tissue polypeptide specific antigen (TPS) is a new tumor marker that indicates tumor proliferative rate rather than tumor burden. The purpose of this study was to assess the clinical value of TPS in patients with renal cell carcinoma (RCC). Serum levels of TPS were measured in 30 patients with locoregional and in 20 patients with advanced disease before and after therapy. The results showed that: (1) the detection sensitivity of TPS for RCC is 60.0%; (2) the detection sensitivity of TPS in advanced RCC (100.0%) was significantly higher than in locoregional RCC (33.3%); (3) the 10 locoregional RCC patients without recurrence had normal serum TPS levels during a follow-up period of 1 year; and (4) the 8 advanced RCC patients with good response during therapy had normal serum TPS levels, while 12 patients with poor disease had significantly elevated serum TPS levels during a follow-up period of 1 year. Our results suggest that TPS may have a potential clinical role as a valuable tumor marker for RCC, especially in advanced diseases and follow-up therapy response.

Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model.

PubMed

Zhong, Zhaoyang; Wu, Wei; Wang, Dong; Wang, Dan; Shan, Jinlu; Qing, Yi; Zhang, Zhimin

2010-06-15

A new, highly sensitive electrochemical immunosensor with a sandwich-type immunoassay format was designed to quantify carcinoembryonic antigen (CEA), as a model tumor marker, using nanogold-enwrapped graphene nanocomposites (NGGNs) as trace labels in clinical immunoassays. The device consisted of a glassy carbon electrode coated with Prussian Blue (PB) on whose surface gold nanoparticles were electrochemically deposited to the further modified with the specific analyte-capturing molecule, anti-CEA antibodies. The immunoassay was performed using horseradish peroxidase (HRP)-conjugated anti-CEA as secondary antibodies attached on the NGGN surface (HRP-anti-CEA-NGGN). The method using HRP-anti-CEA-NGGNs as detection antibodies shows high signal amplification, and exhibits a dynamic working range of 0.05-350 ng/mL with a low detection limit of 0.01 ng/mL CEA (at 3s). The assayed results of serum samples with the sensor received an acceptable agreement with the reference values. Importantly, the methodology provides a promising ultrasensitive assay strategy for clinical applications. Copyright 2010 Elsevier B.V. All rights reserved.

Use of immunoblotting to detect Aspergillus fumigatus antigen in sera and urines of rats with experimental invasive aspergillosis.

PubMed Central

Yu, B; Niki, Y; Armstrong, D

1990-01-01

Immunoblotting was used to detect Aspergillus fumigatus antigen in sera and urines of immunosuppressed rats experimentally infected with A. fumigatus. Organisms were administered by both intravenous and intratracheal injections. Intravenously infected rats developed disseminated aspergillosis, but intratracheally infected rats developed pulmonary disease only. Fungal cultures of blood and urine samples from infected rats were negative. In the urines of intravenously infected rats, antigen was detected 24 to 48 h after infection; in the urines of intratracheally infected animals, antigen was detected on days 4 to 5 after infection. Antigen in serum was detected later than antigen in urine was. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of serum and urine samples, the most strongly reacting antigenic materials were found in the 88-, 40-, 27-, and 20-kilodalton regions. These dominant antigens appeared to be the same as those of control antigens prepared from A. fumigatus grown in vitro. Rabbit antiserum to Aspergillus filtrate antigen was found to be more immunoreactive than antiserum to mycelial or conidial antigen. No mycelium-specific antigens were detected. Images PMID:2199519

Evaluation of the rapid influenza detection tests GOLD SIGN FLU and Quick Navi-Flu for the detection of influenza A and B virus antigens in adults during the influenza season.

PubMed

Akaishi, Yu; Matsumoto, Tetsuya; Harada, Yoshimi; Hirayama, Yoji

2016-11-01

As the characteristics and accuracy of rapid influenza detection tests (RIDTs) vary, the development of a high-performance RIDT has been eagerly anticipated. In this study, the new RIDT GOLD SIGN FLU and the existing RIDT Quick Navi-Flu were evaluated in terms of detecting the antigens of influenza viruses A and B in Japanese adults with influenza-like symptoms. The study was performed from December 2013 to March 2014. Among the 123 patients from whom nasopharyngeal swab specimens were collected, 59 tested positive by viral isolation as the gold standard method (influenza A, n=38; influenza B, n=21). For GOLD SIGN FLU, the sensitivities were 73.7% and 81.0%, and the specificities were 97.6% and 98.0% for influenza A and B, respectively. For Quick Navi-Flu, the sensitivities were 86.8% and 85.7%, and the specificities were 98.8% and 100% for influenza A and B, respectively. The time to the appearance of the line on the test strip was less than 3min for influenza A and less than 2min for influenza B with both RIDTs in more than 90% of cases. GOLD SIGN FLU was useful for diagnosing influenza A, and the result was readily available for influenza B particularly among adult patients. Quick Navi-Flu showed better sensitivities and specificities than GOLD SIGN FLU. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

A one-step duplex rRT-PCR assay for the simultaneous detection of duck hepatitis A virus genotypes 1 and 3.

PubMed

Hu, Qin; Zhu, Dekang; Ma, Guangpeng; Cheng, Anchun; Wang, Mingshu; Chen, Shun; Jia, Renyong; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Chen, Xiaoyue

2016-10-01

Duck hepatitis A virus (DHAV) is a highly infectious pathogen that causes significant bleeding lesions in the viscera of ducklings less than 3 weeks old. There are three serotypes of DHAV: serotype 1 (DHAV-1), serotype 2 (DHAV-2) and serotype 3 (DHAV-3). These serotypes have no cross-antigenicity with each other. To establish an rRT-PCR assay for the rapid detection of a mixed infection of DHAV-1 and DHAV-3, two pairs of primers and a pair of matching TaqMan probes were designed based on conserved regions of DHAV-1 VP0 and DHAV-3 VP3. Finally, we established a one-step duplex rRT-PCR assay with high specificity and sensitivity for the simultaneous detection of DHAV-1 and DHAV-3. This method showed no cross-antigenicity with the other pathogens tested, including duck plague virus, Muscovy duck parvovirus, Riemerella anatipestifer, and pathogenic E. coli from ducks. Sensitivity tests identified the minimum detection limits of this method as 98 (DHAV-1) and 10 (DHAV-3) copies/reaction. To validate the method, thirty-eight clinical samples and thirty artificially infected samples collected from dead duck embryos were studied. Thirty-seven samples were positive for DHAV-1, seventeen samples were positive for DHAV-3, and fourteen samples were positive for a mixed infection using the duplex rRT-PCR method. The method established in this study is specific, sensitive, convenient and timesaving and is a powerful tool for detecting DHAV-1, DHAV-3, and their mixed infection and for conducting surveys of pandemic virus strains. Copyright © 2016. Published by Elsevier B.V.

A simple and inexpensive point-of-care test for hepatitis B surface antigen detection: serological and molecular evaluation.

PubMed

Gish, R G; Gutierrez, J A; Navarro-Cazarez, N; Giang, K; Adler, D; Tran, B; Locarnini, S; Hammond, R; Bowden, S

2014-12-01

Early identification of chronic hepatitis B is important for optimal disease management and prevention of transmission. Cost and lack of access to commercial hepatitis B surface antigen (HBsAg) immunoassays can compromise the effectiveness of HBV screening in resource-limited settings and among marginalized populations. High-quality point-of-care (POC) testing may improve HBV diagnosis in these situations. Currently available POC HBsAg assays are often limited in sensitivity. We evaluated the NanoSign(®) HBs POC chromatographic immunoassay for its ability to detect HBsAg of different genotypes and with substitutions in the 'a' determinant. Thirty-seven serum samples from patients with HBV infection, covering HBV genotypes A-G, were assessed for HBsAg titre with the Roche Elecsys HBsAg II quantification assay and with the POC assay. The POC assay reliably detected HBsAg at a concentration of at least 50 IU/mL for all genotypes, and at lower concentrations for some genotypes. Eight samples with substitutions in the HBV 'a' determinant were reliably detected after a 1/100 dilution. The POC strips were used to screen serum samples from 297 individuals at risk for HBV in local clinical settings (health fairs and outreach events) in parallel with commercial laboratory HBsAg testing (Quest Diagnostics EIA). POC testing was 73.7% sensitive and 97.8% specific for detection of HBsAg. Although the POC test demonstrated high sensitivity over a range of genotypes, false negatives were frequent in a clinical setting. Nevertheless, the POC assay offers advantages for testing in both developed and resource-limited countries due to its low cost (0.50$) and immediately available results. © 2014 John Wiley & Sons Ltd.

Development and evaluation of a highly sensitive immunochromatographic strip test using gold nanoparticle for direct detection of Vibrio cholerae O139 in seafood samples.

PubMed

Pengsuk, Chalinan; Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

2013-04-15

A strip test for the detection of Vibrio cholerae O139 was developed using two monoclonal antibodies (MAbs), namely VC-273 and VC-812, which specifically bind to the lipopolysaccharide and capsular polysaccharide of V. cholerae O139. The MAb VC-273 gold nanoparticle conjugate was sprayed onto a glass fiber pad that was placed adjacent to a sample chamber. MAb VC-812 and the goat anti-mouse immunoglobulin G (GAM) antibody were sprayed onto a nitrocellulose membrane in strips at positions designated as T and C, respectively. The test strips were assessed for their ability to directly detect V. cholerae O139 using samples dispersed in application buffer, and a 100 μL aliquot of sample was applied to the sample chamber. The results were observable within 20 min after application of the sample. In samples containing V. cholerae O139, the antigen was bound to the colloidal gold-conjugated MAb to form an antibody-antigen complex. This complex was captured by the MAbs at the T test line, resulting in the appearance of a reddish-purple band at the T position. The sensitivity of the test was determined to be 10⁴ cfu mL⁻¹. Direct detection of V. cholerae O139 in various fresh seafood samples could be accomplished with similar sensitivities. The detection limit was substantially improved to 1 cfu mL⁻¹ of the original bacterial content after pre-incubation of the sample in alkaline peptone water for 12 h. The V. cholerae strip test provides several advantages over other methods, including the speed and simplicity of use because there is no requirement for sophisticated equipment. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of rapid immunochromatographic test for hemagglutinin antigen of H7 subtype in patients infected with novel avian influenza A (H7N9) virus.

PubMed

Kang, Keren; Chen, Li; Zhao, Xiang; Qin, Chengfeng; Zhan, Zanwu; Wang, Jihua; Li, Wenmei; Dzakah, Emmanuel E; Huang, Weijuang; Shu, Yuelong; Jiang, Tao; Cao, Wuchun; Xie, Mingquan; Luo, Xiaochun; Tang, Shixing

2014-01-01

Since human infection with the novel H7N9 avian influenza virus was identified in China in March 2013, the relatively high mortality rate and possibility of human-to-human transmission have highlighted the urgent need for sensitive and specific assays for diagnosis of H7N9 infection. We developed a rapid diagnostic test for the novel avian influenza A (H7N9) virus using anti-hemagglutinin (HA) monoclonal antibodies specifically targeting H7 in an immunochromatographic assay system. The assay limit of detection was 103.5 pfu/ml or 103TCID50 of H7N9 virus. The assay specifically detected H7N9 viral isolates and recombinant HA proteins of H7 subtypes including H7N7 and H7N9, but did not react with non-H7 subtypes including H1N1, H3N2, H5N1, H5N9, and H9N2. The detection sensitivity was 59.4% (19/32) for H7N9 patients confirmed by RT-PCR. Moreover, the highest sensitivity of 61.5% (16/26) was obtained when testing H7N9 positive sputum samples while 35.7% (5/14) of nasopharyngeal swabs and 20% (2/10) of fecal samples tested positive. No false positive detection was found when testing 180 H7N9 negative samples. Our novel rapid assay can specifically detect H7 HA antigen, facilitating rapid diagnosis for prevention and control of the on-going H7N9 epidemic.

Development of Rapid Immunochromatographic Test for Hemagglutinin Antigen of H7 Subtype in Patients Infected with Novel Avian Influenza A (H7N9) Virus

PubMed Central

Kang, Keren; Chen, Li; Zhao, Xiang; Qin, Chengfeng; Zhan, Zanwu; Wang, Jihua; Li, Wenmei; Dzakah, Emmanuel E.; Huang, Weijuang; Shu, Yuelong; Jiang, Tao; Cao, Wuchun; Xie, Mingquan; Luo, Xiaochun; Tang, Shixing

2014-01-01

Background Since human infection with the novel H7N9 avian influenza virus was identified in China in March 2013, the relatively high mortality rate and possibility of human-to-human transmission have highlighted the urgent need for sensitive and specific assays for diagnosis of H7N9 infection. Methodology/Principal Findings We developed a rapid diagnostic test for the novel avian influenza A (H7N9) virus using anti-hemagglutinin (HA) monoclonal antibodies specifically targeting H7 in an immunochromatographic assay system. The assay limit of detection was 103.5 pfu/ml or 103TCID50 of H7N9 virus. The assay specifically detected H7N9 viral isolates and recombinant HA proteins of H7 subtypes including H7N7 and H7N9, but did not react with non-H7 subtypes including H1N1, H3N2, H5N1, H5N9, and H9N2. The detection sensitivity was 59.4% (19/32) for H7N9 patients confirmed by RT-PCR. Moreover, the highest sensitivity of 61.5% (16/26) was obtained when testing H7N9 positive sputum samples while 35.7% (5/14) of nasopharyngeal swabs and 20% (2/10) of fecal samples tested positive. No false positive detection was found when testing 180 H7N9 negative samples. Conclusions/Significance Our novel rapid assay can specifically detect H7 HA antigen, facilitating rapid diagnosis for prevention and control of the on-going H7N9 epidemic. PMID:24647358

Development and potential applications of microarrays based on fluorescent nanocrystal-encoded beads for multiplexed cancer diagnostics

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Grinevich, Regina; Efimov, Anton E.; Nabiev, Igor; Sukhanova, Alyona

2014-05-01

Advanced multiplexed assays have recently become an indispensable tool for clinical diagnostics. These techniques provide simultaneous quantitative determination of multiple biomolecules in a single sample quickly and accurately. The development of multiplex suspension arrays is currently of particular interest for clinical applications. Optical encoding of microparticles is the most available and easy-to-use technique. This technology uses fluorophores incorporated into microbeads to obtain individual optical codes. Fluorophore-encoded beads can be rapidly analyzed using classical flow cytometry or microfluidic techniques. We have developed a new generation of highly sensitive and specific diagnostic systems for detection of cancer antigens in human serum samples based on microbeads encoded with fluorescent quantum dots (QDs). The designed suspension microarray system was validated for quantitative detection of (1) free and total prostate specific antigen (PSA) in the serum of patients with prostate cancer and (2) carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA 15-3) in the serum of patients with breast cancer. The serum samples from healthy donors were used as a control. The antigen detection is based on the formation of an immune complex of a specific capture antibody (Ab), a target antigen (Ag), and a detector Ab on the surface of the encoded particles. The capture Ab is bound to the polymer shell of microbeads via an adapter molecule, for example, protein A. Protein A binds a monoclonal Ab in a highly oriented manner due to specific interaction with the Fc-region of the Ab molecule. Each antigen can be recognized and detected due to a specific microbead population carrying the unique fluorescent code. 100 and 231 serum samples from patients with different stages of prostate cancer and breast cancer, respectively, and those from healthy donors were examined using the designed suspension system. The data were validated by comparing with the results of the "gold standard" enzyme-linked immunosorbent assay (ELISA). They have shown that our approach is a good alternative to the diagnostics of cancer markers using conventional assays, especially in early diagnostic applications.

Compensating for cross-reactions using avidity and computation in a suspension multiplex immunoassay for serotyping of Zika versus other flavivirus infections.

PubMed

Rönnberg, Bengt; Gustafsson, Åke; Vapalahti, Olli; Emmerich, Petra; Lundkvist, Åke; Schmidt-Chanasit, Jonas; Blomberg, Jonas

2017-10-01

The recent spread of Zika virus (ZIKV) in the Americas and Asia necessitates an increased preparedness for improved maternal and perinatal health and blood safety. However, serological cross-reactions, especially to Dengue virus (DENV), complicate ZIKV antibody serodiagnosis. A novel "pan-Flavi" suspension multiplex immunoassay (PFSMIA) using 25 antigens, whole virus (WV), non-structural protein 1 (NS1), and envelope (E) proteins, from 7 zoonotic flaviviruses for specific detection of ZIKV and DENV IgM and IgG was developed. Patterns of antibody cross-reactivity, avidity, and kinetics were established in 104 sera from returning travelers with known ZIKV and DENV infections. PFSMIA gave IgM- and IgG-sensitivities for both viruses of 96-100%, compared to an immunofluorescence assay. Main IgM cross-reactions were to NS1, for IgG to the E and WV antigens. Infecting virus yielded reactivity to several antigens of the homologous virus, while cross-reactions tended to occur only to a single antigen from heterologous virus(es). A specificity-enhancing computer procedure took into account antibody isotype, number of antibody-reactive antigens per virus, avidity, average degree of cross-reactivity to heterologous flavivirus antigens, and reactivity changes in serial sera. It classified all 50 cases correctly. Applied to sera from 200 pregnant women and 173 blood donors from Sweden, one blood donor was found ZIKV NS1 IgM positive, and another as ZIKV NS1 IgG positive. These samples did not react with other ZIKV antigens and were thereby judged as false-positives. PFSMIA provided sensitive and specific ZIKV and DENV serology, warranting high-throughput serological surveillance and a minimized need for laborious and expensive virus neutralization assays.

Performance of Commercial Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Bordetella pertussis▿

PubMed Central

Riffelmann, M.; Thiel, K.; Schmetz, J.; Wirsing von Koenig, C. H.

2010-01-01

Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of ELISA kits that were commercially available in Germany. Eleven measured IgG antibodies, and nine measured IgA antibodies. An in-house ELISA with purified antigens served as a reference method. Samples included two WHO reference preparations, the former Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research (CBER) reference preparations, serum samples from patients with clinically suspected pertussis, and serum samples from patients having received a combined tetanus, diphtheria, and pertussis (Tdap) vaccination. Kits using pertussis toxin (PT) as an antigen showed linearity compared to the WHO Reference preparation (r2 between 0.82 and 0.99), and these kits could quantify antibodies according to the reference preparation. ELISA kits using mixed antigens showed no linear correlation to the reference preparations. Patient results were compared to results of in-house ELISAs using a dual cutoff of either ≥100 IU/ml anti-PT IgG or ≥40 IU/ml anti-PT IgG together with ≥12 IU/ml anti-PT IgA. The sensitivities of kits measuring IgG antibodies ranged between 0.84 and 1.00. The specificities of kits using PT as an antigen were between 0.81 and 0.93. The specificities of kits using mixed antigens were between 0.51 and 0.59 and were thus not acceptable. The sensitivities of kits measuring IgA antibodies ranged between 0.53 and 0.73, and the specificities were between 0.67 and 0.94, indicating that IgA antibodies may be of limited diagnostic value. Our data suggest that ELISAs should use purified PT as an antigen and be standardized to the 1st International Reference preparation. PMID:20943873

Performance of the Alere Determine™ HIV-1/2 Ag/Ab Combo Rapid Test with algorithm-defined acute HIV-1 infection specimens.

PubMed

Parker, Monica M; Bennett, S Berry; Sullivan, Timothy J; Fordan, Sally; Wesolowski, Laura G; Wroblewski, Kelly; Gaynor, Anne M

2018-05-14

The capacity of HIV Antigen/Antibody (Ag/Ab) immunoassays (IA) to detect HIV-1 p24 antigen has resulted in improved detection of HIV-1 infections in comparison to Ab-only screening assays. Since its introduction in the US, studies have shown that the Determine HIV-1/2 Ag/Ab Combo assay (Determine Ag/Ab) detects HIV infection earlier than laboratory-based IgM/IgG-sensitive IAs, but its sensitivity for HIV-1 p24 Ag detection is reduced compared to laboratory-based Ag/Ab assays. However, further evaluation is needed to assess its capacity to detect acute HIV-1 infection. To assess the performance of Determine Ag/Ab in serum from acute HIV-1 infections. Select serum specimens that screened reactive on a laboratory-based Ag/Ab IA or IgM/IgG Ab-only IA, with a negative or indeterminate supplemental antibody test and detectable HIV-1 RNA were retrospectively tested with Determine Ag/Ab. Results were compared with those of the primary screening immunoassay to evaluate concordance within this set of algorithm-defined acute infections. Of 159 algorithm-defined acute HIV-1 specimens, Determine Ag/Ab was reactive for 105 resulting in 66.0% concordance. Of 125 that were initially detected by a laboratory-based Ag/Ab IA, 81 (64.8%) were reactive by Determine Ag/Ab. A total of 34 acute specimens were initially detected by a laboratory-based IgM/IgG Ab-only IA and 24 (70.6%) of those were reactive by Determine Ag/Ab. Due to their enhanced sensitivity, laboratory-based Ag/Ab IAs continue to be preferred over the Determine Ag/Ab as the screening method used by laboratories conducting HIV diagnostic testing on serum and plasma specimens. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.