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.

[Contribution of HCV core antigen testing in HCV diagnosis by test from the company Abbott Laboratories].

PubMed

Trbusek, J

2009-11-01

Detection of HCV core antigen as direct marker of hepatitis C infection clearly improves diagnosis of this disease (especially reduction of window period) and brings broad clinical utilization. The company Abbott Laboratories offers fully automated laboratory test for measurement of HCV core antigen on ARCHITECT analyzers.

Multiple Strategy Bio-Detection Sensor Platforms Made From Carbon and Polymer Materials

DTIC Science & Technology

2006-08-10

binding of antigens. Further investigations were conducted on the antibody-antigen detection scheme using known test antigens (i.e. bacteria ). While...this method worked in preliminary studies, the use of antibodies was determined not to be feasible for detecting many different types of bacteria in...solution. Also, as the number of bacteria in a solution increased, it became necessary to wash the detection surface more extensively to favor specific

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.

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.

[Comparison and evaluation of the Binax EIA and Biotest EIA urinary antigen kits for detection of Legionella pneumophila antigen in urine samples].

PubMed

Rastawicki, Waldemar; Rokosz, Natalia; Jagielski, Marek

2011-01-01

The Binax and the Biotest urinary antigen kits for detection of L. pneumophila antigen were compared by testing of selected 67 urine samples obtained from EWGLI as reference samples in External Quality Assessment Scheme. Thirty nine were positive with the Binax kit (100% of sensitivity), and 33 were positive with the Biotest (84.6% of sensitivity). The test specificities were 100% for the both kits. It was concluded that the Binax kit was more suitable for the routine diagnosis of Legionella infections than the Biotest kit.

Cross-reactions of sera from dogs infected with Angiostrongylus vasorum in commercially available Dirofilaria immitis test kits.

PubMed

Schnyder, Manuela; Deplazes, Peter

2012-11-13

Dirofilaria immitis and Angiostrongylus vasorum are both important potentially fatal canine nematodes with overlapping endemic areas, especially in Europe. The preadult and adult stages of both species are living in the Arteria pulmonalis and the right heart, and diagnostically detectable circulating parasite antigens have been demonstrated for both species. For the detection of D. immitis infections, a variety of commercial tests have been developed, however, they have not been evaluated for cross-reactions against circulating antigens of A. vasorum. In this study, potential cross-reactions of sera from 16 dogs, which were experimentally infected with A. vasorum and which had circulating antigens as confirmed by a species-specific ELISA, were evaluated for the detection of A. vasorum antigen in six commercially available D. immitis test kits. In three fast tests (Witness® Dirofilaria, SensPERT® Canine Heartworm, SNAP® 4Dx® Plus), all sera were negative. One fast membrane ELISA (SNAP® HTWM RT Test) was positive with four sera (25%), and one serum delivered a non-valid result twice. In the PetChek® HTWM PF Test, depending on the interpretation protocol, 5 or 8 dogs (31.2 - 50%) were positive. With the DiroCHEK®-ELISA, a single A. vasorum-infected dog (6.2%) tested positive. Due to potential cross-reactions with A. vasorum in commercially available test kits for the detection of D. immitis antigen, the simultaneous use of highly specific diagnostic methods for the differentiation of these two canine heart worms is recommended.

78 FR 16513 - Application of Advances in Nucleic Acid and Protein Based Detection Methods to Multiplex...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-15

... Transfusion-Transmissible Agents and Blood Cell Antigens in Blood Donations; Public Workshop AGENCY: Food and... Methods to Multiplex Detection of Transfusion- Transmissible Agents and Blood Cell Antigens in Blood... and the use of these tests in blood donor screening and blood cell antigen typing. The public workshop...

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 co-agglutination test in diagnosis of experimental microsporidiosis.

PubMed

Gaafar, Maha R

2011-05-01

Microsporidiosis is an emerging and opportunistic infection associated with wide range of clinical syndromes in humans. Confirmation of the presence of microsporidia in different samples is laborious, costly and often difficult. The present study was designed to evaluate the utility of the Co-agglutination test (Co-A test) for detection of urinary, fecal and circulating microsporidial antigens in experimentally infected mice. One hundred and twenty male Swiss albino mice were divided into non infected control and infected experimental groups which were further subdivided into two equal subgroups; immunosuppressed and immunocompetent. Microsporidial spores were isolated from human stools and identified to be Encephalitozoon intestinalis by the molecular methods. They were used to infect each subgroup of mice, then their urine, stools and sera were collected at the 1st, 3rd, 5th, 7th and 9th days post-infection (PI). Co-A test, using prepared hyperimmune serum, was used to detect antigens in all samples collected. The cross reactivity of microsporidial hyperimmune sera with antigens of Cyclospora cyatenensis and Cryptosporidium parvum was investigated by Co-A test. The results showed that Co-A test was effective in detecting microsporidial antigen in stool of immunosuppressed infected mice from the 1st day PI, and in urine and serum from the 3rd day PI till the end of the study. In the immunocompetent subgroup, Co-A test detected microsporidial antigens in stool, serum and urine of mice from the 1st day, 3rd day and the 5th day PI, respectively till the end of the study, without cross reactivity with C. cyatenensis or C. parvum in both subgroups. Co-A test proved to be simple and suitable tool for detecting microsporidial antigen in different specimens and did not need sophisticated equipment. It is very practical under field or rural conditions and in poorly equipped clinical laboratories. Copyright © 2011 Elsevier Inc. All rights reserved.

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.

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.

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.

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

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

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.

Cross-reactions of sera from dogs infected with Angiostrongylus vasorum in commercially available Dirofilaria immitis test kits

PubMed Central

2012-01-01

Background Dirofilaria immitis and Angiostrongylus vasorum are both important potentially fatal canine nematodes with overlapping endemic areas, especially in Europe. The preadult and adult stages of both species are living in the Arteria pulmonalis and the right heart, and diagnostically detectable circulating parasite antigens have been demonstrated for both species. For the detection of D. immitis infections, a variety of commercial tests have been developed, however, they have not been evaluated for cross-reactions against circulating antigens of A. vasorum. Methods In this study, potential cross-reactions of sera from 16 dogs, which were experimentally infected with A. vasorum and which had circulating antigens as confirmed by a species-specific ELISA, were evaluated for the detection of A. vasorum antigen in six commercially available D. immitis test kits. Results In three fast tests (Witness® Dirofilaria, SensPERT® Canine Heartworm, SNAP® 4Dx® Plus), all sera were negative. One fast membrane ELISA (SNAP® HTWM RT Test) was positive with four sera (25%), and one serum delivered a non-valid result twice. In the PetChek® HTWM PF Test, depending on the interpretation protocol, 5 or 8 dogs (31.2 – 50%) were positive. With the DiroCHEK®-ELISA, a single A. vasorum-infected dog (6.2%) tested positive. Conclusions Due to potential cross-reactions with A. vasorum in commercially available test kits for the detection of D. immitis antigen, the simultaneous use of highly specific diagnostic methods for the differentiation of these two canine heart worms is recommended. PMID:23148786

The impact of commercial rapid respiratory virus diagnostic tests on patient outcomes and health system utilization.

PubMed

Ko, Fiona; Drews, Steven J

2017-10-01

Acute respiratory tract infections due to influenza A/B and respiratory syncytial virus (RSV) are major causes of morbidity and mortality globally. Rapid tests for detection of these pathogens include antigen detection point of care tests (POC) and newer easy to use molecular tests. From experience, these assays improve both laboratory workflow and assay interpretation issues. However, the question of the benefits of using rapid test technology compared to routine laboratory testing for respiratory viral pathogens is still often asked. Areas covered: Specifically, this review aims to; 1) identify clinical/patient indicators that can be measured prior to and following the implementation of rapid diagnostic test for influenza and RSV, 2) provide multiple perspectives on the extent of impact of a rapid diagnostic test, including direct and indirect outcomes, and 3) identify the technological advancements in the development of rapid testing, demonstrating a timeline that transitions from antigen-based assays to molecular assays. Expert commentary: Key benefits to the use of either antigen-based or molecular rapid tests for patient care, patient flow within institutions, as well as laboratory utilization are identified. Due to improved test characteristics, the authors feel that rapid molecular tests have greater benefits than antigen-based detection methods.

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.

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

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.

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

Application of the dengue virus NS1 antigen rapid test for on-site detection of imported dengue cases at airports.

PubMed

Shu, Pei-Yun; Yang, Cheng-Fen; Kao, Jeng-Fong; Su, Chien-Ling; Chang, Shu-Fen; Lin, Chien-Chou; Yang, Wen-Chih; Shih, Hsiu; Yang, Shih-Yan; Wu, Ping-Fuai; Wu, Ho-Sheng; Huang, Jyh-Hsiung

2009-04-01

We used the dengue virus NS1 antigen (Ag) rapid test for on-site detection of imported dengue cases at airports. Among 22 positive cases of dengue identified from 850 patients with a fever suspected to have dengue, 17 were NS1 Ag test positive. These findings demonstrate the usefulness of the NS1 Ag rapid test in screening imported dengue cases at airports.

Application of the Dengue Virus NS1 Antigen Rapid Test for On-Site Detection of Imported Dengue Cases at Airports▿

PubMed Central

Shu, Pei-Yun; Yang, Cheng-Fen; Kao, Jeng-Fong; Su, Chien-Ling; Chang, Shu-Fen; Lin, Chien-Chou; Yang, Wen-Chih; Shih, Hsiu; Yang, Shih-Yan; Wu, Ping-Fuai; Wu, Ho-Sheng; Huang, Jyh-Hsiung

2009-01-01

We used the dengue virus NS1 antigen (Ag) rapid test for on-site detection of imported dengue cases at airports. Among 22 positive cases of dengue identified from 850 patients with a fever suspected to have dengue, 17 were NS1 Ag test positive. These findings demonstrate the usefulness of the NS1 Ag rapid test in screening imported dengue cases at airports. PMID:19193828

21 CFR 660.43 - Potency test.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect the...

21 CFR 660.43 - Potency test.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect the...

21 CFR 660.43 - Potency test.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect the...

21 CFR 660.43 - Potency test.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect the...

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

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

The sensitivity and the specifity of rapid antigen test in streptococcal upper respiratory tract infections.

PubMed

Gurol, Yesim; Akan, Hulya; Izbirak, Guldal; Tekkanat, Zuhal Tazegun; Gunduz, Tehlile Silem; Hayran, Osman; Yilmaz, Gulden

2010-06-01

It is aimed to detect the sensitivity and specificity of rapid antigen detection of group A beta hemolytic streptococci from throat specimen compared with throat culture. The other goal of the study is to help in giving clinical decisions in upper respiratory tract infections according to the age group, by detection of sensitivity and positive predictive values of the rapid tests and throat cultures. Rapid antigen detection and throat culture results for group A beta hemolytic streptococci from outpatients attending to our university hospital between the first of November 2005 and 31st of December 2008 were evaluated retrospectively. Throat samples were obtained by swabs from the throat and transported in the Stuart medium and Quickvue Strep A [Quidel, San Diego, USA] cassette test was applied and for culture, specimen was inoculated on 5% blood sheep agar and identified according to bacitracin and trimethoprim-sulphametaxazole susceptibility from beta hemolytic colonies. During the dates between the first of November 2005 and 31st of December 2008, from 453 patients both rapid antigen detection and throat culture were evaluated. Rapid antigen detection sensitivity and specificity were found to be 64.6% and 96.79%, respectively. The positive predictive value was 80.95% whereas negative predictive value was 92.82%. Kappa index was 0.91. When the results were evaluated according to the age groups, the sensitivity and the positive predictive value of rapid antigen detection in children were 70%, 90.3% and in adults 59.4%, 70.4%. When bacterial infection is concerned to prevent unnecessary antibiotic use, rapid streptococcal antigen test (RSAT) is a reliable method to begin immediate treatment. To get the maximum sensitivity of RSAT, the specimen collection technique used and education of the health care workers is important. While giving clinical decision, it must be taken into consideration that the sensitivity and the positive predictive value of the RSAT is quite lower in adult age group than in pediatric age group. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Benchtop Antigen Detection Technique using Nanofiltration and Fluorescent Dyes

NASA Technical Reports Server (NTRS)

Scardelletti, Maximilian C.; Varaljay, Vanessa

2009-01-01

The designed benchtop technique is primed to detect bacteria and viruses from antigenic surface marker proteins in solutions, initially water. This inclusive bio-immunoassay uniquely combines nanofiltration and near infrared (NIR) dyes conjugated to antibodies to isolate and distinguish microbial antigens, using laser excitation and spectrometric analysis. The project goals include detecting microorganisms aboard the International Space Station, space shuttle, Crew Exploration Vehicle (CEV), and human habitats on future Moon and Mars missions, ensuring astronaut safety. The technique is intended to improve and advance water contamination testing both commercially and environmentally as well. Lastly, this streamlined technique poses to greatly simplify and expedite testing of pathogens in complex matrices, such as blood, in hospital and laboratory clinics.

Detection of genome, antigen, and antibodies in oral fluids from pigs infected with foot-and-mouth disease virus.

PubMed

Senthilkumaran, Chandrika; Yang, Ming; Bittner, Hilary; Ambagala, Aruna; Lung, Oliver; Zimmerman, Jeffrey; Giménez-Lirola, Luis G; Nfon, Charles

2017-04-01

Virus nucleic acids and antibody response to pathogens can be measured using swine oral fluids (OFs). Detection of foot-and-mouth disease virus (FMDV) genome in swine OFs has previously been demonstrated. Virus isolation and viral antigen detection are additional confirmatory assays for diagnosing FMDV, but these methods have not been evaluated using swine OF. The objectives of this study were to further validate the molecular detection of FMDV in oral fluids, evaluate antigen detection and FMDV isolation from swine OFs, and develop an assay for isotypic anti-FMDV antibody detection in OFs. Ribonucleic acid (RNA) from FMDV was detected in OFs from experimentally infected pigs by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) from 1 day post-infection (dpi) to 21 dpi. Foot-and-mouth disease virus (FMDV) was isolated from OFs at 1 to 5 dpi. Additionally, FMDV antigens were detected in OFs from 1 to 6 dpi using a lateral flow immunochromatographic strip test (LFIST), which is a rapid pen-side test, and from 2 to 3 dpi using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA). Furthermore, FMDV-specific immunoglobulin A (IgA) was detected in OFs using an isotype-specific indirect ELISA starting at dpi 14. These results further demonstrated the potential use of oral fluids for detecting FMDV genome, live virus, and viral antigens, as well as for quantifying mucosal IgA antibody response.

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.

Diagnosis of AIDS-Related Intestinal Parasites

DTIC Science & Technology

1990-06-20

previously in ELISA tests to detect antigens of either Giardia lamblia or Entamoeba histolytica in human fecal specimens (67,68), the mean optical...techniques. Eight other intestinal parasites were idtritified Giardia lamblia (67 specimens), Endoimax nana (38 specimens), Entamoeba coli (34 specimens...tests to detect antigens of Entamoeba histolytica or Giardia lamblia in fecal specimens are now truly well-established and have been developed by at

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.

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

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.

High prevalence of HIV p24 antigen among HIV antibody negative prospective blood donors in Ile-Ife, Nigeria.

PubMed

Japhet, Margaret Oluwatoyin; Adewumi, Moses Olubusuyi; Adesina, Olufisayo Adeyemi; Donbraye, Emmanuel

2016-01-01

Blood transfusion service centers in Nigeria screen donated blood for markers of HIV infection using antibody- (Ab) based rapid test and in some centers, positives are re-tested using Ab-based ELISA. Paucity of data exists on p24 antigen prevalence among HIV Ab-negative donors in Nigeria. This study aims at detecting HIV p24 antigen among prospective blood donors in Osun State, Nigeria. Prospective blood donors negative for HIV antibodies using Determine test kit were re-tested using BIORAD GENSCREEN Ultra Ag-Ab ELISA kit, a fourth-generation ELISA kit that detects HIV antibodies/p24 antigen. Of the 169 HIV Ab-negative prospective donors, 10 (5.9%) were positive for HIV p24 antigen and 70% (7/10) of them were in the age range 18-30 years. Results of this study show that blood transfusion is still one of the major routes of HIV transmission in Nigeria and a higher proportion is among youth. Inclusion of p24 antigen testing into the blood donor screening will help reduce transfusion associated HIV in Nigeria if Nucleic Acid Testing (NAT) of all blood donor samples is not affordable; also, HIV enlightenment programs tailored toward youth may help reduce this rate among donors since more young people donate blood in low/middle-income countries than in high-income countries.

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.

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.

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.

[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.

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

Tests for the measurement of factor VII-activating protease (FSAP) activity and antigen levels in citrated plasma, their correlation to PCR testing, and utility for the detection of the Marburg I-polymorphism of FSAP.

PubMed

Stephan, Sina; Schwarz, Herbert; Borchert, Anja; Bussfeld, Delia; Quak, Elfriede; Simshaeuser-Knaub, Beate; Teigelkamp, Stefan; Behrens, Fritz; Vitzthum, Frank

2008-01-01

The single nucleotide Marburg I (MRI) polymorphism of the factor VII-activating protease (FSAP) gene, the prourokinase-activating activity of FSAP, and antigen levels of FSAP in plasma have been associated with incidence and progression of carotid stenosis and venous thromboembolism. However, more information on the extent of these associations, potential further ones, and respective clinical utilities remain to be determined. At present, testing is performed mainly by PCR assays based on probes or SYBR Green I. Some studies include testing for antigen levels of total FSAP and its ability to activate prourokinase. To test large cohorts, it is beneficial to rely on assays that are cost-effective, reliable, easy to use, rapid to perform, and that may eventually be automated. In addition, it appears advantageous to use functional tests or tests that determine antigen levels as they may relate more closely to the phenotype than the genotype does. Tests for the measurements of antigen levels of FSAP and its prourokinase-activating activity were improved and performance characteristics assessed. To determine the FSAP genotypes, an amplification created restriction site (ACRS) PCR test was developed. Key performance characteristics of the FSAP activity and antigen tests were as follows: measuring range: 350-1400 mPEU/mL and 1.8-120 ng/mL, total coefficients of variation (CV): 5%-20% and 5%-14%, within-run CV: 4%-11% and 2.3%-12%, and run-to-run CV: 2%-17% and 4.3%-8.3%, respectively. The ratio of the activity and antigen level of FSAP correctly identified the FSAP genotypes of 126 samples tested. The ACRS PCR test is useful for laboratories that do not have the equipment to perform probe or SYBR Green I based real-time PCR. Furthermore, the tests developed for the determination of FSAP activity and antigen levels are convenient for determining clinical correlations, even for large population studies. The ratio of activity and antigen level of FSAP appears to be a promising and efficient alternative to molecular diagnostic techniques to detect the MRI polymorphism of FSAP.

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.

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

Mass Spectrometry for Paper-Based Immunoassays: Toward On-Demand Diagnosis.

PubMed

Chen, Suming; Wan, Qiongqiong; Badu-Tawiah, Abraham K

2016-05-25

Current analytical methods, either point-of-care or centralized detection, are not able to meet recent demands of patient-friendly testing and increased reliability of results. Here, we describe a two-point separation on-demand diagnostic strategy based on a paper-based mass spectrometry immunoassay platform that adopts stable and cleavable ionic probes as mass reporter; these probes make possible sensitive, interruptible, storable, and restorable on-demand detection. In addition, a new touch paper spray method was developed for on-chip, sensitive, and cost-effective analyte detection. This concept is successfully demonstrated via (i) the detection of Plasmodium falciparum histidine-rich protein 2 antigen and (ii) multiplexed and simultaneous detection of cancer antigen 125 and carcinoembryonic antigen.

Detection of Non-hazardous, Fluorescent Ricin-B Via an Immunoassay on Simulated Plastic Wings

DTIC Science & Technology

2012-09-01

biological weapon . Large quantities of the toxin could potentially wind up in the wrong hands and be armed with relative ease. This danger has spawned many...classified as a toxin), which poses as the antigen (target) molecule. A relatively dilute solution of the tagged antigen complex was aerosolized and...validates the antibody- antigen binding sequence. A test station was developed to further evaluate hazard detection capability in an experimental setup

[One example of false negative hepatitis B surface antigen (EIA) result due to variant S area strain and reagment reactiveness related to hepatitis B surface antigen].

PubMed

Matsuda, Chikashi; Moriyama, Hidehiko; Taketani, Takeshi; Shibata, Hiroshi; Nagai, Atsushi

2011-01-01

The presence in serum of the Hepatitis B surface antigen (HBsAg), the outer envelope of the hepatitis B virus (HBV), indicates viral infection, used in laboratory tests to confirm this. We report a case of discrepancy among HBsAg test results detected between measurements in a subject with HB infection. Gene analysis demonstrated several S region gene mutations, not detected previously. We tested 12 measurements e.g., EIA, CLIA, CLEIA, F-EIA, MAT, and IC for whether they could detect our subject's HBsAg and found that it was not recognized by a method using only a single monoclonal antibody to detect HBsAg in two detection processes, in contrast to the 11 other measurements, which used two different antibodies. This case shows that amino acid substitution may cause a false negative result for HBsAg. Gene mutations known to occur in HBV, should thus trigger an awareness of the need to keep in mind that false negative results can happen in case such as ours.

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.

Detection of Multiple Pathogens in Serum Using Silica-Encapsulated Nanotags in a Surface-Enhanced Raman Scattering-Based Immunoassay.

PubMed

Neng, Jing; Li, Yina; Driscoll, Ashley J; Wilson, William C; Johnson, Patrick A

2018-06-06

A robust immunoassay based on surface-enhanced Raman scattering (SERS) has been developed to simultaneously detect trace quantities of multiple pathogenic antigens from West Nile virus, Rift Valley fever virus, and Yersinia pestis in fetal bovine serum. Antigens were detected by capture with silica-encapsulated nanotags and magnetic nanoparticles conjugated with polyclonal antibodies. The magnetic pull-down resulted in aggregation of the immune complexes, and the silica-encapsulated nanotags provided distinct spectra corresponding to each antigen captured. The limit of detection was ∼10 pg/mL in 20% fetal bovine serum, a significant improvement over previous studies in terms of sensitivity, level of multiplexing, and medium complexity. This highly sensitive multiplex immunoassay platform provides a promising method to detect various antigens directly in crude serum samples without the tedious process of sample preparation, which is desirable for on-site diagnostic testing and real-time disease monitoring.

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.

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.

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

Coproantigens in taeniasis and echinococcosis.

PubMed

Allan, James C; Craig, Philip S

2006-01-01

The application of modern immunodiagnostic or molecular diagnostic techniques has improved the diagnosis of the taeniid cestode infections, echinococcosis and taeniasis. One particularly promising approach is the detection of parasite-specific antigens in faeces (coproantigens). This approach has been applied to both Echinoccocus and Taenia species and it has gained increasingly widespread use. Taeniid coproantigen tests are based on either monoclonal or polyclonal antibodies raised against adult tapeworm antigens. These tests have the following common characteristics; they are largely genus-specific, specificity is high (>95%), parasite antigen can be detected in faeces weeks prior to patency, levels of coproantigen are independent of egg output, coproantigen is stable for days at a range of temperatures (-80 degrees C to 35 degrees C), for several months in formalin-fixed faecal samples, and coproantigen levels drop rapidly (1-5 days) following successful treatment. In the genus Taenia, most work has been done on Taenia solium and coproantigen tests have reliably detected many more tapeworm carriers than microscopy. For Echinococcus species, there is a broad positive correlation between test sensitivity and worm burden with a reliable threshold level for the test of >50 worms. Characterisation of taeniid coproantigens in order to further improve the tests is ongoing. Studies indicate taeniid coproantigens to include high molecular weight (>150 kDa), heavily glycosylated molecules with carbohydrate moieties contributing substantially to the levels of antigen detected in faeces. Application of the existing coproantigen tests in epidemiological and control programmes for Echinococcus and Taenia species infection has begun to contribute to an improved understanding of transmission and of surveillance of these important zoonotic cestodes.

Kinetic-dependent enzyme-linked immunosorbent assay for detection of antibodies to Legionella pneumophila.

PubMed Central

Sampson, J S; Wilkinson, H W; Tsang, V C; Brake, B J

1983-01-01

A semiautomated, kinetic-dependent, enzyme-linked immunosorbent assay (K-ELISA) was adapted to detect serum antibodies to Legionella pneumophila. In a comparative study, 158 human serum samples (79 pairs) were tested by K-ELISA and the standard indirect immunofluorescence assay for determination of antibody levels to L. pneumophila serogroup 1. K-ELISA determinations were made by using a serogroup-specific antigen or a preparation (unfractionated antigen) which contained both common antigen and serogroup-specific reactivity. There was good correlation between the immunofluorescence assay and the K-ELISA by using either antigen, although greater correlation was achieved with the unfractionated antigen (coefficients of correlation, 0.894 with unfractionated antigen and 0.841 with serogroup-specific antigen). These results indicate that the K-ELISA is a reliable alternative to the immunofluorescence assay for serologically diagnosing legionellosis. PMID:6361052

Kinetic-dependent enzyme-linked immunosorbent assay for detection of antibodies to Legionella pneumophila.

PubMed

Sampson, J S; Wilkinson, H W; Tsang, V C; Brake, B J

1983-12-01

A semiautomated, kinetic-dependent, enzyme-linked immunosorbent assay (K-ELISA) was adapted to detect serum antibodies to Legionella pneumophila. In a comparative study, 158 human serum samples (79 pairs) were tested by K-ELISA and the standard indirect immunofluorescence assay for determination of antibody levels to L. pneumophila serogroup 1. K-ELISA determinations were made by using a serogroup-specific antigen or a preparation (unfractionated antigen) which contained both common antigen and serogroup-specific reactivity. There was good correlation between the immunofluorescence assay and the K-ELISA by using either antigen, although greater correlation was achieved with the unfractionated antigen (coefficients of correlation, 0.894 with unfractionated antigen and 0.841 with serogroup-specific antigen). These results indicate that the K-ELISA is a reliable alternative to the immunofluorescence assay for serologically diagnosing legionellosis.

Ovarian tumor antigens.

PubMed

Bhattacharya, M; Barlow, J J

1978-09-01

Evidence has been reported for at least two common tumor-associated antigens, or antigenic determinants, in human cystadenocarcinomas of the ovary that are apparently absent in tissues of normal reproductive organs. These antigenic determinants are immunologically distinct from carcinoembryonic antigen, alpha-fetoprotein, ferritins and histocompatibility antigens. One of these two ovarian cystadenocarcinoma-associated antigens (OCAA) is not detectable in any ovarian carcinomas except serous or mucinous types, other gynecologic or nongynecologic malignancies thus far tested, while the second antigen is present in about 90% of all gynecologic tumors and occasionally in breast and colon tumors. OCAA has been purified and partially characterized. It is a high molecular weight glycoprotein which carries the unique ovarian tumor-specific antigenic determinant along with some normal cross-reacting determinants. High levels of this glycoprotein antigen have been detected in the sera of ovarian cancer patients with advanced disease by the radioimmunoassay inhibition technique. The serial determination of circulating OCAA appeared to correlate with tumor volume as well as the clinical status of the patients.

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

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.

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.

Combination of DNA-based and conventional methods to detect human leukocyte antigen polymorphism and its use for paternity testing.

PubMed

Kereszturya, László; Rajczya, Katalin; Lászikb, András; Gyódia, Eva; Pénzes, Mária; Falus, András; Petrányia, Gyõzõ G

2002-03-01

In cases of disputed paternity, the scientific goal is to promote either the exclusion of a falsely accused man or the affiliation of the alleged father. Until now, in addition to anthropologic characteristics, the determination of genetic markers included human leukocyte antigen gene variants; erythrocyte antigens and serum proteins were used for that reason. Recombinant DNA techniques provided a new set of highly variable genetic markers based on DNA nucleotide sequence polymorphism. From the practical standpoint, the application of these techniques to paternity testing provides greater versatility than do conventional genetic marker systems. The use of methods to detect the polymorphism of human leukocyte antigen loci significantly increases the chance of validation of ambiguous results in paternity testing. The outcome of 2384 paternity cases investigated by serologic and/or DNA-based human leukocyte antigen typing was statistically analyzed. Different cases solved by DNA typing are presented involving cases with one or two accused men, exclusions and nonexclusions, and tests of the paternity of a deceased man. The results provide evidence for the advantage of the combined application of various techniques in forensic diagnostics and emphasizes the outstanding possibilities of DNA-based assays. Representative examples demonstrate the strength of combined techniques in paternity testing.

Polymorphic Expression of a Human Superficial Bladder Tumor Antigen Defined by Mouse Monoclonal Antibodies

NASA Astrophysics Data System (ADS)

Fradet, Yves; Islam, Nazrul; Boucher, Lucie; Parent-Vaugeois, Carmen; Tardif, Marc

1987-10-01

Three mouse monoclonal antibodies (mAbs), which define a highly restricted antigen, were obtained by simultaneous immunizations with superficial papillary bladder tumor cells and mouse polyclonal serum against normal urothelium. The antigen was detected by the avidin/biotin/peroxidase method in 30/44 superficial bladder tumors (68%) but in only 4/27 infiltrating urothelial cancers (with much less intensity). No normal adult or fetal tissues tested expressed the antigen, including normal urothelium from 40 individuals, 13 of whom had a bladder tumor positive for the antigen. Only 1 of 45 nonbladder tumors showed some reactivity with one of the three mAbs. Serological tests on a large panel of human cancer cell lines and normal cultured cells were negative. The antigen is highly stable and well preserved on paraffin-embedded tissues. Electrophoretic transfer blot experiments with fresh tumor extracts showed that all three mAbs react with a determinant on a component of 300,000 Mr (pI 9.5) and 62,000 Mr (pI 6.5). The antigen shows polymorphic expression at the cellular level on tissue sections and also at a molecular level on immunoblots where the two bands are differentially detected on extracts of a series of tumors but are not visualized on normal urothelium extracts. The characteristics of this antigenic system suggest that it may provide some insights about the biology of bladder cancer. Specific detection of the antigen on 70% of superficial bladder tumors with normal cytology may be useful for their diagnosis and follow-up.

Human herpesvirus 8 infection among various population groups in southern Israel.

PubMed

Margalith, Miriam; Chatlynne, Louise G; Fuchs, Efrat; Owen, Carole; Lee, Cheng-Rei Ruth; Yermiyahu, Tikva; Whitman, James E; Ablashi, Dharam V

2003-12-15

To compare the prevalence of antibodies to human herpesvirus 8 (HHV-8) or Kaposi sarcoma-associated herpesvirus among Israeli and Ethiopian subjects. Serum samples were obtained from 98 Israeli Jewish students aged 18-30 years, 100 HIV-1-seronegative Ethiopian immigrants to Israel of the same age, and 100 HIV-1-seronegative Ethiopian children 1-12 years old upon their arrival in southern Israel. Plasma samples were obtained from 3 hospitalized patients with multicentric Castleman disease (MCD) as positive controls. All serum samples were tested for antibodies to both latent and lytic antigens. Antibodies to the lytic antigens and the latency-associated nuclear antigen (LANA) of HHV-8 were detected by enzyme linked immunosorbent assay and by immunofluorescence assay. HHV-8 DNA from serum or plasma samples was detected by polymerase chain reaction analysis. Antibodies to HHV-8 LANA were detected in 2.9% of the Israeli subjects aged 18-30 years and in 26% of the Ethiopian subjects from both age groups tested. Antibodies to the lytic antigens were detected in all 3 MCD patients, in 4% of the Ethiopian children, and in 2% of the 18- to 30-year-old Ethiopians. No antibodies to the lytic antigens were detected in the Israeli students. HHV-8 DNA was detected in all 3 MCD patients and in 2 of 4 of the Ethiopian children positive for the lytic antigens. HHV-8 is highly prevalent in Ethiopian immigrants to Israel as compared with Israeli students. Antibodies to HHV-8 in Ethiopia are acquired before puberty. The results of this study indicate the association of HHV-8 with MCD, as has been documented by many other researchers.

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.

Structural and functional studies of a 50 kDa antigenic protein from Salmonella enterica serovar Typhi.

PubMed

Choong, Yee Siew; Lim, Theam Soon; Chew, Ai Lan; Aziah, Ismail; Ismail, Asma

2011-04-01

The high typhoid incidence rate in developing and under-developed countries emphasizes the need for a rapid, affordable and accessible diagnostic test for effective therapy and disease management. TYPHIDOT®, a rapid dot enzyme immunoassay test for typhoid, was developed from the discovery of a ∼50 kDa protein specific for Salmonella enterica serovar Typhi. However, the structure of this antigen remains unknown till today. Studies on the structure of this antigen are important to elucidate its function, which will in turn increase the efficiency of the development and improvement of the typhoid detection test. This paper described the predictive structure and function of the antigenically specific protein. The homology modeling approach was employed to construct the three-dimensional structure of the antigen. The built structure possesses the features of TolC-like outer membrane protein. Molecular docking simulation was also performed to further probe the functionality of the antigen. Docking results showed that hexamminecobalt, Co(NH(3))(6)(3+), as an inhibitor of TolC protein, formed favorable hydrogen bonds with D368 and D371 of the antigen. The single point (D368A, D371A) and double point (D368A and D371A) mutations of the antigen showed a decrease (single point mutation) and loss (double point mutations) of binding affinity towards hexamminecobalt. The architecture features of the built model and the docking simulation reinforced and supported that this antigen is indeed the variant of outer membrane protein, TolC. As channel proteins are important for the virulence and survival of bacteria, therefore this ∼50 kDa channel protein is a good specific target for typhoid detection test. Copyright © 2011 Elsevier Inc. All rights reserved.

Heteroassembled gold nanoparticles with sandwich-immunoassay LSPR chip format for rapid and sensitive detection of hepatitis B virus surface antigen (HBsAg).

PubMed

Kim, Jinwoon; Oh, Seo Yeong; Shukla, Shruti; Hong, Seok Bok; Heo, Nam Su; Bajpai, Vivek K; Chun, Hyang Sook; Jo, Cheon-Ho; Choi, Bong Gill; Huh, Yun Suk; Han, Young-Kyu

2018-06-01

This study aimed to develop a more sensitive method for the detection of hepatitis B surface antigen (HBsAg) using heteroassembled gold nanoparticles (AuNPs). A single layered localized surface plasmon resonance (LSPR) chip format was developed with antigen-antibody reaction-based detection symmetry using AuNPs, which detected HBsAg at 10 pg/mL. To further improve the detection limit, a modified detection format was fabricated by fixing a secondary antibody (to form a heteroassembled sandwich format) to the AuNP monolayer, which enhanced the detection sensitivity by about 100 times. The developed heteroassembled AuNPs sandwich-immunoassay LSPR chip format was able to detect as little as 100 fg/mL of HBsAg within 10-15 min. In addition, the heteroassembled AuNPs sandwich-immunoassay LSPR chip format did not show any non-specific binding to other tested antigens, including alpha fetoprotein (AFP), C-reactive protein (CRP), and prostate-specific antigen (PSA). These findings confirm that the proposed detection strategy of heteroassembled AuNPs sandwich-immunoassay LSPR chip format may provide a new platform for early diagnosis of various human diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

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.

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

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.

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

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

[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.

Detection of urinary Vi antigen as a diagnostic test for typhoid fever.

PubMed Central

Taylor, D N; Harris, J R; Barrett, T J; Hargrett, N T; Prentzel, I; Valdivieso, C; Palomino, C; Levine, M M; Blake, P A

1983-01-01

Since Vi antigen is limited primarily to Salmonella typhi, it has been thought that detection of the antigen may be a useful method for diagnosing acute typhoid fever. The slide coagglutination method and enzyme-linked immunosorbent assay have recently been suggested as ways to detect small quantities of Vi antigen in urine. In Santiago, Chile, we compared the results of these two methods in patients with acute typhoid fever, paratyphoid fever, and other febrile illnesses and in afebrile control subjects. Using a cut-off value that maximally separated typhoid patients from controls, the enzyme-linked immunosorbent assay was positive in 62.4% of 141 patients with culture-proven typhoid infections and in 13.2% of 159 afebrile control subjects. The enzyme-linked immunosorbent assay was false positive in 64.7% of 34 culture-proven paratyphoid A or B patients and 47.1% of 21 patients with other nontyphoidal febrile illnesses. The coagglutination test was positive in 34% of typhoid patients, 14% of afebrile control subjects, and 46% of febrile control subjects. We conclude that these tests when performed with the Vi antibodies employed in this study are of little value for the diagnosis of typhoid fever in this setting. PMID:6630465

Detection of Circulating Paracoccidioides brasiliensis Antigen in Urine of Paracoccidioidomycosis Patients before and during Treatment

PubMed Central

Salina, Margarete Aparecida; Shikanai-Yasuda, Maria Aparecida; Mendes, Rinaldo Poncio; Barraviera, Benedito; Mendes Giannini, Maria José Soares

1998-01-01

For the diagnosis and follow-up of paracoccidioidomycosis patients undergoing therapy, we evaluated two methods (immunoblotting and competition enzyme immunoassay) for the detection of circulating antigen in urine samples. A complex pattern of reactivity was observed in the immunoblot test. Bands of 70 and 43 kDa were detected more often in urine samples from patients before treatment. The immunoblot method detected gp43 and gp70 separately or concurrently in 11 (91.7%) of 12 patients, whereas the competition enzyme immunoassay detected antigenuria in 9 (75%) of 12 patients. Both tests appeared to be highly specific (100%), considering that neither fraction detectable by immunoblotting was present in urine samples from the control group. gp43 remained present in the urine samples collected during the treatment period, with a significant decrease in reactivity in samples collected during clinical recovery and increased reactivity in samples collected during relapses. Reactivity of some bands was also detected in urine specimens from patients with “apparent cure.” The detection of Paracoccidioides brasiliensis antigens in urine appears to be a promising method for diagnosing infection, for evaluating the efficacy of treatment, and for detecting relapse. PMID:9620407

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.

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.

[The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

PubMed

Ganova, L A; Kovtoniuk, G V; Korshun, L N; Kiseleva, E K; Tereshchenko, M I; Vudmaska, M I; Moĭsa, L N; Shevchuk, V A; Spivak, N Ia

2014-08-01

The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").

Challenge with Bovine viral diarrhea virus by exposure to persistently infected calves: protection by vaccination and negative results of antigen testing in nonvaccinated acutely infected calves

PubMed Central

Johnson, Bill J.; Briggs, Robert E.; Ridpath, Julia F.; Saliki, Jeremiah T.; Confer, Anthony W.; Burge, Lurinda J.; Step, Douglas L.; Walker, Derek A.; Payton, Mark E.

2006-01-01

Abstract Calves persistently infected (PI) with Bovine viral diarrhea virus (BVDV) represent an important source of infection for susceptible cattle. We evaluated vaccine efficacy using calves PI with noncytopathic BVDV2a for the challenge and compared tests to detect BVDV in acutely or transiently infected calves versus PI calves. Vaccination with 2 doses of modified live virus vaccine containing BVDV1a and BVDV2a protected the calves exposed to the PI calves: neither viremia nor nasal shedding occurred. An immunohistochemistry test on formalin-fixed ear notches and an antigen-capture enzyme-linked immunosorbent assay on fresh notches in phosphate-buffered saline did not detect BVDV antigen in any of the acutely or transiently infected calves, whereas both tests had positive results in all the PI calves. PMID:16639944

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.

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.

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

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.

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

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.

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

Expression and Purification of a Novel Computationally Designed Antigen for Simultaneously Detection of HTLV-1 and HBV Antibodies.

PubMed

Heydari Zarnagh, Hafez; Ravanshad, Mehrdad; Pourfatollah, Ali Akbar; Rasaee, Mohammad Javad

2015-04-01

Computational tools are reliable alternatives to laborious work in chimeric protein design. In this study, a chimeric antigen was designed using computational techniques for simultaneous detection of anti-HTLV-I and anti-HBV in infected sera. Databases were searched for amino acid sequences of HBV/HLV-I diagnostic antigens. The immunodominant fragments were selected based on propensity scales. The diagnostic antigen was designed using these fragments. Secondary and tertiary structures were predicted and the B-cell epitopes were mapped on the surface of built model. The synthetic DNA coding antigen was sub-cloned into pGS21a expression vector. SDS-PAGE analysis showed that glutathione fused antigen was highly expressed in E. coli BL21 (DE3) cells. The recombinant antigen was purified by nickel affinity chromatography. ELISA results showed that soluble antigen could specifically react with the HTLV-I and HBV infected sera. This specific antigen could be used as suitable agent for antibody-antigen based screening tests and can help clinicians in order to perform quick and precise screening of the HBV and HTLV-I infections.

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

Detection of hMPV antigen by EIA in clinical specimens.

PubMed

Pancer, Katarzyna; Ciaćka, Agnieszka; Gut, Włodzimierz; Lipka, Bozena; Mierzejewska, Justyna; Milewska-Bobula, Bogumiła; Smorczewska-Kiljan, Anna; Jahnz-Rózyk, Karina; Litwińska, Bogumiła

2011-01-01

Human Metapneumovirus (hMPV) is one of the latest discovered viruses. It has been classified to Paramyxoviridae family. It is the second viral etiological agent, after RSV, which causes respiratory tract infections (RTI) in children, especially children below 5 years old. It is estimated that 5-25% of RTI in children is due to hMPV. In adults hMPV reinfections are bounded to upper respiratory tract infections. The aim of the study was to establish usefulness of ELISA test in detecting hMPV antigen and to analyze hMPV infection in connection to clinical diagnosis. 273 nasopharyngeal swabs from children (189 swabs) and adults (84 swabs) with respiratory tract infections collected from 2008 to 2010 were examined. Due to similarity of hMPV and RSV viruses and overlapping of their epidemic season rapid immunochromatographic test for RSV antigen detection was also performed in case of 120 samples, hMPV antigen was detected in 24.5% of all swabs (n = 67): in 0.0% probes in 2008, 29.0% in 2009 and 36.8% in first quarter of 2010. The highest rate ofhMPV infection was detected from summer of 2009 till the end of March 2010 (VIII-IX 2009 - 62.5%, X-XII 2009 - 44.1% and I-III 2010 -36.8%). We analyzed respiratory tract diseases reported in patients with hMPV infection. Infection due to hMPV was found in 26.5% of children and 24.0% of adults with recognized pneumonia, respectively in 28.4 and 17.6% of patients with bronchitis. Bronchiolitis was diagnosed in two children with hMPV. RSV and hMPV coinfections were confirmed in 15 out of 120 examined probes. Cross reaction pattern was excluded thanks to ELISA hMPV antigen test which was performed with suspension of RSV and thanks to statistical analysis. Coinfections were confirmed in 8% of pneumonia, 11% of bronchitis and 24.2% of the rest concomitant diagnoses. We found hMPV infection as the significant agent ofpneumonia not only in children but also in adults. ELISA hMPV antigen test can be used in diagnosis of etiological agent of respiratory infections in children and adults and in coinfections as well.

Immunodetection of Fasciola gigantica Circulating Antigen in Sera of Infected Individuals for Laboratory Diagnosis of Human Fascioliasis

PubMed Central

Attallah, Abdelfattah M.; Bughdadi, Faisal A.; El-Shazly, Atef M.

2013-01-01

Currently, the laboratory diagnosis of human fascioliasis is based on the parasitological examination of parasite eggs in stool specimens and serological detection of specific antibodies in serum samples, which are often unreliable diagnostic approaches. Ideally, a sensitive and specific diagnostic test for Fasciola infection should be based on the detection of circulating Fasciola antigen, which implies active infection. Here, a 27-kDa-molecular-mass antigen was identified in a Fasciola gigantica adult worm antigen preparation, excretory-secretory products, and sera from F. gigantica-infected individuals, and it was not detected in antigenic extracts of other parasites and sera from noninfected individuals. The target antigen was isolated and partially characterized as a protein. Immunoperoxidase staining located the target epitope within teguments and guts of F. gigantica adult worms. The performance characteristics of a newly developed enzyme-linked immunosorbent assay (ELISA) based on F. gigantica circulating antigen detection in serum (FgCA-27 ELISA) were investigated using sera of 120 parasitologically diagnosed F. gigantica-infected individuals and 80 noninfected individuals. The area under the receiving operating characteristic (ROC) curve (AUC) for ELISA was significantly high (AUC = 0.961, P < 0.0001) for discriminating Fasciola-infected and noninfected individuals. The developed assay showed high degrees of sensitivity, specificity, and efficiency (>93%), and a significant correlation (r = 0.715, P < 0.0001) between antigen level and parasite egg count was shown. In conclusion, a 27-kDa Fasciola antigen was identified in sera of F. gigantica-infected individuals. A highly sensitive and specific Fasciola antigen detection assay, FgCA-27 ELISA, was developed for laboratory diagnosis of human fascioliasis. PMID:23945158

Immunodetection of Fasciola gigantica circulating antigen in sera of infected individuals for laboratory diagnosis of human fascioliasis.

PubMed

Attallah, Abdelfattah M; Bughdadi, Faisal A; El-Shazly, Atef M; Ismail, Hisham

2013-10-01

Currently, the laboratory diagnosis of human fascioliasis is based on the parasitological examination of parasite eggs in stool specimens and serological detection of specific antibodies in serum samples, which are often unreliable diagnostic approaches. Ideally, a sensitive and specific diagnostic test for Fasciola infection should be based on the detection of circulating Fasciola antigen, which implies active infection. Here, a 27-kDa-molecular-mass antigen was identified in a Fasciola gigantica adult worm antigen preparation, excretory-secretory products, and sera from F. gigantica-infected individuals, and it was not detected in antigenic extracts of other parasites and sera from noninfected individuals. The target antigen was isolated and partially characterized as a protein. Immunoperoxidase staining located the target epitope within teguments and guts of F. gigantica adult worms. The performance characteristics of a newly developed enzyme-linked immunosorbent assay (ELISA) based on F. gigantica circulating antigen detection in serum (FgCA-27 ELISA) were investigated using sera of 120 parasitologically diagnosed F. gigantica-infected individuals and 80 noninfected individuals. The area under the receiving operating characteristic (ROC) curve (AUC) for ELISA was significantly high (AUC = 0.961, P < 0.0001) for discriminating Fasciola-infected and noninfected individuals. The developed assay showed high degrees of sensitivity, specificity, and efficiency (>93%), and a significant correlation (r = 0.715, P < 0.0001) between antigen level and parasite egg count was shown. In conclusion, a 27-kDa Fasciola antigen was identified in sera of F. gigantica-infected individuals. A highly sensitive and specific Fasciola antigen detection assay, FgCA-27 ELISA, was developed for laboratory diagnosis of human fascioliasis.

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

PrimaTB STAT-PAK Assay, a Novel, Rapid Lateral-Flow Test for Tuberculosis in Nonhuman Primates▿

PubMed Central

Lyashchenko, Konstantin P.; Greenwald, Rena; Esfandiari, Javan; Greenwald, David; Nacy, Carol A.; Gibson, Susan; Didier, Peter J.; Washington, Marc; Szczerba, Peter; Motzel, Sherri; Handt, Larry; Pollock, John M.; McNair, James; Andersen, Peter; Langermans, Jan A. M.; Verreck, Frank; Ervin, Sean; Ervin, Frank; McCombs, Candace

2007-01-01

Tuberculosis (TB) is the most important zoonotic bacterial disease in nonhuman primates (NHP). The current diagnostic method, the intradermal palpebral tuberculin test, has serious shortcomings. We characterized antibody responses in NHP against Mycobacterium tuberculosis to identify immunodominant antigens and develop a rapid serodiagnostic test for TB. A total of 422 NHP were evaluated, including 243 rhesus (Macaca mulatta), 46 cynomolgus (Macaca fascicularis), and 133 African green (Cercopithecus aethiops sabaeus) monkeys at five collaborative centers. Of those, 50 monkeys of the three species were experimentally inoculated with M. tuberculosis. Antibody responses were monitored every 2 to 4 weeks for up to 8 months postinfection by MultiAntigen Print ImmunoAssay with a panel of 12 recombinant antigens. All of the infected monkeys produced antibodies at various levels and with different antigen recognition patterns. ESAT-6 and MPB83 were the most frequently recognized proteins during infection. A combination of selected antigens which detected antibodies in all of the infected monkeys was designed to develop the PrimaTB STAT-PAK assay by lateral-flow technology. Serological evaluation demonstrated high diagnostic sensitivity (90%) and specificity (99%). The highest rate of TB detection was achieved when the skin test was combined with the PrimaTB STAT-PAK kit. This novel immunoassay provides a simple, rapid, and accurate test for TB in NHP. PMID:17652522

Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis.

PubMed

Paredes, Adriana; Sáenz, Patricia; Marzal, Miguel W; Orrego, Miguel A; Castillo, Yesenia; Rivera, Andrea; Mahanty, Siddhartha; Guerra-Giraldez, Cristina; García, Hector H; Nash, Theodore E

2016-07-01

Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections. Copyright © 2016 Elsevier Inc. 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.

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

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.

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

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.

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

PubMed Central

Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

1990-01-01

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

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

[Prokaryotic expression of the major antigenic domain of equine arteritis virus GL protein and the establishment of putative indirect ELISA assay].

PubMed

Liang, Cheng-Zhu; Cao, Rui-Bing; Wei, Jian-Chao; Zhu, Lai-Hua; Chen, Pu-Yan

2006-06-01

According to the antigenic analysis of equine arteritis virus (EAV) GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH I and Xho I and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21(DE3) and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was confirmed by Western blot. The recombinant GL1 protein was purified by the means of His * Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against EAV with the purified GL1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 9.65 microg/mL and the optimal dilution of serum was 1:80. The positive criterion of this ELISA assay is OD (the tested serum) > 0.4 and OD (the tested serum) /OD (the negative serum) > 2.0. The iGL-ELISA was evaluated versus micro-virus neutralization test. The ELISA was performed on 900 sera from which were preserved by this lab during horse entry/exit inspection, the agreement (94.1%) of these test were considered suitable for individual serological detection. In another test which 180 sera samples were detected by iGL-ELISA and INGEZIM ELISA kit respectively. The agreement ratio between the two methods is 95.6%.

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.

Comparative study of radiation, chemical, and aging effects on viral transformation. Annual progress report, 1975

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

Coggin, J.H. Jr.

Progress is reported on the following research projects: evaluation of isotopic antiglobulin test (IAT) to detect tumor associated antigens using antisera induced by x-irradiated tumor cells; development of cytotoxic antibody for embryonic antigens (EA); acrylamide gel cell culture assay for transformation; and evaluation of 3-MCA induced sarcomas for TSTA and cross-reacting antigens. (HLW)

Disparate host immunity to Mycobacterium avium subsp. paratuberculosis antigens in calves inoculated with M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis

USDA-ARS?s Scientific Manuscript database

Cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and criticism of current tests for the detection of paratuberculosis. In the present study, host immune responses to antigen preparations of Mycobacterium avium subsp. paratuberculosis (MAP), consis...

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

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.

Evaluation of new monoclonal antibody-based latex agglutination test for detection of cryptococcal polysaccharide antigen in serum and cerebrospinal fluid.

PubMed Central

Kiska, D L; Orkiszewski, D R; Howell, D; Gilligan, P H

1994-01-01

We evaluated the performance of CRYPTO-LEX (Trinity Laboratories, Inc., Raleigh, N. C.), a new mouse immunoglobulin M monoclonal antibody latex agglutination reagent which reacts with the capsular polysaccharide of the four serogroups of Cryptococcus neoformans. This test was compared with CALAS (Meridian Diagnostics, Cincinnati, Ohio) for the ability to detect cryptococcal antigen in serum and cerebrospinal fluid (CSF). A total of 580 clinical specimens (327 serum and 253 CSF samples), primarily from human immunodeficiency virus-infected patients, were tested in this study. Sixty-seven specimens (44 serum and 23 CSF samples) were positive for cryptococcal antigen with both tests, and 511 (282 serum and 229 CSF samples) were negative. The two latex reagents agreed for 326 of 327 serum specimens (44 positives and 282 negatives). One serum specimen with a titer of 1:2 was CALAS positive but CRYPTO-LEX negative. The titer correlation coefficient for the two tests was 0.884 when two highly discordant serum specimens were eliminated from analysis of the data. The two latex tests agreed for 252 of 253 CSF specimens (23 positives and 229 negatives). One specimen with a titer of 1:2 was positive with CALAS and negative by CRYPTO-LEX. The correlation coefficient of the two tests for CSF titers was 0.886. The sensitivity and specificity of CRYPTO-LEX were 97 and 100%, respectively, with a 99.6% correlation with CALAS. These data show that the performance of CRYPTO-LEX is comparable to that of CALAS for detection of cryptococcal antigen in serum and CSF. PMID:7814566

Detection of specific IgE antibodies to major and minor antigenic determinants in sera of penicillin allergic patients.

PubMed

Zhao, Yongxing; Qiao, Hailing

2003-12-01

To investigate the mechanism(s) of penicillins allergic reaction. The radioallergosorbent test (RAST) was used to detect 9 specific IgE antibodies, including major antigenic determinants: benzylpenicilloyl (BPO), ampicilloyl (APO), amoxicilloyl (AXO), phenoxomethylpenicilloyl (PVO) and flucloxacilloyl (FLUO), and minor antigenic determinants: benzylpenicillanyl (BPA), amoxicillanyl (AXA), 6-aminopenicillanic (APA) and phenoxomethylpenicillany (PVA), in the sera of 32 penicillin allergic patients. The relationship between specific IgE antibodies and penicillins chemical structures was studied by radioallergosorbent inhibition test. Nineteen of 32 patients (59.4%) were RAST positive, among whom, five cases were positive only to one or two antigenic minor determinants, and three cases were positive only to one or three major antigenic determinants. The remaining 11 patients were positive not only to major antigenic determinants but also minor antigenic determinants. In 9 specific IgE antibodies, the positive rate of PVA-IgE was the highest (34.38%), followed by BPO-IgE (31.25%). The positive rate of FLUO-IgE was the lowest (15.63%). Of the total patient group, 53.13% were positive to one or more minor antigenic determinants, while 37.5% (12/32) were positive to one or more major antigenic determinants. The percentage of patients with urticarial reactions who were positive to minor antigenic determinants (63.16%) was significantly higher than observed in the anaphylactic shock group (38.5%, P < 0.05). The minor antigenic determinant was important in allergic reaction. The combining sites of the specific IgE antibodies were likely to be the side-chain of drug or the overwhelming drug molecule.

Performance of polymerase chain reaction for the diagnosis of cystic echinococcosis using serum, urine, and cyst fluid samples

PubMed Central

Chaya, DR; Parija, Subhash Chandra

2014-01-01

Introduction: Cystic echinococcosis (CE) is a chronic zoonosis which presents with variable clinical manifestations. Currently the diagnosis of this disease is based on radiological findings and serological tests which lack specificity. Although antigen detection from the cyst fluid is the most specific, it is seldom done due to the complications involved. Detecting the presence of Echinococcus granulosus specific deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR) could provide a definitive diagnosis of CE. Materials and Methods: An in-house PCR assay was devised to detect E. granulosus specific DNA in serum, urine and hydatid cyst fluid. The ability of the PCR to detect E. granulosus in the above mentioned samples were observed in comparison with other antigen and antibody detection tests. Results: Serum samples from surgically confirmed patients of CE with ruptured cysts contained the corresponding DNA while the in the majority of cases who had an intact cyst had no DNA of E. granulosus in their serum. DNA of E. granulosus was not found to be excreted in urine. PCR performed equal to antigen detection ELISA while testing hydatid cyst fluid samples. Conclusions: Serum and urine might not serve as useful samples for the molecular diagnosis of cystic echinococcosis. However, PCR can be useful on serum samples to detect ruptured hydatid cysts and on hydatid cyst fluid to confirm the parasitic diagnosis. PMID:24754027

Performance of polymerase chain reaction for the diagnosis of cystic echinococcosis using serum, urine, and cyst fluid samples.

PubMed

Chaya, Dr; Parija, Subhash Chandra

2014-01-01

Cystic echinococcosis (CE) is a chronic zoonosis which presents with variable clinical manifestations. Currently the diagnosis of this disease is based on radiological findings and serological tests which lack specificity. Although antigen detection from the cyst fluid is the most specific, it is seldom done due to the complications involved. Detecting the presence of Echinococcus granulosus specific deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR) could provide a definitive diagnosis of CE. An in-house PCR assay was devised to detect E. granulosus specific DNA in serum, urine and hydatid cyst fluid. The ability of the PCR to detect E. granulosus in the above mentioned samples were observed in comparison with other antigen and antibody detection tests. Serum samples from surgically confirmed patients of CE with ruptured cysts contained the corresponding DNA while the in the majority of cases who had an intact cyst had no DNA of E. granulosus in their serum. DNA of E. granulosus was not found to be excreted in urine. PCR performed equal to antigen detection ELISA while testing hydatid cyst fluid samples. Serum and urine might not serve as useful samples for the molecular diagnosis of cystic echinococcosis. However, PCR can be useful on serum samples to detect ruptured hydatid cysts and on hydatid cyst fluid to confirm the parasitic diagnosis.

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).

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.

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

Performances of fourth generation HIV antigen/antibody assays on filter paper for detection of early HIV infections.

PubMed

Kania, Dramane; Truong, Tam Nguyen; Montoya, Ana; Nagot, Nicolas; Van de Perre, Philippe; Tuaillon, Edouard

2015-01-01

Point-of-care testing and diagnosis of HIV acute infections play important roles in preventing transmission, but HIV rapid diagnosis tests have poor capacity to detect early infections. Filter paper can be used for capillary blood collection and HIV testing using 4th generation immunoassays. Antigen/antibody combined immunoassays were evaluated for their capacity to identify early HIV infections using filter paper in comparison with rapid test. Thirty nine serum samples collected from HIV seroconverters were spotted onto filter paper and tested by the Roche Elecsys(®) HIV Combi PT test and the DiaSorin Liaison XL Murex HIV Ab/Ag assay. Fourth generation immunoassays identified 34 out of 39 HIV early infections using dried serum spot, whereas the Determine™ HIV-1/2 rapid test detected 24 out of 39 HIV positive serum (87.2% vs 61.5% respectively, p = 0.009). p24 antigen was detected by the Liaison XL in 19 dried serum samples (48.7%). In the group characterized by a negative western blot, 7 out of 8 (87.5%) and 6 out of 8 (75.0%) samples were found positive for HIV using the Elecsys and the Liaison XL, respectively. None of these eight samples classified in this group of early acute infections were found positive by the rapid test. Fourth generation Ag/Ab immunoassays performed on dried serum spot had good performance for HIV testing during the early phases of HIV infection. This method may be useful to detect HIV early infections in hard-to-reach populations and individuals living in remote areas before rapid tests become positive. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Clinical report: Detection and management of bovine viral diarrhea virus Type 1b in a large dairy herd

USDA-ARS?s Scientific Manuscript database

Case Description: 1,081 newborn calves from a commercial dairy were tested for bovine viral diarrhea virus antigen by pooled RT-PCR as part of a screening program. Ear tissue from twenty six calves initially tested positive and 14 confirmed positive with antigen capture ELISA two weeks later (1.3...

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

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.

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

The fluorescent treponemal antibody-absorption (FTA-ABS) test for syphilis.

PubMed

Hunter, E F

1975-01-01

The FTA test was developed at a time when immunofluorescence procedures were not well-defined. Through technique control and research, a modification of the FTA test, the FTA-ABS, has attained a position as one of the leading treponemal tests to confirm the reagin tests for syphilis. In this review of the FTA-ABS test, attention has been focused on reagent development, with the anticipation that reagent standardization may soon become a reality. The T. pallidum antigen obtained by extracting infected rabbit testicular tissue has evolved from a preparation in which the treponemes remained in the initial extracting fluid to a reagent that can be free of rabbit tissue and globulin. These washed antigen preparations improve visibility of the treponemes on the microscope slide, reduce background fluorescence, and reduce or prevent from occurring nonspecific reactions that are a result of tissue and globulin components. Both washed and nonwashed antigens are available commercially, and, to date, little differentiation has appeared on the product label. The predominant immunoglobulin that reacts with T. pallidum in the indirect fluorescent antibody tests appears to be IgG. This is the major immunoglobulin detected in the FTA-ABS test. IgM, although increased in early syphilis, is also increased in other clinical conditions. Several reports suggest that adult IgM detection in the present FTA-ABS test would be nonspecific. Until specific IgM antibody in adult syphilis can be detected without a risk to test specificity, the conjugate for the FTA-ABS test should continue to be an anti-IgG reagent. Class-specific, anti-IgG reagents are more expensive than other reagents; however, their use may eliminate the problem of nonspecificity resulting from IgM detection. Additionally, micromethods can be used to reduce cost, and this possibility should be investigated. The sorbent that contains an antigen to the Reiter treponeme may or may not specifically absorb the reactivity that occurs in normal sera; certainly, there are questionable aspects about this reagent. Group antibodies not related to Reiter treponemes may be responsible for some nonspecific reactivity; additionally, antiglobulin factors have been reported to participate in the reaction. Antigens free of rabbit serum factors and class-specific, antiimmunoglobulin reagents are available, and may lead to a better understanding of nonspecific reactions. These reagents should allow resolution of the possible multiplicity of reactivity. In this interim period, the sorbent, with its possible nonspecific nature, appears to maintain a biological balance between natural or group and immune antibodies when used to detect IgG antibody.

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

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.

Detection of brown-rot antigens in southern pine

Treesearch

Carol A. Clausen

1996-01-01

Brown-rot fungal antigens were detected by particle capture immunoassay(PCI) in southern pine 2 X 4’s beyond visible or culturable hyphal growth. Further analysis of test samples revealed changes along the 2 X 4’s that could be grouped into zones. Zone 1, the point of inoculation through 6 cm, showed low pH, measurable oxalic acid, high moisture, and high protein. Zone...

Detection of Chlamydophila psittaci from feral pigeons in environmental samples: problems with currently available techniques.

PubMed

Geigenfeind, Ila; Haag-Wackernagel, Daniel

2010-03-01

Chlamydophila psittaci (Lillie, 1930) Everett et al., 1999, the pathogenic agent of human ornithosis, is widespread in feral pigeon populations and many cases of transmission from feral pigeons to humans have been reported. The aim of the present study was to detect C. psittaci in environmental samples to find out more about possible transmission routes and, therefore, to assess the zoonotic risk for humans. Fecal samples were collected from nest boxes in a feral pigeon loft. Additionally, samples were taken from the feather dust film covering the water surface of public fountains where pigeons regularly bathe. The samples were tested for the presence of chlamydial antigen using an antigen enzyme-linked immunosorbent assay to prove shedding of C. psittaci by feral pigeons. This test detects a genus specific lipopolysaccharide in the outer membrane of the chlamydial bacteria. Samples were tested using the IDEIA PCE Chlamydia Test kit (DakoCytomation) and positive results were verified with IDEIA Chlamydia Blocking Reagents (DakoCytomation). The IDEIA PCE Chlamydia Test yields a high proportion of positive results. However, when IDEIA Chlamydia Blocking was performed, most of the positive results turned out to be negative or could not be interpreted. We conclude that antigen-enzyme-linked immunosorbent assay tests are not suitable for detecting C. psittaci in environmental samples. Previous publications where no blocking test was used should be reconsidered critically. © 2010 ISZS, Blackwell Publishing and IOZ/CAS.

Acute HIV Discovered During Routine HIV Screening With HIV Antigen-Antibody Combination Tests in 9 US Emergency Departments.

PubMed

White, Douglas A E; Giordano, Thomas P; Pasalar, Siavash; Jacobson, Kathleen R; Glick, Nancy R; Sha, Beverly E; Mammen, Priya E; Hunt, Bijou R; Todorovic, Tamara; Moreno-Walton, Lisa; Adomolga, Vincent; Feaster, Daniel J; Branson, Bernard M

2018-01-05

Newer combination HIV antigen-antibody tests allow detection of HIV sooner after infection than previous antibody-only immunoassays because, in addition to HIV-1 and -2 antibodies, they detect the HIV-1 p24 antigen, which appears before antibodies develop. We determine the yield of screening with HIV antigen-antibody tests and clinical presentations for new diagnoses of acute and established HIV infection across US emergency departments (EDs). This was a retrospective study of 9 EDs in 6 cities with HIV screening programs that integrated laboratory-based antigen-antibody tests between November 1, 2012, and December 31, 2015. Unique patients with newly diagnosed HIV infection were identified and classified as having either acute HIV infection or established HIV infection. Acute HIV infection was defined as a repeatedly reactive antigen-antibody test result, a negative HIV-1/HIV-2 antibody differentiation assay, or Western blot result, but detectable HIV ribonucleic acid (RNA); established HIV infection was defined as a repeatedly reactive antigen-antibody test result and a positive HIV-1/HIV-2 antibody differentiation assay or Western blot result. The primary outcomes were the number of new HIV diagnoses and proportion of patients with laboratory-defined acute HIV infection. Secondary outcomes compared reason for visit and the clinical presentation of acute HIV infection. In total, 214,524 patients were screened for HIV and 839 (0.4%) received a new diagnosis, of which 122 (14.5%) were acute HIV infection and 717 (85.5%) were established HIV infection. Compared with patients with established HIV infection, those with acute HIV infection were younger, had higher RNA and CD4 counts, and were more likely to have viral syndrome (41.8% versus 6.5%) or fever (14.3% versus 3.4%) as their reason for visit. Most patients with acute HIV infection displayed symptoms attributable to acute infection (median symptom count 5 [interquartile range 3 to 6]), with fever often accompanied by greater than or equal to 3 other symptoms (60.7%). ED screening using antigen-antibody tests identifies previously undiagnosed HIV infection at proportions that exceed the Centers for Disease Control and Prevention's screening threshold, with the added yield of identifying acute HIV infection in approximately 15% of patients with a new diagnosis. Patients with acute HIV infection often seek ED care for symptoms related to seroconversion. Copyright © 2017 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.

Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

NASA Astrophysics Data System (ADS)

Samuelsen, Simone V.; Solov'Yov, Ilia A.; Balboni, Imelda M.; Mellins, Elizabeth; Nielsen, Christoffer Tandrup; Heegaard, Niels H. H.; Astakhova, Kira

2016-10-01

New techniques to detect and quantify antibodies to nucleic acids would provide a significant advance over current methods, which often lack specificity. We investigate the potential of novel antigens containing locked nucleic acids (LNAs) as targets for antibodies. Particularly, employing molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best oligonucleotide binders in surface plasmon resonance studies to analyze binding and kinetic aspects of interactions between antigens and target DNA. These DNA and LNA/DNA sequences showed improved binding in enzyme-linked immunosorbent assay using human samples of pediatric lupus patients. Our results suggest that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies.

Epitope Mapping of Avian Influenza M2e Protein: Different Species Recognise Various Epitopes

PubMed Central

Hasan, Noor Haliza; Ignjatovic, Jagoda; Tarigan, Simson; Peaston, Anne; Hemmatzadeh, Farhid

2016-01-01

A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e) protein of avian influenza virus (AIV) as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA) is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs) recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb) and chicken antibodies (cAbs) recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development. PMID:27362795

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

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.

[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.

42 CFR 493.911 - Bacteriology.

Code of Federal Regulations, 2012 CFR

2012-10-01

... laboratories: (1) Those that interpret Gram stains or perform primary inoculation, or both; and refer cultures...; (2) Those that use direct antigen techniques to detect an organism and may also interpret Gram stains... interpreting Gram stains, performing primary inoculations, and using direct antigen tests, also isolate and...

42 CFR 493.911 - Bacteriology.

Code of Federal Regulations, 2014 CFR

2014-10-01

... laboratories: (1) Those that interpret Gram stains or perform primary inoculation, or both; and refer cultures...; (2) Those that use direct antigen techniques to detect an organism and may also interpret Gram stains... interpreting Gram stains, performing primary inoculations, and using direct antigen tests, also isolate and...

42 CFR 493.911 - Bacteriology.

Code of Federal Regulations, 2013 CFR

2013-10-01

... laboratories: (1) Those that interpret Gram stains or perform primary inoculation, or both; and refer cultures...; (2) Those that use direct antigen techniques to detect an organism and may also interpret Gram stains... interpreting Gram stains, performing primary inoculations, and using direct antigen tests, also isolate and...

[Detection of antigen structures in blood cells in various prepared plasma transfusions].

PubMed

Barz, D

1994-01-01

We investigated the content of antigen-bearing cells and cell fragments in Fresh Frozen Plasma (FFP) from blood centers, in Octaplas (virus-inactivated fresh plasma produced with the solvent/detergent technique by the Octapharma Company) and in MB-plasma (virus-inactivated fresh plasma after photodynamic treatment with methylen blue coming from the German Red Cross in Springe, Lower Saxony). With the aid of an immunoassay (MAIPA-test) these plasmas were tested regarding Rhesus-D-antigen, HLA-class-I- and HLA-class-II-antigens, platelet specific antigens HPA-1a/HPA-1b and granulocyte specific antigens NA1/NA2. In Octaplas (n = 10) we did not find cells or cell fragments and no antigen-bearing blood cell structures. In FFP (n = 28) there were platelet specific antigens in 27 cases (96.4%) and HLA-class-I-antigens in 4 cases (14.3%). In MB-plasma (n = 14) we found platelet specific antigens in all cases, HLA-class-I-antigens in 4 cases (18.6%), HLA-class-II-antigens and granulocyte specific antigens in 1 case (7.1%) and Rhesus-D-antigen in 3 cases (21.4%). Plasma derived from whole blood showed lower levels of cells and antigens than plasma which was produced with the aid of the cell separator.

DNA sequences and proteic antigens of H. pylori in cholecystic bile and tissue of patients with gallstones.

PubMed

Neri, V; Margiotta, M; de Francesco, V; Ambrosi, A; Valle, N Della; Fersini, A; Tartaglia, N; Minenna, M F; Ricciardelli, C; Giorgio, F; Panella, C; Ierardi, E

2005-10-15

Although Helicobacter pylori DNA sequences have been detected in cholecystic bile and tissue of patients with gallstones, controversial results are reported from different geographic areas. To detect H. pylori in cholecystic bile and tissue of patients with gallstones from a previously uninvestigated geographic area, southern Italy. Detection included both the bacterial DNA and the specific antigen (H. pylori stool antigen) identified in the stools of infected patients for diagnostic purposes. The study enclosed 33 consecutive patients undergoing laparoscopic cholecystectomy for gallstones. DNA sequences of H. pylori were detected by polymerase chain reaction in both cholecystic bile and tissue homogenate. Moreover, we assayed H.pylori stool antigen on gall-bladder cytosolic and biliary proteins after their extraction. Bacterial presence in the stomach was assessed by urea breath test in all patients and Deltadelta13CPDB value assumed as marker of intragastric load. Fisher's exact probability and Student's t-tests were used for statistical analysis. DNA sequences of H. pylori in bile were found in 51.5% and significantly correlated with its presence in cholecystic tissue homogenate (P<0.005), H. pylori stool antigen in gall-bladder (P=0.0013) and bile (P=0.04) proteins, gastric infection (P<0.01) and intragastric bacterial load (P<0.001). No correlation was found, however, with sex and age of the patients. Our prevalence value of bacterial DNA in bile and gall-bladder of patients with gallstones agreed with that of the only other Italian study. The simultaneous presence of both bacterial DNA and proteic antigen suggests that the same prototype of bacterium could be located at both intestinal and cholecystic level and, therefore, the intestine represents the source of biliary contagion.

False-positive cryptococcal antigen latex agglutination caused by disinfectants and soaps.

PubMed Central

Blevins, L B; Fenn, J; Segal, H; Newcomb-Gayman, P; Carroll, K C

1995-01-01

Five disinfectants or soaps were tested to determine if any could be responsible for false-positive results obtained with the Latex-Crypto Antigen Detection System kit (Immuno-Mycologics, Inc., Norman, Okla.). Three disinfectants or soaps (Derma soap, 7X, and Bacdown) produced false-positive agglutination after repeated washing of ring slides during testing of a known negative cerebrospinal fluid specimen. PMID:7650214

Red blood cell antigen genotype analysis for 9087 Asian, Asian American, and Native American blood donors.

PubMed

Delaney, Meghan; Harris, Samantha; Haile, Askale; Johnsen, Jill; Teramura, Gayle; Nelson, Karen

2015-10-01

There has yet to be a comprehensive analysis of blood group antigen prevalence in Asian Americans and Native Americans. There may be ethnic differences in blood group frequencies that would result in clinically important mismatches through transfusion. Blood donors who self-identified as Asian or Native American were tested using a single-nucleotide polymorphism (SNP) DNA array (HEA BeadChip kit, Bioarray Solutions Ltd) that predicts expression of 38 human erythrocyte antigens (HEAs) and by serology for ABO, D, C, M, N, Jk(a) , and Jk(b) . The prevalence of blood group antigens was compared to published European prevalence. Discrepancies between SNP-predicted and serology-detected antigens were tallied. A total of 9087 blood donors were tested from nine Asian and Native American heritages. The predicted prevalence of selected antigens in the RHCE, JK, FY, MNS, LU, CO, and DO blood group systems were variable between Asian populations, but overall not significantly different than Europeans. Compared to European frequencies, Kell blood group allele frequencies were significantly different in the Chinese, Native American, Hawaiian/Pacific Islander, South Asian, and Southeast Asian heritage blood donors; Diego antigens Di(a) and Di(b) were different in donors of Native American and South Asian ancestries (p < 0.05). Of the donors tested, 4.5% showed a SNP-serology discrepancy that segregated within specific ethnic groups. This study provides HEA allele frequency and antigen prevalence data in a cohort of Asian and Native Americans donors. Several ethnic groups exhibited differences in HEA frequencies compared to Europeans. Genotype-serotype discrepancies were detected in all systems studied. © 2015 AABB.

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

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.

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.

[Contribution of urinary pneumococcal antigen detection combined with the research of legionella antigen for diagnosis of pneumonia in hospitalized patients].

PubMed

Honoré, S; Trillard, M; Ould-Hocine, Z; Lesprit, P; Deforges, L; Legrand, P

2004-10-01

Bacteriological confirmation of pneumonia (PNM) in hospitalized patients is often erratic or belated. Because of importance of prognosis, early adaptation of treatment requires an empirical antimicrobial therapy (generally aminopenicillin and macrolide combination). The starting therapeutic strategy should profit by a fast and reliable test asserting a pneumococcal etiology. The Binax Now S. pneumoniae (BNP) test allows an urinary pneumococcal antigen (UPA) detection using an immunochromatographic membrane assay within 15 minutes. We first evaluated the BNP test for 28 patients with pneumococcal PNM proved by culture, and 118 negative control patients without PNM. The BNP test was then evaluated by testing urine from 158 hospitalized patients with a clinical picture of PNM (community-acquired: 90, nosocomial: 68) for whom a research of urinary Legionella antigen (Binax Now) was prescribed and was positive for only two cases. 57 patients (36.1%) were hospitalized in ICU. The sensitivity was 71.4% (85.7% for the 21 bacteriemic PNM), and the specificity was 98.3%; that is consistent with previous published data. Among the 158 patients with PNM, UPA was detected in 17 cases (10.8%): 15 within the community-acquired PNM (16.7%) and 2 (2.9%) within the nosocomial cases. The pneumococcal etiology was confirmed by bacteriological samples in 7/17 patients (6 by blood cultures). The 10 others showed clinical and radiological features in agreement with a pneumococcal PNM. Among the 141 patients with negative AUP, S. pneumoniae was isolated from 6 of them (2 in blood cultures). The Binax Now S. pneumoniae test allowed a fast and reliable etiological diagnosis in 10.8% of hospitalized PNM (16.7% of the community-acquired cases) having a research of urinary Legionella antigen (conceiving with severity factors). So it could conduce to an improved adjustment of the starting antimicrobial therapy of hospitalized adult patients with PNM.

Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

PubMed Central

Tiwari, Sapana; Kumar, Ashu; Mangalgi, Smita; Rathod, Vedika; Prakash, Archana; Barua, Anita; Arora, Sonia; Sathyaseelan, Kannusamy

2013-01-01

Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and promising results for further use in clinical screening and serodiagnosis of human brucellosis. PMID:23761658

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.

Identification of the soluble HVP-associated antigen of the lymphoblastoid cell line established from lymphomatous baboon (Papio hamadryas).

PubMed

Voevodin, A F; Lapin, B A; Agrba, V Z; Timanovskaya, V V

1978-01-01

A new technique (indirect double immunodiffusion) for detection of EBV-associated soluble antigen and corresponding antibodies has been developed. This technique includes three steps: 1) simple double immunodiffusion with extracts of Raji cells (or other EBV-genome positive cells) and human sera containing antibodies against EBV-associated soluble antigen; 2) extensive washing and treatment with anti-human globulin; 3) extensive washing and treatment with tannic acid. Using this test it was shown that the soluble antigen indistinguishable from EBV-associated soluble antigen was present in KMPG-1 cells producing HVP.

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

Enzyme-linked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent for salmonid bacterial kidney disease

USGS Publications Warehouse

Pascho, R.J.; Mulcahy, D.

1987-01-01

A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of a soluble fraction of Renibacterium salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. The Costar® Serocluster™ EIA microplate gave the highest absorbance and signal-to-noise ratios among seven types tested. Including Tween 80 in the wash buffer resulted in higher absorbances than Tween 20 when antigen was present. Background absorbance did not increase when Tween 80 was added to the wash buffer, but did when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37 °C and 16 h at 4 °C. Antigen attachment to antibody-coated microplate wells depended more on incubation temperature than duration; we adopted a 3-h incubation at 25 °C. Conjugate incubation for longer than 1 h at 37 °C or 3 h at 25 °C resulted in unacceptable background levels. No cross-reactions resulted from heat-extracted antigens of 10 other species of bacteria. The optimized ELISA is a 6-h test that enables detection of levels of soluble antigen as low as 2–20 ng.

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.

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.

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.

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.

Rotavirus Antigenemia in Children Is Associated with Viremia

PubMed Central

Blutt, Sarah E; Matson, David O; Crawford, Sue E; Staat, Mary Allen; Azimi, Parvin; Bennett, Berkeley L; Piedra, Pedro A; Conner, Margaret E

2007-01-01

Background Antigenemia is commonly detected in rotavirus-infected children. Although rotavirus RNA has been detected in serum, definitive proof of rotavirus viremia has not been shown. We aimed to analyze a defined patient population to determine if infectious virus could be detected in sera from children with rotavirus antigenemia. Methods and Findings Serum samples obtained upon hospitalization from children with gastroenteritis (57 stool rotavirus-positive and 41 rotavirus-negative), children with diagnosed bronchiolitis of known (n = 58) or unknown (n = 17) viral etiology, children with noninfectious, nonchronic conditions (n = 17), and healthy adults (n = 28) were tested for rotavirus antigen by enzyme immunoassay (EIA). Results of serum antigen testing were assessed for association with clinical and immunological attributes of the children. Rotavirus antigenemia was detected in 90% (51/57) of children with rotavirus-positive stools, in 89% (8/9) of children without diarrhea but with rotavirus-positive stools, in 12% (2/17) of children with bronchiolitis of unknown etiology without gastroenteritis, and in 12% (5/41) of children with gastroenteritis but with rotavirus-negative stools. Antigenemia was not detected in sera from children with noninfectious nonchronic conditions, children with bronchiolitis of known etiology and no gastroenteritis, or healthy adults. Neither age nor timing of serum collection within eight days after onset of gastroenteritis significantly affected levels of antigenemia, and there was no correlation between antigenemia and viral genotype. However, there was a negative correlation between serum rotavirus antigen and acute rotavirus-specific serum IgA (r = −0.44, p = 0.025) and IgG (r = −0.40, p = 0.01) titers. We examined 11 antigen-positive and nine antigen-negative sera for infectious virus after three blind serial passages in HT-29 cells using immunofluorescence staining for rotavirus structural and nonstructural proteins. Infectious virus was detected in 11/11 (100%) sera from serum antigen-positive children and in two out of nine (22%) sera samples from antigen-negative children (p = 0.002). Conclusions Most children infected with rotavirus are viremic. The presence of viremia is directly related to the detection of antigenemia and is independent of the presence of diarrhea. Antigenemia load is inversely related to the titer of antirotavirus antibody in the serum. The finding of infectious rotavirus in the blood suggests extraintestinal involvement in rotavirus pathogenesis; however, the impact of rotavirus viremia on clinical manifestations of infection is unknown. PMID:17439294

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.

[Development of an optimal scheme for calculating results of the use of an immunoenzyme test-system for determining the antigenic activity of a cultured antirabies vaccine].

PubMed

Tsetlin, E M; Volkova, R A

1996-01-01

Ninety-eight lots of commercial antirabies vaccine manufactured by Immunopreparat Research and Production Amalgamation have been tested using enzyme immunoassay system for the detection of rabies virus antigens. Comparison of different variants of interpreting and expressing the results helped define the optimal method for assessment of vaccine titer and reference values: optical density value equal to 0.2 is taken as the cut-off. Antigenic activity of the vaccine may be expressed in international units, similarly as immunogenic activity.

Molecular diagnosis of Legionella infections--Clinical utility of front-line screening as part of a pneumonia diagnostic algorithm.

PubMed

Gadsby, Naomi J; Helgason, Kristjan O; Dickson, Elizabeth M; Mills, Jonathan M; Lindsay, Diane S J; Edwards, Giles F; Hanson, Mary F; Templeton, Kate E

2016-02-01

Urinary antigen testing for Legionella pneumophila serogroup 1 is the leading rapid diagnostic test for Legionnaires' Disease (LD); however other Legionella species and serogroups can also cause LD. The aim was to determine the utility of front-line L. pneumophila and Legionella species PCR in a severe respiratory infection algorithm. L. pneumophila and Legionella species duplex real-time PCR was carried out on 1944 specimens from hospitalised patients over a 4 year period in Edinburgh, UK. L. pneumophila was detected by PCR in 49 (2.7%) specimens from 36 patients. During a LD outbreak, combined L. pneumophila respiratory PCR and urinary antigen testing had optimal sensitivity and specificity (92.6% and 98.3% respectively) for the detection of confirmed cases. Legionella species was detected by PCR in 16 (0.9%) specimens from 10 patients. The 5 confirmed and 1 probable cases of Legionella longbeachae LD were both PCR and antibody positive. Front-line L. pneumophila and Legionella species PCR is a valuable addition to urinary antigen testing as part of a well-defined algorithm. Cases of LD due to L. longbeachae might be considered laboratory-confirmed when there is a positive Legionella species PCR result and detection of L. longbeachae specific antibody response. Copyright © 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

A pathological study of the tongues of rabid dogs in the Philippines.

PubMed

Shiwa, Nozomi; Kimitsuki, Kazunori; Manalo, Daria Llenaresas; Inoue, Satoshi; Park, Chun-Ho

2018-06-01

During rabies virus infections, the minor salivary glands are one of the important organs for virus replication and excretion into the oral cavity. However, details of pathological findings and viral antigen distribution in the minor salivary glands remain poorly understood. In this study, we conducted pathological tests on the tongues of 71 rabid dogs in the Philippines; the minor salivary glands (von Ebner's glands, lingual glands), circumvallate papilla, autonomic ganglia, and skeletal muscles were evaluated. Inflammatory changes were observed in the von Ebner's glands of 20/71 dogs, in the circumvallate papilla of 10/71, and in the tongue muscle of 1/71. Conversely, no morphological changes were observed in the lingual glands and autonomic ganglia. Viral antigens were detected via immunohistochemistry-based methods in the cytoplasm of the acinar epithelium in the von Ebner's glands of all 71 dogs. Virus particles were confirmed in the intercellular canaliculi and acinar lumen via electron microscopy. In the autonomic ganglia, viral antigens were detected in 67/71 rabid dogs. Viral antigens were detected in the taste buds of all 71 dogs, and were distributed mainly in type II and III taste bud cells. In tongue muscle fibers, viral antigens were detected in 11/71 dogs. No virus antigens were detected in lingual glands. These findings suggest that rabies virus descends in the tongue along the glossopharyngeal nerve after proliferation in the brain, and von Ebner's glands and taste buds are one of the portals of virus excretion into the saliva in rabid dogs.

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

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.

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

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.

Performance of Dengue Diagnostic Tests in a Single-Specimen Diagnostic Algorithm.

PubMed

Hunsperger, Elizabeth A; Muñoz-Jordán, Jorge; Beltran, Manuela; Colón, Candimar; Carrión, Jessica; Vazquez, Jesus; Acosta, Luz Nereida; Medina-Izquierdo, Juan F; Horiuchi, Kalanthe; Biggerstaff, Brad J; Margolis, Harold S

2016-09-15

Anti-dengue virus (DENV) immunoglobulin M (IgM) seroconversion has been the reference standard for dengue diagnosis. However, paired specimens are rarely obtained, and the interval for this testing negates its usefulness in guiding clinical case management. The presence of DENV viremia and appearance of IgM during the febrile phase of dengue provides the framework for dengue laboratory diagnosis by using a single serum specimen. Archived paired serum specimens (n = 1234) from patients with laboratory-confirmed dengue from 2005 through 2011 were used to determine the diagnostic performance of real-time reverse transcription polymerase chain reaction (RT-PCR), for detection of DENV serotypes 1-4, and enzyme-linked immunosorbent assays (ELISAs), for detection of DENV nonstructural protein 1 (NS1) antigen and anti-DENV IgM. During 1-3 days after illness onset, real-time RT-PCR and NS1 antigen testing detected 82%-69% and 90%-84% of cases, respectively, as viremia levels declined, while anti-DENV IgM ELISA detected 5%-41% of cases as antibody appeared. Over the 10-day period of the febrile phase of dengue, the cumulative effect of using these 3 types of tests in a diagnostic algorithm confirmed ≥90% of dengue cases. The use of molecular or NS1 antigen tests to detect DENV and one to detect anti-DENV IgM in a single serum specimen collected during the first 10 days of illness accurately identified ≥90% of dengue primary and secondary cases. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Rift Valley fever on the east coast of Madagascar.

PubMed

Morvan, J; Saluzzo, J F; Fontenille, D; Rollin, P E; Coulanges, P

1991-01-01

In March 1990, a Rift Valley fever virus (RVFV) outbreak was suspected in the district of Fenerive on the east coast of Madagascar after an abnormally high incidence of abortions and disease in livestock. Sera from humans and cattle were tested for RVFV antibodies by immunofluorescence assay (IFA) and ELISA-IgM capture. Sera and mosquitoes collected in the same area were tested for virus isolation by tissue culture and suckling mouse intracerebral inoculation, and for antigen detection by an ELISA antigen capture assay. Among cattle from the area, RVFV antibody prevalence was 58.6% by IFA and 29.6% by ELISA-IgM. In contrast, human populations in the same area had a lower RVFV antibody prevalence, with 8.01% IFA and 5.4% IgM-positive sera. No RVFV antigen was detected and virus isolation was unsuccessful from the sera and mosquito pools tested. Different hypotheses concerning the emergence and diffusion of RVFV in this area and the occurrence of the outbreak are discussed.

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

Increasing Early Detection of Prostate Cancer in African American Men Through a Culturally Targeted Print Intervention

DTIC Science & Technology

2006-03-01

of a protein called prostate-specific antigen (PSA). Normally, PSA is found in the blood at very low levels. Elevated PSA readings can be a sign of...cancer. ♦ Prostate Specific Antigen test (also called PSA test) - This simple blood test measures the level of a protein called prostate- specific...meat ♦ Lycopene, a compound in cooked tomato products and watermelon . 9 A number of Black men say they have problems with their

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.

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.

Macrophage migration inhibition test in untreated syphilis.

PubMed

Bowszyc, J

1975-01-01

Two modifications of macrophage migration inhibition test, one of George and Vaughan and the other one of Svejcar, were performed on a total of 78 cases of untreated syphilis at various stages. As specific antigens were used: Treponema Pallidum ultrasonate and cardiolipin. Inhibition of migration was observed in 87 percent patients with primary syphillis and in all patients with late and late congenital syphilis. After improving and standardisation of the technique of Treponema Pallidum antigen-the migration inhibition test may be recommended as a specific in vitro-test for detection of cell mediated immunity in syphilis.

Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents.

PubMed Central

Nato, F; Mazie, J C; Fournier, J M; Slizewicz, B; Sagot, N; Guibourdenche, M; Postic, D; Riou, J Y

1991-01-01

Polyclonal and monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, B, and C were produced in order to develop immunological reagents allowing both the detection of soluble antigens during meningococcal meningitis and antigenic serogrouping of N. meningitidis cultures. The performance characteristics of monoclonal and polyclonal antibody latex reagents were compared. For the detection of soluble polysaccharide antigen, polyclonal antibody latex reagent was selected for N. meningitidis A and C. The latex reagent prepared with polyclonal antibodies against N. meningitidis B could not detect capsular polysaccharide even at 1 mg/ml. The monoclonal antibody B latex reagent which detected 100 ng of polysaccharide per ml was therefore chosen. For the serogroup identification of N. meningitidis, the use of a confirmatory test results in an overall specificity of 100% with polyclonal or monoclonal antibody latex reagents. PMID:1909346

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.

Revisiting the Roles of Culture and Culture-Independent Detection Tests for Campylobacter.

PubMed

Couturier, Marc Roger

2016-05-01

Culture-independent detection tests (CIDTs) for Campylobacter have become an area of intense controversy and confusion among laboratorians in the field of clinical microbiology. To date, the true analytical and clinical performance of stool antigen CIDTs versus truly optimized culture conditions is unknown. In this issue of the Journal of Clinical Microbiology, Fitzgerald and colleagues (C. Fitzgerald et al., J Clin Microbiol 54:1209-1215, 2016, http://dx.doi.org/10.1128/JCM.01925-15) report comprehensive performance data for four Campylobacter stool antigen CIDTs versus culture and molecular diagnostics. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

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.

Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1985 Annual Report.

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

Kaattari, Stephen L.

1986-06-01

Bacterial kidney disease (BRD) has been and remains a chronic contributory problem limiting the productivity of salmon in the Columbia River Basin. Control of this disease will not come easily, but it would lead to a tremendous increase in the health and numbers of salmon populations. Vaccination of salmon to Renibacterium salmoninarum (KDB) is a potentially successful method of controlling this disease. To date, however, no successful vaccine has been developed for general use. A possible solution to this problem, and thus the goal of this research, is to isolate the antigenic components of KDB and enhance their ability tomore » activate the host defenses. This will be accomplished by the chemical modification of these antigens with potent immunomodulatory substances. These modified antigens will then be tested for their effectiveness in inducing immunity to BKD and thereby preventing the disease. The goal of the project's second year was to chemically modify the major antigens of Renibacteirium salmoninarum, immunize coho salmon (Oncorhynchus kisutch), and to test the immunogenicity of the preparations used. Immunogenicity of the antigenic material was tested by (1) admixture experiments, using whole KD cells with muramyl dipepetide, Vibrio anguillarum extract, E. coli lipopolysaccharide, or Mycobacterium tuberculosis in Freund's complete adjuvant. In addition to these goals a number of important techniques have been developed in order to facilitate the production of the vaccine. These procedures include: (1) the use of the soluble antigen for diagnosis in the ELISA and Western blot analysis, (2) detection of salmonid anti-KD antibodies by an ELISA technique, (3) detection of cellular immune responses to the soluble antigen, and (4) development of immersion challenge procedures for bacterial kidney disease (BKD).« less

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.

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.

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

PubMed

Khan, Mohsin; Dhanwani, Rekha; Kumar, Jyoti S; Rao, P V Lakshmana; Parida, Manmohan

2014-07-01

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous. © 2013 Wiley Periodicals, Inc.

[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.

Serological diagnosis of Taenia solium in pigs: No measurable circulating antigens and antibody response following exposure to Taenia saginata oncospheres.

PubMed

Dorny, P; Dermauw, V; Van Hul, A; Trevisan, C; Gabriël, S

2017-10-15

Taenia solium taeniasis/cysticercosis is a zoonosis included in the WHO's list of neglected tropical diseases. Accurate diagnostic tools for humans and pigs are needed to monitor intervention outcomes. Currently used diagnostic tools for porcine cysticercosis all have drawbacks. Serological tests are mainly confronted with problems of specificity. More specifically, circulating antigen detecting tests cross-react with Taenia hydatigena and the possibility of transient antigens as a result of aborted infections is suspected. Furthermore, the hypothesis has been raised that hatched ingested eggs of other Taenia species may lead to a transient antibody response or to the presence of circulating antigen detectable by serological tests used for porcine cysticercosis. Here we describe the results of a study that consisted of oral administration of Taenia saginata eggs to five piglets followed by serological testing during five weeks and necropsy aiming at studying possible cross reactions in serological tests used for porcine cysticercosis. The infectivity of the eggs was verified by in vitro hatching and by experimental infection of a calf. One piglet developed acute respiratory disease and died on day 6 post infection. The remaining four piglets did not show any clinical signs until euthanasia. None of the serum samples from four piglets collected between days 0 and 35 post infection gave a positive reaction in the B158/B60 Ag-ELISA and in a commercial Western blot for antibody detection. In conclusion, this study showed that experimental exposure of four pigs to T. saginata eggs did not result in positive serologies for T. solium. These results may help interpreting serological results in monitoring of T. solium control programmes. Copyright © 2017 Elsevier B.V. All rights reserved.

Potential for rapid antibody detection to identify tuberculous cattle with non-reactive tuberculin skin test results.

PubMed

Waters, W Ray; Vordermeier, H Martin; Rhodes, Shelley; Khatri, Bhagwati; Palmer, Mitchell V; Maggioli, Mayara F; Thacker, Tyler C; Nelson, Jeffrey T; Thomsen, Bruce V; Robbe-Austerman, Suelee; Bravo Garcia, Doris M; Schoenbaum, Mark A; Camacho, Mark S; Ray, Jean S; Esfandiari, Javan; Lambotte, Paul; Greenwald, Rena; Grandison, Adrian; Sikar-Gang, Alina; Lyashchenko, Konstantin P

2017-06-07

Bovine tuberculosis (TB) control programs generally rely on the tuberculin skin test (TST) for ante-mortem detection of Mycobacterium bovis-infected cattle. Present findings demonstrate that a rapid antibody test based on Dual-Path Platform (DPP ® ) technology, when applied 1-3 weeks after TST, detected 9 of 11 and 34 of 52 TST non-reactive yet M. bovis-infected cattle from the US and GB, respectively. The specificity of the assay ranged from 98.9% (n = 92, US) to 96.0% (n = 50, GB) with samples from TB-free herds. Multi-antigen print immunoassay (MAPIA) revealed the presence of antibodies to multiple antigens of M. bovis in sera from TST non-reactors diagnosed with TB. Thus, use of serologic assays in series with TST can identify a significant number of TST non-reactive tuberculous cattle for more efficient removal from TB-affected herds.

A Novel Technique for Detecting Antibiotic-Resistant Typhoid from Rapid Diagnostic Tests

PubMed Central

Nic Fhogartaigh, Caoimhe; Dance, David A. B.; Davong, Viengmon; Tann, Pisey; Phetsouvanh, Rattanaphone; Turner, Paul; Newton, Paul N.

2015-01-01

Fluoroquinolone-resistant typhoid is increasing. An antigen-detecting rapid diagnotic test (RDT) can rapidly diagnose typhoid from blood cultures. A simple, inexpensive molecular technique performed with DNA from positive RDTs accurately identified gyrA mutations consistent with phenotypic susceptibility testing results. Field diagnosis combined with centralized molecular resistance testing could improve typhoid management and surveillance in low-resource settings. PMID:25762768

Evaluation of a computer-assisted, kinetics-based enzyme-linked immunosorbent assay for detection of coronavirus antibodies in cats.

PubMed Central

Barlough, J E; Jacobson, R H; Downing, D R; Marcella, K L; Lynch, T J; Scott, F W

1983-01-01

A computer-assisted, kinetics-based enzyme-linked immunosorbent assay was adapted for the detection of coronavirus antibodies in feline serum. An alkaline antigen diluent (carbonate-bicarbonate buffer, pH 9.6) used in initial experiments produced diffuse, nonspecific color reactions in both viral and control antigen cuvettes which were correlated, paradoxically, with coronavirus antibody levels in test sera. These interfering reactions were minimized by use of lower-pH antigen diluents such as water and phosphate-buffered saline. Background kinetics-based enzyme-linked immunosorbent assay reactivity directed against a noncoronaviral component of antigen tissue culture fluids could then detected in numerous sera, particularly in samples with lower titers. Much of this reactivity was shown to be associated with bovine gamma globulins in cell culture fluid. It was not serum lot or species specific, since a variety of bovine serum lots as well as individual lots of serum from other mammalian and avian species reacted. Reactivity was markedly reduced when cells for antigen preparation were grown in gamma globulin-free bovine serum. Generation of corrected slope values from the kinetics-based enzyme-linked immunosorbent assay made it possible to correct for residual background reactivity in individual test sera and thus eliminate a potentially major source of false-positive reactions. Collectively, these studies indicated that the control of nonspecific reactivity in feline coronavirus serology is absolutely essential to obtain useful estimates of specific antibody responses. PMID:6300184

Evaluation of a computer-assisted, kinetics-based enzyme-linked immunosorbent assay for detection of coronavirus antibodies in cats.

PubMed

Barlough, J E; Jacobson, R H; Downing, D R; Marcella, K L; Lynch, T J; Scott, F W

1983-02-01

A computer-assisted, kinetics-based enzyme-linked immunosorbent assay was adapted for the detection of coronavirus antibodies in feline serum. An alkaline antigen diluent (carbonate-bicarbonate buffer, pH 9.6) used in initial experiments produced diffuse, nonspecific color reactions in both viral and control antigen cuvettes which were correlated, paradoxically, with coronavirus antibody levels in test sera. These interfering reactions were minimized by use of lower-pH antigen diluents such as water and phosphate-buffered saline. Background kinetics-based enzyme-linked immunosorbent assay reactivity directed against a noncoronaviral component of antigen tissue culture fluids could then detected in numerous sera, particularly in samples with lower titers. Much of this reactivity was shown to be associated with bovine gamma globulins in cell culture fluid. It was not serum lot or species specific, since a variety of bovine serum lots as well as individual lots of serum from other mammalian and avian species reacted. Reactivity was markedly reduced when cells for antigen preparation were grown in gamma globulin-free bovine serum. Generation of corrected slope values from the kinetics-based enzyme-linked immunosorbent assay made it possible to correct for residual background reactivity in individual test sera and thus eliminate a potentially major source of false-positive reactions. Collectively, these studies indicated that the control of nonspecific reactivity in feline coronavirus serology is absolutely essential to obtain useful estimates of specific antibody responses.

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.

Prostate-specific antigen (PSA) blood test

MedlinePlus

... very early. But there is debate over the value of the PSA test for detecting prostate cancer. No single answer fits all men. Before having the test, talk to your provider about the pros and cons of having a PSA test. Ask ...

Varied distribution of RhD epitopes in the Indian population.

PubMed

Kulkarni, S S; Gupte, S C; Vasantha, K; Mohanty, D; Ghosh, K

2007-01-01

Inhabited by more than 4000 caste and tribal groups, India has an extremely heterogenous population. For thousands of years many tribal groups have practised endogamy and are practically genetically isolated. Traditionally, polyclonal anti-D reagent has been used for RhD typing; though monoclonal antibodies are increasingly being used. As a result, blood banks find it difficult to assign the RhD status to an increasing number of people. As monoclonal anti-D typing reagents may not detect all RhD antigen epitopes, we studied the RhD antigen epitope heterogeneity in different population groups in India. Red cells of 5315 RhD-positive individuals belonging to different castes and tribes of India were tested with 30 different epitope-specific monoclonal anti-D antibodies. No single monoclonal antibody could detect all RhD-positive red cells detected by polyclonal antisera. The highest proportion of D antigen was detected by LHM 76/55 and BRAD-8 (98%) monoclonal antibodies. We need to determine the correct mix of monoclonal antibodies that will detect nearly all RhD antigens detected by polyclonal anti-D sera. Similarly, before accepting monoclonal anti-D for therapeutic use, it would be necessary to determine the appropriate ones for use in the Indian population.

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.

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.

Immunohistochemical Demonstration of Specific Antigens in the Human Brain Fixed in Zinc-ethanol-Formaldehyde

PubMed Central

Korzhevskii, D.E.; Sukhorukova, E.G.; Kirik, O.V.; Grigorev, I.P.

2015-01-01

Tissue fixation is critical for immunohistochemistry. Recently, we developed a zinc-ethanol-formalin fixative (ZEF), and the present study was aimed to assess the applicability of the ZEF for the human brain histology and immunohistochemistry and to evaluate the detectability of different antigens in the human brain fixed with ZEF. In total, 11 antigens were tested, including NeuN, neuron-specific enolase, GFAP, Iba-1, calbindin, calretinin, choline acetyltransferase, glutamic acid decarboxylase (GAD65), tyrosine hydroxylase, synaptophysin, and α-tubulin. The obtained data show that: i) the ZEF has potential for use in general histological practice, where detailed characterization of human brain morphology is needed; ii) the antigens tested are well-preserved in the human brain specimens fixed in the ZEF. PMID:26428887

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.

Comparison of four diagnostic tests for the identification of serum antibodies in small ruminants infected with Mycoplasma agalactiae.

PubMed

Kittelberger, R; O'Keefe, J S; Meynell, R; Sewell, M; Rosati, S; Lambert, M; Dufour, P; Pépin, M

2006-02-01

To determine the diagnostic capability of a newly developed Western blot (WB) assay for the detection of serum antibodies against Mycoplasma agalactiae compared with conventional serological tests, and to identify the best test for routine diagnostic use. The serological test methods used were: two commercial indirect enzyme-linked immunosorbent assays (ELISA), viz ELISA-1, using a bacterial antigen preparation, and ELISA-2, using a recombinant protein (lipoprotein p48) antigen; the complement fixation test (CFT); and a newly developed WB assay, the latter both using a bacterial antigen preparation. Thirty sera from goats infected with M. agalactiae and 97 sera from non-infected sheep were tested using all four methods. Staining patterns in the WB were quite variable. An immuno-dominant band of 41 kDa was detected in 63% of sera from infected animals. The same band also appeared, although mostly very weakly, in 10% of sera from non-infected animals. When suspicious or very weak reactors were omitted, the diagnostic sensitivity (DSE) and diagnostic specificity (DSP), respectively, for the four assays were: WB=56.7%, 97.9%; ELISA-1=76.7%, 99.0%; ELISA-2=56.7%, 100%; and CFT=40.0%, 94.8%. ELISA-1 performed best in this comparison. While the WB can be used, it did not have a technical advantage over the ELISA. The CFT should be discouraged as the primary screening method for contagious agalactia and should be replaced by ELISA-1. Results from this study confirm that serological test methods for contagious agalactia are useful for the detection of infected flocks but will not detect every individual infected animal.

ATP diphosphohydrolase from Schistosoma mansoni egg: characterization and immunocytochemical localization of a new antigen.

PubMed

Faria-Pinto, P; Meirelles, M N L; Lenzi, H L; Mota, E M; Penido, M L O; Coelho, P M Z; Vasconcelos, E G

2004-07-01

The fact that the Schistosoma mansoni egg has two ATP diphosphohydrolase (EC 3.6.1.5) isoforms with different net charges and an identical molecular weight of 63,000, identified by non-denaturing polyacrylamide gel electrophoresis and immunological cross-reactivity with potato apyrase antibodies, is shown. In soluble egg antigen (SEA), only the isoform with the lower net negative charge was detected and seemed to be the predominant species in this preparation. By confocal fluorescence microscopy, using anti-potato apyrase antibodies, the S. mansoni egg ATP diphosphohydrolase was detected on the external surface of miracidium and in von Lichtenberg's envelope. Intense fluorescence was also seen in the outer side of the egg-shell, entrapped by the surface microspines, suggesting that a soluble isoform is secreted. ATP diphosphohydrolase antigenicity was tested using the vegetable protein as antigen. The purified potato apyrase was recognized in Western blots by antibodies present in sera from experimentally S. mansoni-infected mice. In addition, high levels of IgG anti-ATP diphosphohydrolase antibodies were detected by ELISA in the same sera. This work represents the first demonstration of antigenic properties of S. mansoni ATP diphosphohydrolase and immunological cross-reactivity between potato apyrase and sera from infected individuals.

Laboratory Surveillance of Rabies in Humans, Domestic Animals, and Bats in Madagascar from 2005 to 2010

PubMed Central

Reynes, Jean-Marc; Andriamandimby, Soa Fy; Razafitrimo, Girard Marcelin; Razainirina, Josette; Jeanmaire, Elisabeth Marie; Bourhy, Hervé; Heraud, Jean-Michel

2011-01-01

Background. Rabies virus (RABV) has circulated in Madagascar at least since the 19th century. Objectives. To assess the circulation of lyssavirus in the island from 2005 to 2010. Materials and Methods. Animal (including bats) and human samples were tested for RABV and other lyssavirus using antigen, ribonucleic acid (RNA), and antibodies detection and virus isolation. Results. Half of the 437 domestic or tame wild terrestrial mammal brains tested were found RABV antigen positive, including 54% of the 341 dogs tested. This percentage ranged from 26% to 75% across the period. Nine of the 10 suspected human cases tested were laboratory confirmed. RABV circulation was confirmed in 34 of the 38 districts sampled. No lyssavirus RNA was detected in 1983 bats specimens. Nevertheless, antibodies against Lagos bat virus were detected in the sera of 12 among 50 Eidolon dupreanum specimens sampled. Conclusion. More than a century after the introduction of the vaccine, rabies still remains endemic in Madagascar. PMID:21991442

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.

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.

Assessment of the repeatability and border-plate effects of the B158/B60 enzyme-linked-immunosorbent assay for the detection of circulating antigens (Ag-ELISA) of Taenia saginata.

PubMed

Jansen, Famke; Dorny, Pierre; Berkvens, Dirk; Van Hul, Anke; Van den Broeck, Nick; Makay, Caroline; Praet, Nicolas; Gabriël, Sarah

2016-08-30

The monoclonal antibody-based circulating antigen detecting ELISA (B158/B60 Ag-ELISA) has been used elaborately in several studies for the diagnosis of human, bovine and porcine cysticercosis. Interpretation of test results requires a good knowledge of the test characteristics, including the repeatability and the effect of the borders of the ELISA plates. Repeatability was tested for 4 antigen-negative and 5 antigen-positive reference bovine serum samples by calculating the Percentage Coefficient of Variation (%CV) within and between plates, within and between runs, overall, for two batches of monoclonal antibodies and by 2 laboratory technicians. All CV values obtained were below 20% (except one: 24.45%), which indicates a good repeatability and a negligible technician error. The value of 24.45% for indicating the variability between batches of monoclonal antibodies for one positive sample is still acceptable for repeatability measures. Border effects were determined by calculating the %CV values between the inner and outer wells of one plate for 2 positive serum samples. Variability is a little more present in the outer wells but this effect is very small and no significant border effect was found. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of an Indirect ELISA Based on a Recombinant Chimeric Protein for the Detection of Antibodies against Bovine Babesiosis

PubMed Central

Jaramillo Ortiz, José Manuel; Montenegro, Valeria Noely; de la Fournière, Sofía Ana María; Sarmiento, Néstor Fabián; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

2018-01-01

The current method for Babesia spp. serodiagnosis based on a crude merozoite antigen is a complex and time-consuming procedure. An indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant multi-antigen of Babesia bovis (rMABbO) was developed for detection of antibodies in bovines suspected of infection with this parasite. The multi-antigen comprises gene fragments of three previously characterized B. bovis antigens: MSA-2c, RAP-1 and the Heat Shock protein 20 that are well-conserved among geographically distant strains. The cutoff value for the new rMABbo-iELISA was determined using 75 known—positive and 300 known—negative bovine sera previously tested for antibodies to B. bovis by the gold-standard ELISA which uses a merozoite lysate. A cutoff value of ≥35% was determined in these samples by receiver operator characteristic (ROC) curve analysis, showing a sensitivity of 95.9% and a specificity of 94.3%. The rMABbo-iELISA was further tested in a blind trial using an additional set of 263 field bovine sera from enzootic and tick-free regions of Argentina. Results showed a good agreement with the gold standard test with a Cohen’s kappa value of 0.76. Finally, the prevalence of bovine babesiosis in different tick enzootic regions of Argentina was analyzed where seropositivity values among 68–80% were obtained. A certain level of cross reaction was observed when samples from B. bigemina infected cattle were analyzed with the new test, which can be attributed to shared epitopes between 2 of the 3 antigens. This new rMABbo-iELISA could be considered a simpler alternative to detect anti Babesia spp. antibodies and appears to be well suited to perform epidemiological surveys at the herd level in regions where ticks are present. PMID:29360801

Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease

PubMed Central

Weiner, Zachary P.; Crew, Rebecca M.; Brandt, Kevin S.; Ullmann, Amy J.; Schriefer, Martin E.; Molins, Claudia R.

2015-01-01

Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing. PMID:26376927

Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease.

PubMed

Weiner, Zachary P; Crew, Rebecca M; Brandt, Kevin S; Ullmann, Amy J; Schriefer, Martin E; Molins, Claudia R; Gilmore, Robert D

2015-11-01

Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Development of an Indirect ELISA Based on a Recombinant Chimeric Protein for the Detection of Antibodies against Bovine Babesiosis.

PubMed

Jaramillo Ortiz, José Manuel; Montenegro, Valeria Noely; de la Fournière, Sofía Ana María; Sarmiento, Néstor Fabián; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

2018-01-23

The current method for Babesia spp. serodiagnosis based on a crude merozoite antigen is a complex and time-consuming procedure. An indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant multi-antigen of Babesia bovis (rMABbO) was developed for detection of antibodies in bovines suspected of infection with this parasite. The multi-antigen comprises gene fragments of three previously characterized B. bovis antigens: MSA-2c, RAP-1 and the Heat Shock protein 20 that are well-conserved among geographically distant strains. The cutoff value for the new rMABbo-iELISA was determined using 75 known-positive and 300 known-negative bovine sera previously tested for antibodies to B. bovis by the gold-standard ELISA which uses a merozoite lysate. A cutoff value of ≥35% was determined in these samples by receiver operator characteristic (ROC) curve analysis, showing a sensitivity of 95.9% and a specificity of 94.3%. The rMABbo-iELISA was further tested in a blind trial using an additional set of 263 field bovine sera from enzootic and tick-free regions of Argentina. Results showed a good agreement with the gold standard test with a Cohen's kappa value of 0.76. Finally, the prevalence of bovine babesiosis in different tick enzootic regions of Argentina was analyzed where seropositivity values among 68-80% were obtained. A certain level of cross reaction was observed when samples from B. bigemina infected cattle were analyzed with the new test, which can be attributed to shared epitopes between 2 of the 3 antigens. This new rMABbo-iELISA could be considered a simpler alternative to detect anti Babesia spp. antibodies and appears to be well suited to perform epidemiological surveys at the herd level in regions where ticks are present.

Detection of Leptospira interrogans DNA and antigen in fixed equine eyes affected with end-stage equine recurrent uveitis.

PubMed

Pearce, Jacqueline W; Galle, Laurence E; Kleiboeker, Steve B; Turk, James R; Schommer, Susan K; Dubielizig, Richard R; Mitchell, William J; Moore, Cecil P; Giuliano, Elizabeth A

2007-11-01

Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on clinical signs and histologic lesions. Sections were subjected to immunohistochemical staining with an array of rabbit anti-Leptospira polyclonal antibodies. DNA extractions were performed by using a commercial kit designed for fixed tissue. Real-time PCR analysis was completed on extracted DNA. The target sequence for PCR was designed from alignments of available Leptospira 16S rDNA partial sequences obtained from GenBank. Two of 10 test samples were positive for Leptospira antigen by immunohistochemical assay. Zero of 20 controls were positive for Leptospira antigen. All test samples and controls were negative for Leptospira DNA by real-time PCR analysis. Leptospira was detected at a lower frequency than that previously reported for fresh ERU-affected aqueous humor and vitreous samples. Leptospira is not frequently detectable in fixed ocular tissues of horses affected with ERU when using traditional immunohistochemical and real-time PCR techniques.

A new paradigm for Aedes spp. surveillance using gravid ovipositing sticky trap and NS1 antigen test kit.

PubMed

Lau, Sai Ming; Chua, Tock H; Sulaiman, Wan-Yussof; Joanne, Sylvia; Lim, Yvonne Ai-Lian; Sekaran, Shamala Devi; Chinna, Karuthan; Venugopalan, Balan; Vythilingam, Indra

2017-03-21

Dengue remains a serious public health problem in Southeast Asia and has increased 37-fold in Malaysia compared to decades ago. New strategies are urgently needed for early detection and control of dengue epidemics. We conducted a two year study in a high human density dengue-endemic urban area in Selangor, where Gravid Ovipositing Sticky (GOS) traps were set up to capture adult Aedes spp. mosquitoes. All Aedes mosquitoes were tested using the NS1 dengue antigen test kit. All dengue cases from the study site notified to the State Health Department were recorded. Weekly microclimatic temperature, relative humidity (RH) and rainfall were monitored. Aedes aegypti was the predominant mosquito (95.6%) caught in GOS traps and 23% (43/187 pools of 5 mosquitoes each) were found to be positive for dengue using the NS1 antigen kit. Confirmed cases of dengue were observed with a lag of one week after positive Ae. aegypti were detected. Aedes aegypti density as analysed by distributed lag non-linear models, will increase lag of 2-3 weeks for temperature increase from 28 to 30 °C; and lag of three weeks for increased rainfall. Proactive strategy is needed for dengue vector surveillance programme. One method would be to use the GOS trap which is simple to setup, cost effective (below USD 1 per trap) and environmental friendly (i.e. use recyclable plastic materials) to capture Ae. aegypti followed by a rapid method of detecting of dengue virus using the NS1 dengue antigen kit. Control measures should be initiated when positive mosquitoes are detected.

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

PubMed Central

Nakatsuma, Akira; Kaneda, Mugiho; Kodama, Hiromi; Morikawa, Mika; Watabe, Satoshi; Nakaishi, Kazunari; Yamashita, Masakane; Yoshimura, Teruki; Miura, Toshiaki; Ninomiya, Masaki; Ito, Etsuro

2015-01-01

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10-18 moles of the p24/assay corresponds to ca. 103 copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (102 copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA. PMID:26098695

Antiserum Preparation For Immunodiffusion In Southern Pine Beetle Predation Studies

Treesearch

M.C. Miller; W. Adrian Chappell; William C. Gamble; J. Robert Bridges

1978-01-01

An anti-adult southern pine beetle serum was produced by subcutaneous injection of rabbits with southern pine beetle (SPB) adult antigen. Initial tests demonstrated the ability of the anti-adult SPB serum to detect adult SPB antigen in the body of the adult predator, Thanasimus dubius (F.). Cross reactivity was found between the anti-adult serum...

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

Diagnostic, therapeutic and economic consequences of a positive urinary antigen test for Legionella spp. in patients admitted with community-acquired pneumonia: a 7-year retrospective evaluation.

PubMed

Engel, M F; van Manen, L; Hoepelman, A I M; Thijsen, S; Oosterheert, J J

2013-09-01

A positive urinary antigen test for Legionella spp. (Legionella urinary antigen test; LUAT) allows an early switch from empiric to targeted treatment (TT) in hospitalised, community-acquired pneumonia (CAP) patients. We aimed to evaluate the diagnostic, therapeutic and economic consequences of this frequently used test 7 years after its implementation. We retrospectively evaluated LUATs performed between 2005 and 2011 in two teaching hospitals. All tests performed in hospitalised CAP patients were used in the economic evaluation and positive tests were included in the treatment evaluation. Data on patient characteristics, admission and outcome were retrieved from the patients' files. The number of days gained by making a rapid aetiological diagnosis, the number of days TT could be provided and their costs were calculated. Of 4485 LUATs, 2504 (56%) were performed for CAP including 55 (1%) positive tests (€1041/positive test). In 26 (60%) of the 43 included positive tests, LUAT was the only test showing Legionella spp. Subsequently, earlier TT was possible in the remaining cases during 209 cumulative admission days (€274/TT day). LUAT led to detection of Legionella spp. 13 days earlier per case (€203/day) as compared with culture/serology alone. Timely LUAT use in accordance with current guidelines allows early detection and treatment of CAP caused by Legionella spp. at considerable expense.

Combination of culture, antigen and toxin detection, and cytotoxin neutralization assay for optimal Clostridium difficile diagnostic testing

PubMed Central

Alfa, Michelle J; Sepehri, Shadi

2013-01-01

BACKGROUND: There has been a growing interest in developing an appropriate laboratory diagnostic algorithm for Clostridium difficile, mainly as a result of increases in both the number and severity of cases of C difficile infection in the past decade. A C difficile diagnostic algorithm is necessary because diagnostic kits, mostly for the detection of toxins A and B or glutamate dehydrogenase (GDH) antigen, are not sufficient as stand-alone assays for optimal diagnosis of C difficile infection. In addition, conventional reference methods for C difficile detection (eg, toxigenic culture and cytotoxin neutralization [CTN] assays) are not routinely practiced in diagnostic laboratory settings. OBJECTIVE: To review the four-step algorithm used at Diagnostic Services of Manitoba sites for the laboratory diagnosis of toxigenic C difficile. RESULT: One year of retrospective C difficile data using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenic C difficile. Sixty per cent (310 of 517) of toxigenic C difficile stools were detected following the first two steps of the algorithm. CTN confirmation of GDH-positive, toxin A- and B-negative assays resulted in detection of an additional 37.7% (198 of 517) of toxigenic C difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenic C difficile samples. DISCUSSION: Using GDH antigen as the screening and toxin A and B as confirmatory test for C difficile, 85% of specimens were reported negative or positive within 4 h. Without CTN confirmation for GDH antigen and toxin A and B discordant results, 37% (195 of 517) of toxigenic C difficile stools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted for C difficile testing. CONCLUSION: The overview of the data illustrated the significance of each stage of this four-step C difficile algorithm and emphasized the value of using CTN assay and culture as parts of an algorithm that ensures accurate diagnosis of toxigenic C difficile. PMID:24421808

Diagnosis of haematological disease using anti-i. II. Distinction between acute myeloblastic and acute lymphoblastic leukaemia.

PubMed

Shumak, K H; Rachkewich, R A; Beldotti, L E

1979-03-01

Leukaemic blast cells were obtained from the blood of six patients with acute lymphoblastic leukaemia (ALL) and 15 patients with acute myeloblastic leukaemia (AML). The blasts were compared with lymphocytes from normal subjects in cytotoxicity and 125I-labelled antibody binding tests using several examples of anti-i. As much i antigen was detected on ALL blasts as on normal lymphocytes; much less i antigen was detected on AML blasts. Studies of three patients with morphologically undifferentiated acute leukaemia suggest that, in tests with anti-i, blasts from such patients react either like lymphoblasts or myeloblasts despite the absence of the corresponding morphological features.

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.

Retrospective swine influenza serological surveillance in the four highest pig density provinces of Thailand before the introduction of the 2009 pandemic Influenza A virus subtype H1N1 using various antibody detection assays.

PubMed

Sreta, Donruethai; Jittimanee, Suphattra; Charoenvisal, Nataya; Amonsin, Alongkorn; Kitikoon, Pravina; Thanawongnuwech, Roongroje

2013-01-01

Genetic characterization of the hemagglutinin gene of the 6 selected Thai Swine influenza virus (SIV) isolates (4 H1 and 2 H3 isolates) used in the establishment of a hemagglutination inhibition (HI) assay was analyzed. Based on the phylogenetic analysis, Thai SIVs could be divided into 3 clusters of the H1 viruses (clusters I and II belonging to classical swine H1α, and cluster III belonging to classical swine H1γ), and 2 clusters of the H3 viruses both belonging to human-like 1970s. The serological results indicated that swH1N1-06 (H1 cluster I) is a suitable representative SIV for the HI test antigen to detect H1 SIV-specific antibodies in the Thai swine population, while both swH3N2-05 and swH3N2-07 should be used for Thai H3 SIV-specific antibody detection. The HI test results of swine sera collected from pigs in the 4 highest pig population provinces of Thailand indicated that the percentage of pigs seropositive to swH3N2-07 was highest compared to swH1N1-06, swH1N1-09, and swH3N2-05 (85.4%, 50.1%, 18.6%, and 15.8%, respectively). It should be noted that countries lacking SIV genetic information should be concerned with determining the most suitable HI test antigens to use when performing the tests due to the genetic variation and limited cross-reaction of SIVs. The results of the current study demonstrated that HI tests should be implemented with the suitable field strains as the representative test antigen to ascertain accurate SIV serostatus in Thailand and that test antigens should be genetically analyzed and compared with circulating strains regularly.

Clinical score and rapid antigen detection test to guide antibiotic use for sore throats: randomised controlled trial of PRISM (primary care streptococcal management).

PubMed

Little, Paul; Hobbs, F D Richard; Moore, Michael; Mant, David; Williamson, Ian; McNulty, Cliodna; Cheng, Ying Edith; Leydon, Geraldine; McManus, Richard; Kelly, Joanne; Barnett, Jane; Glasziou, Paul; Mullee, Mark

2013-10-10

To determine the effect of clinical scores that predict streptococcal infection or rapid streptococcal antigen detection tests compared with delayed antibiotic prescribing. Open adaptive pragmatic parallel group randomised controlled trial. Primary care in United Kingdom. Patients aged ≥ 3 with acute sore throat. An internet programme randomised patients to targeted antibiotic use according to: delayed antibiotics (the comparator group for analyses), clinical score, or antigen test used according to clinical score. During the trial a preliminary streptococcal score (score 1, n=1129) was replaced by a more consistent score (score 2, n=631; features: fever during previous 24 hours; purulence; attends rapidly (within three days after onset of symptoms); inflamed tonsils; no cough/coryza (acronym FeverPAIN). Symptom severity reported by patients on a 7 point Likert scale (mean severity of sore throat/difficulty swallowing for days two to four after the consultation (primary outcome)), duration of symptoms, use of antibiotics. For score 1 there were no significant differences between groups. For score 2, symptom severity was documented in 80% (168/207 (81%) in delayed antibiotics group; 168/211 (80%) in clinical score group; 166/213 (78%) in antigen test group). Reported severity of symptoms was lower in the clinical score group (-0.33, 95% confidence interval -0.64 to -0.02; P=0.04), equivalent to one in three rating sore throat a slight versus moderate problem, with a similar reduction for the antigen test group (-0.30, -0.61 to -0.00; P=0.05). Symptoms rated moderately bad or worse resolved significantly faster in the clinical score group (hazard ratio 1.30, 95% confidence interval 1.03 to 1.63) but not the antigen test group (1.11, 0.88 to 1.40). In the delayed antibiotics group, 75/164 (46%) used antibiotics. Use of antibiotics in the clinical score group (60/161) was 29% lower (adjusted risk ratio 0.71, 95% confidence interval 0.50 to 0.95; P=0.02) and in the antigen test group (58/164) was 27% lower (0.73, 0.52 to 0.98; P=0.03). There were no significant differences in complications or reconsultations. Targeted use of antibiotics for acute sore throat with a clinical score improves reported symptoms and reduces antibiotic use. Antigen tests used according to a clinical score provide similar benefits but with no clear advantages over a clinical score alone. ISRCTN32027234.

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.

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.

Development of a Fish Stress Protein Antibody/Antigen-Based Approach for Biomonitoring of Water Quality.

DTIC Science & Technology

1993-01-25

10 DISCUSSION ............................................... 14 FIELD TESTS OF ANTIBODY DETECTION OF HEAT SHOCK PROTEIN ACCUMULATION IN... TESTS OF ANTIBODY DETECTION OF HEAT SHOCK PROTEIN ACCUMULATION IN ASIAN CLAMS (CORBICULA FLUMINEA) INTRODUCTION The Trinity River flows through...the utility of induction of heat shock proteins as an indicator of stress in another test organism, the Asian clam (Corbicula fluminea). This organism

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

Biomarkers for prostate cancer detection and progression: Beyond prostate-specific antigen.

PubMed

Berney, Daniel M

2010-04-01

Prostate cancer is a major health problem with an incompletely understood pathogenesis and etiology. The advent of the prostate-specific antigen (PSA) test in the 1980s caused a revolution in how the disease was detected, but the evidence for PSA as a screening test is deficient. Biomarkers have been investigated, both for detection and discrimination of indolent from aggressive cancers. Refinements to the PSA test have been proposed but for practical and evidence-based reasons none have translated through to widespread clinical use. Of the novel biomarkers, the most promising is the prostate cancer antigen 3 (PCA3) test. New biomarkers to predict aggressive disease are even more contentious. The pathological grade of tumor remains the most powerful biomarker of prognosis. Other proven variables include tumor extent on biopsy and serum PSA. Tissue biomarkers have proven unhelpful due to a variable and biased literature with multiple methodological flaws, but Ki-67 probably shows more promise than any other current tissue biomarker. The recent discovery of a family of fusion genes in the prostate has led to considerable discussion on their prognostic role. Dissection of the genetic basis of the disease may lead to discoveries that will enhance our understanding and aid the search for prognostically valuable biomarkers. Copyright 2010 Prous Science, S.A.U. or its licensors. All rights reserved.

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.

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.

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections.

PubMed

Bentes, Gentil Arthur; Lanzarini, Natália Maria; Lima, Lyana Rodrigues Pinto; Manso, Pedro Paulo de Abreu; da Silva, Alexandre Dos Santos; Mouta Junior, Sergio da Silva E; Guimarães, Juliana Rodrigues; de Moraes, Marcia Terezinha Baroni; Pelajo-Machado, Marcelo; Pinto, Marcelo Alves

2015-06-01

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

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.

A rapid field test for sylvatic plague exposure in wild animals.

PubMed

Abbott, Rachel C; Hudak, Robert; Mondesire, Roy; Baeten, Laurie A; Russell, Robin E; Rocke, Tonie E

2014-04-01

Plague surveillance is routinely conducted to predict future epizootics in wildlife and exposure risk for humans. The most common surveillance method for sylvatic plague is detection of antibodies to Yersinia pestis F1 capsular antigen in sentinel animals, such as coyotes (Canis latrans). Current serologic tests for Y. pestis, hemagglutination (HA) test and enzyme-linked immunosorbent assay (ELISA), are expensive and labor intensive. To address this need, we developed a complete lateral flow device for the detection of specific antibodies to Y. pestis F1 and V antigens. Our test detected anti-F1 and anti-V antibodies in serum and Nobuto filter paper samples from coyotes, and in serum samples from prairie dogs (Cynomys ludovicianus), lynx (Lynx canadensis), and black-footed ferrets (Mustela nigripes). Comparison of cassette results for anti-F1 and anti-V antibodies with results of ELISA or HA tests showed correlations ranging from 0.68 to 0.98. This device provides an affordable, user-friendly tool that may be useful in plague surveillance programs and as a research tool.

A rapid field test for sylvatic plague exposure in wild animals

USGS Publications Warehouse

Abbott, Rachel C.; Hudak, Robert; Mondesire, Roy; Baeten, Laurie A.; Russell, Robin E.; Rocke, Tonie E.

2014-01-01

Plague surveillance is routinely conducted to predict future epizootics in wildlife and exposure risk for humans. The most common surveillance method for sylvatic plague is detection of antibodies to Yersinia pestis F1 capsular antigen in sentinel animals, such as coyotes (Canis latrans). Current serologic tests for Y. pestis, hemagglutination (HA) test and enzyme-linked immunosorbent assay (ELISA), are expensive and labor intensive. To address this need, we developed a complete lateral flow device for the detection of specific antibodies to Y. pestis F1 and V antigens. Our test detected anti-F1 and anti-V antibodies in serum and Nobuto filter paper samples from coyotes, and in serum samples from prairie dogs (Cynomys ludovicianus), lynx (Lynx canadensis), and black-footed ferrets (Mustela nigripes). Comparison of cassette results for anti-F1 and anti-V antibodies with results of ELISA or HA tests showed correlations ranging from 0.68 to 0.98. This device provides an affordable, user-friendly tool that may be useful in plague surveillance programs and as a research tool.

The first Korean case report of anti-Gerbich.

PubMed

Jeon, You La; Park, Tae Sung; Cho, Sun Young; Oh, Seung Hwan; Kim, Myeong Hee; Kang, So Young; Lee, Woo-In

2012-11-01

In this study, we report the first Korean case of an anti-Gerbich (Ge) alloantibody to a high-incidence antigen that belongs to the Ge blood group system. The alloantibody was detected in a middle-aged Korean woman who did not have a history of transfusion. Her blood type was B+, and findings from the antibody screening test revealed 1+ reactivity in all panels except the autocontrol. The cross-matching test showed incompatible results with all 5 packed red blood cells. Additional blood type antigen and antibody tests confirmed the anti-Ge alloantibody. While rare, cases of hemolytic transfusion reaction or hemolytic disease in newborns due to anti-Ge have been recently reported in the literature. Therefore, additional further studies on alloantibodies to high-incidence antigens, including anti-Ge, are necessary in the future.

A survey of feline leukaemia virus infection of domestic cats from selected areas in Harare, Zimbabwe.

PubMed

Muchaamba, Francis; Mutiringindi, Takudzwa H; Tivapasi, Musavenga T; Dhliwayo, Solomon; Matope, Gift

2014-11-14

A cross-sectional study was conducted to detect the feline leukaemia virus (FeLV) p27 antigen and to determine risk factors and the haematological changes associated with infection in domestic cats in Zimbabwe. Sera were collected for detection of the p27 antigen, urea, creatinine, alanine aminotransferase and gamma-glutamyl transferase levels, whilst whole blood was collected for haematology. FeLV p27 antigen was detected using a rapid enzyme-linked immunosorbent assay (ELISA) test kit. Data on risk factors were analysed using a logistic regression model. Of the 100 cats tested, 41% (95% CI: 31.19% - 50.81%) (41/100) were positive for the FeLV p27 antigen. Sex and health status of cats were not significantly (p > 0.05) associated with infection. Intact cats (OR = 9.73), those living in multicat housing (OR = 5.23) and cats that had access to outdoor life (OR = 35.5) were found to have higher odds of infection compared with neutered cats, those living in single-cat housing, and without access to outdoor life, respectively. Biochemistry and haematology revealed no specific changes. The results showed that FeLV infection was high in sampled cats, providing evidence of active infection. Thus, it would be prudent to introduce specific control measures for FeLV infection in Zimbabwe.

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.

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.

Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients.

PubMed

Shehat, Michael G; Bahey-El-Din, Mohammed; Kassem, Mervat A; Farghaly, Faten A; Abdul-Rahman, Medhat H; Fanaki, Nourhan H

2015-08-01

HCV is a single-stranded RNA virus with a single open reading frame (ORF) that is translated into a polyprotein that is then processed to form 10 viral proteins. An additional eleventh viral protein, the alternative reading frame protein (ARFP), was discovered relatively recently. This protein results from a translational frameshift in the core region during the expression of the viral proteins. Recombinant expression of different forms of ARFP was previously done for HCV genotypes 1 and 2, and more recently, genotype 3. However, none of the previous studies addressed the expression of ARFP of HCV genotype 4a, which is responsible for 80 % of HCV infections in the Middle East and Africa. Moreover, the direct detection of the ARFP antigen in HCV-infected patients was never studied before for any HCV genotype. In the present study, recombinant ARFP derived from HCV genotype 4a was successfully expressed in E. coli and purified using metal affinity chromatography. The recombinant ARFP protein and anti-ARFP antibodies were used for detection of ARFP antigen in patients' sera, employing competitive enzyme-linked immunosorbent assay (ELISA) procedures. Furthermore, the recombinant antigen was also used to detect and quantify anti-ARFP antibodies in HCV-infected Egyptian patients at different stages of pegylated interferon/ribavirin therapy, using an ELISA assay. The ARFP antigen was detectable in 69.4 % of RNA-positive sera, indicating that ARFP antigen is produced during the natural course of HCV infection. In addition, significant levels of anti-ARFP antibodies were present in 41 % of the serum samples tested. The important diagnostic value of the recombinant ARFP antigen was also demonstrated.

Diagnosis and treatment of streptococcal pharyngitis.

PubMed

Choby, Beth A

2009-03-01

Common signs and symptoms of streptococcal pharyngitis include sore throat, temperature greater than 100.4 degrees F (38 degrees C), tonsillar exudates, and cervical adenopathy. Cough, coryza, and diarrhea are more common with viral pharyngitis. Available diagnostic tests include throat culture and rapid antigen detection testing. Throat culture is considered the diagnostic standard, although the sensitivity and specificity of rapid antigen detection testing have improved significantly. The modified Centor score can be used to help physicians decide which patients need no testing, throat culture/rapid antigen detection testing, or empiric antibiotic therapy. Penicillin (10 days of oral therapy or one injection of intramuscular benzathine penicillin) is the treatment of choice because of cost, narrow spectrum of activity, and effectiveness. Amoxicillin is equally effective and more palatable. Erythromycin and first-generation cephalosporins are options in patients with penicillin allergy. Increased group A beta-hemolytic streptococcus (GABHS) treatment failure with penicillin has been reported. Although current guidelines recommend first-generation cephalosporins for persons with penicillin allergy, some advocate the use of cephalosporins in all nonallergic patients because of better GABHS eradication and effectiveness against chronic GABHS carriage. Chronic GABHS colonization is common despite appropriate use of antibiotic therapy. Chronic carriers are at low risk of transmitting disease or developing invasive GABHS infections, and there is generally no need to treat carriers. Whether tonsillectomy or adenoidectomy decreases the incidence of GABHS pharyngitis is poorly understood. At this time, the benefits are too small to outweigh the associated costs and surgical risks.

A new kind of highly sensitive competitive lateral flow immunoassay displaying direct analyte-signal dependence. Application to the determination of the mycotoxin deoxynivalenol.

PubMed

Urusov, Alexandr E; Gubaidullina, Miliausha K; Petrakova, Alina V; Zherdev, Anatoly V; Dzantiev, Boris B

2017-12-06

A new kind of competitive immunochromatographic assay is presented. It is based on the use of a test strip loaded with (a) labeled specific antibodies, (b) a hapten-protein conjugate at the control zone, and (c) antibodies interacting with the specific antibodies in the analytical zone. In the case where a sample does not contain the target antigen (hapten), all labeled antibodies remain in the control zone because of the selected ratio of reactants. The analytical zone remains colorless because the labeled antibodies do not reach it. If an antigen is present in the sample, it interferes with the binding of the specific antibodies in the control zone and knocks them out. Some of these antibodies pass the control zone to form a colored line in the analytical zone. The intensity of the color is directly proportional to the amount of the target antigen in the sample. The assay has an attractive feature in that an appearance in coloration is more easily detected visually than a decoloration. Moreover, the onset of coloration is detectable at a lower concentration than a decoloration. The new detection scheme was applied to the determination of the mycotoxin deoxynivalenol. The visual limit of detection is 2 ng·mL -1 in corn extracts (35 ng per gram of sample). With the same reagents, this is lower by a factor of 60 than the established test strip. The assay takes only 15 min. This new kind of assay has wide potential applications for numerous low molecular weight analytes. Graphical abstract Competitive immunochromatography with direct analyte-signal dependence is proposed. It provides a 60-fold decrease of the detection limit for mycotoxin deoxynivalenol. The analyte-antibody-label complexes move along the immobilized antigen (control zone) and bind with anti-species antibodies (test zone).

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

Comparative assessment of two commonly used commercial ELISA tests for the serological diagnosis of contagious agalactia of small ruminants caused by Mycoplasma agalactiae

PubMed Central

2012-01-01

Background Contagious agalactia (CA) of sheep and goats caused by Mycoplasma agalactiae is a widely occurring economically important disease that is difficult to control. The ELISA is commonly used for the serological detection of CA but it has some limitations and the performance of the available tests have not been properly evaluated. Two commercial ELISA kits are widely used, one involving a fusion protein as target antigen and the other a total antigen. The objectives were to compare these tests by evaluating: i. Their diagnostic sensitivity and specificity, the relevance of the recommended cut-off points, the correlation between the two tests, and, the correlation between serology data and the milk shedding of M. agalatiae; ii. The influence of extrinsic factors such as the targeted animal species, geographical origin of the samples, intra-specific variability of M. agalactiae and concurrent mycoplasma infections. A sample of 5900 animals from 211 farms with continuous CA monitoring for 20 years and no prior vaccination history was used. The infection status was known from prior bacteriological, epidemiological and serological monitoring with a complementary immunoblotting test. Results The average diagnostic sensitivity was 56% [51.8–59.8] for the fusion protein ELISA and 84% [81.3–87.2] for the total antigen ELISA, with noteworthy flock-related variations. The average diagnostic specificity for the fusion protein ELISA was 100% [99.9–100], and for the total antigen ELISA differed significantly between goats and sheep: 99.3% [97.4–99.9] and 95.7% [93.8–97.2] respectively. Experimental inoculations with different M. agalactiae strains revealed that the ELISA kits poorly detected the antibody response to certain strains. Furthermore, test performances varied according to the host species or geographical origin of the samples. Finally, the correlation between milk shedding of M. agalactiae and the presence of detectable antibodies in the blood was poor. Conclusions These serological tests are not interchangeable. The choice of a test will depend on the objectives (early detection of infection or disease control program), on the prevalence of infection and the control protocol used. Given the variety of factors that may influence performance, a preliminary assessment of the test in a given situation is recommended prior to widespread use. PMID:22776779

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

Apel, William A; Thompson, Vicki S

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 immunemore » 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.« less

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.

Rapid classification of biological components

DOEpatents

Thompson, Vicki S.; Barrett, Karen B.; Key, Diane E.

2013-10-15

A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein the 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 antigens in the array, thereby forming immune complexes; washing away antibodies that do not form immune complexes; and detecting the immune complexes, thereby forming 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 a subject's identity.

Rapid classification of biological components

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

Thompson, Vicki S.; Barrett, Karen B.; Key, Diane E.

A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, cocaine, methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens to the surface of a solid support in a preselected pattern to form an array wherein the 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 antigens in the array, thereby forming immunemore » complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, thereby forming 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.« less

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.

[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.

The challenge of producing skin test antigens with minimal resources suitable for human application against a neglected tropical disease; leprosy.

PubMed

Rivoire, Becky L; TerLouw, Stephen; Groathouse, Nathan A; Brennan, Patrick J

2014-01-01

True incidence of leprosy and its impact on transmission will not be understood until a tool is available to measure pre-symptomatic infection. Diagnosis of leprosy disease is currently based on clinical symptoms, which on average take 3-10 years to manifest. The fact that incidence, as defined by new case detection, equates with prevalence, i.e., registered cases, suggests that the cycle of transmission has not been fully intercepted by implementation of multiple drug therapy. This is supported by a high incidence of childhood leprosy. Epidemiological screening for pre-symptomatic leprosy in large endemic populations is required to facilitate targeted chemoprophylactic interventions. Such a test must be sensitive, specific, simple to administer, cost-effective, and easy to interpret. The intradermal skin test method that measures cell-mediated immunity was explored as the best option. Prior knowledge on skin testing of healthy subjects and leprosy patients with whole or partially fractionated Mycobacterium leprae bacilli, such as Lepromin or the Rees' or Convit' antigens, has established an acceptable safety and potency profile of these antigens. These data, along with immunoreactivity data, laid the foundation for two new leprosy skin test antigens, MLSA-LAM (M. leprae soluble antigen devoid of mycobacterial lipoglycans, primarily lipoarabinomannan) and MLCwA (M. leprae cell wall antigens). In the absence of commercial interest, the challenge was to develop these antigens under current good manufacturing practices in an acceptable local pilot facility and submit an Investigational New Drug to the Food and Drug Administration to allow a first-in-human phase I clinical trial.

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.

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.

Performance Characteristics and Utilization of Rapid Antigen Test, DNA Probe, and Culture for Detection of Group A Streptococci in an Acute Care Clinic

PubMed Central

Chapin, Kimberle C.; Blake, Patricia; Wilson, Claire D.

2002-01-01

Group A streptococcus (GAS) antigen testing has become a routine point-of-care (POC) test in acute care settings. Concern about performance parameters (PP) of these tests as well as inappropriate antibiotic use has resulted in various recommendations regarding diagnosis of GAS. There were two objectives in this study. The first was to evaluate the rapid GAS antigen test presently in use (Thermo BioStar, Boulder, Colo.) and the GAS Direct probe test (Gen-Probe, San Diego, Calif.) compared to culture. The second was to define the optimal use of these technologies in a large acute care pediatric clinic. A total of 520 consecutive pediatric patients presenting with symptoms of pharyngitis at any of three Lahey Clinic acute care facilities were evaluated. Pharyngeal specimens were collected using a double-swab collection device (Copan, Corona, Calif.). One swab was used for the antigen test, the second was used for the probe test, and the pledget was placed in the collection device for culture on 5% sheep blood agar, incubated for 48 h anaerobically, and subsequently placed in Todd-Hewitt broth. After discrepant analysis, sensitivity, specificity, and positive and negative predictive values were as follows: 94.8, 100, 100, and 96.9% for the probe test and 86.1, 97.1, 93.7, and 93.4% for the antigen test, respectively. Sensitivity using an enhanced culture technique was 99.4% (163 of 164). False-positive (FP) antigen results were often seen from patients previously diagnosed and/or treated for GAS. No FP results were seen with the probe test. Colony counts for the false-negative (FN) antigen tests were higher than those for the FN probe tests. Compared to culture and DNA probe, the rapid antigen test (RAT) offered a result at the time of the patient's visit, with acceptable PP when prevalence of disease is high. Follow-up testing with the RAT of GAS patients who previously tested as positive should be avoided due to increased FP results. The probe test was comparable to culture in performance. Results indicate the probe test can be used as the primary test or as a backup to negative antigen tests. The probe test offers the advantage over culture of same-day reporting of a final result but, in contrast to a POC test, necessitates follow-up communication to the patient. Preliminary data show the specificity of the probe test to be greater than that of the RAT for patients previously diagnosed with GAS. PMID:12409399

[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.

Persistent ICT Malaria P.f/P.v Panmalarial and HRP2 Antigen Reactivity after Treatment of Plasmodium falciparum Malaria Is Associated with Gametocytemia and Results in False-Positive Diagnoses of Plasmodium vivax in Convalescence

PubMed Central

Tjitra, Emiliana; Suprianto, Sri; McBroom, James; Currie, Bart J.; Anstey, Nicholas M.

2001-01-01

A problem with rapid Plasmodium falciparum-specific antigen histidine-rich protein 2 (HRP2) detection tests for malaria is the persistence of antigen in blood after the disappearance of asexual-stage parasitemia and clinical symptoms, resulting in false-positive (FP) test results following treatment. The ICT P.f/P.v immunochromatographic test detects both HRP2 and a panmalarial antigen (PMA) found in both P. falciparum and Plasmodium vivax. To examine posttreatment antigen persistence with this test and whether persistent sexual-stage forms (gametocytes) are a cause of FP tests after treatment, we compared serial antigen test results with microscopy results from patients symptomatic with P. falciparum malaria in Indonesia for 28 days following treatment with chloroquine (CQ; n = 66), sulfadoxine-pyrimethamine (SP; n = 36), and artesunate plus sulfadoxine-pyrimethamine (ART + SP; n = 15). Persistent FP antigenemia following SP treatment occurred in 29% (HRP2) and 42% (PMA) of the patients on day 7 and in 10% (HRP2) and 23% (PMA) on day 14. The high rates of persistent HRP2 and PMA antigenemia following CQ and SP treatment were strongly associated with the presence of gametocytemia, with the proportion with gametocytes on day 7 posttreatment being significantly greater in those with FP results than in those with true-negative PMA and HRP2 results. Gametocyte frequency on day 14 post-SP treatment was also greater in those with FP PMA results. Following SP treatment, PMA persisted longer than HRP2, giving an FP diagnosis of P. vivax in up to 16% of patients on day 14, with all FP P. vivax diagnoses having gametocytemia. In contrast, PMA was rapidly cleared following ART + SP treatment in association with rapid clearance of gametocytemia. Gametocytes appear to be an important cause of persistent posttreatment panmalarial antigenemia in areas of endemicity and may also contribute in part to persistent HRP2 antigenemia following treatment. PMID:11230422

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.

No evidence for transmission of SIVwrc from western red colobus monkeys (piliocolobus badius badius) to wild west african chimpanzees (pan troglodytes verus) despite high exposure through hunting

PubMed Central

2011-01-01

Background Simian Immunodeficiency Viruses (SIVs) are the precursors of Human Immunodeficiency Viruses (HIVs) which have lead to the worldwide HIV/AIDS pandemic. By studying SIVs in wild primates we can better understand the circulation of these viruses in their natural hosts and habitat, and perhaps identify factors that influence susceptibility and transmission within and between various host species. We investigated the SIV status of wild West African chimpanzees (Pan troglodytes verus) which frequently hunt and consume the western red colobus monkey (Piliocolobus badius badius), a species known to be infected to a high percentage with its specific SIV strain (SIVwrc). Results Blood and plasma samples from 32 wild chimpanzees were tested with INNO-LIA HIV I/II Score kit to detect cross-reactive antibodies to HIV antigens. Twenty-three of the samples were also tested for antibodies to 43 specific SIV and HIV lineages, including SIVwrc. Tissue samples from all but two chimpanzees were tested for SIV by PCRs using generic SIV primers that detect all known primate lentiviruses as well as primers designed to specifically detect SIVwrc. Seventeen of the chimpanzees showed varying degrees of cross-reactivity to the HIV specific antigens in the INNO-LIA test; however no sample had antibodies to SIV or HIV strain - and lineage specific antigens in the Luminex test. No SIV DNA was found in any of the samples. Conclusions We could not detect any conclusive trace of SIV infection from the red colobus monkeys in the chimpanzees, despite high exposure to this virus through frequent hunting. The results of our study raise interesting questions regarding the host-parasite relationship of SIVwrc and wild chimpanzees in their natural habitat. PMID:21284842

Performance of Leishmania PFR1 recombinant antigen in serological diagnosis of asymptomatic canine leishmaniosis by ELISA.

PubMed

Ledesma, Darién; Berriatua, Eduardo; Thomas, M Carmen; Bernal, Luis Jesús; Ortuño, María; Benitez, Celia; Egui, Adriana; Papasouliotis, Kostas; Tennant, Bryn; Chambers, Julia; Infante, Juan José; López, Manuel Carlos

2017-10-23

Leishmania infantum is a protozoan parasite transmitted by phlebotomine sand flies that causes life-threatening disease in humans and dogs. The dog is the primary reservoir of the parasite and early diagnosis of canine leishmaniosis is crucial at the clinical and epidemiological level. The currently available serological tests for CanL diagnostic show limitations therefore the aim of the present study was to investigate the diagnostic performance of an indirect antibody ELISA based on the Leishmania infantum recombinant antigen PFR1 in asymptomatically infected dogs. One hundred fifty-six dogs including Leishmania-free experimental Beagles and pet dogs from England, Scotland and Leishmania-endemic Murcia in Spain, were tested with the assay. The later were also tested with two commercial L. infantum crude antigen ELISAs (INgezim and Civtest, respectively) and a real-time kinetoplast PCR test. Anti-PFR1 antibodies were detected in the four groups of dogs, and the mean log-transformed optical density (OD) values were lowest in Beagles and in dogs from England and highest among dogs from Murcia (p < 0.05). Using the highest OD in beagles as the PFR1 ELISA cut-off point, the estimated seroprevalence was 27% (14-40%) in dogs from Murcia, 4% (0-9%) in dogs from Scotland and 3% (0-8%) in dogs from England (p < 0.05). Seroprevalence in dogs from Murcia according to the INgezim and Civtest ELISAs were 24% (12-37%) and 31% (18-45%), respectively, whilst the prevalence of infection based on PCR in these dogs was 73% (60-86). The percentages of PFR1-positive dogs that tested negative on the INgezim and Civtest ELISAs were 30% and 35%, respectively, and all of them tested positive on the PCR test. Relative to the PCR, the specificity, sensitivity and area under the ROC curve of the PFR1 ELISA were 100%, 36% and 0.74 (0.63-0.86), respectively. The ability shown by the PFR1 ELISA to detect infected dogs that go undetected by the crude antigen ELISAs is clinically and epidemiologically useful and PFR1 could be considered a candidate for a multi-antigen-based immunoassay for early detection of L. infantum infected dogs.

Antigen detection and immunological typing of Haemophilus ducreyi with a specific rabbit polyclonal serum.

PubMed Central

Roggen, E L; Pansaerts, R; Van Dyck, E; Piot, P

1993-01-01

A rabbit polyclonal serum was raised against the 29-kDa species-specific marker, as well as the 30- to 34-kDa immunotype-specific markers of Haemophilus ducreyi described elsewhere (E. Roggen, S. De Breucker, E. Van Dyck, and P. Piot, Infect. Immun. 60:590-595, 1992). These antigens were purified from a cocktail of H. ducreyi isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immune serum reacted in enzyme-linked immunosorbent assay (ELISA) preferentially with H. ducreyi, at a titer as high as 50,000. To make it specific to H. ducreyi, nonspecific antibodies were removed by adsorption on a mixture of Haemophilus spp., Escherichia coli, Candida albicans, and Corynebacterium spp. In the 29- to 34-kDa region of immunoblot profiles from H. ducreyi isolates (n = 450), the adsorbed serum revealed essentially the same antigens as did a pool of well-characterized human sera. Yet, eight different immunotypes were observed. With this rabbit polyclonal serum, an ELISA-based antigen detection test was developed. The adsorbed serum reacted specifically with all H. ducreyi isolates tested (n = 450), but not with other bacterial species (n = 15). This test was evaluated with a limited number of clinical specimens from African patients with culture-proven chancroid and no evidence for any other ulcerating etiology (n = 10) and a number of chancroid-negative control patients from Belgium (n = 20). Within this context, the test yielded a sensitivity and specificity of 100%. Images PMID:8349759

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.

Immunocytochemical methods to study the distribution of Orientia tsutsugamushi in Leptotrombidium (Acari: Trombiculidae) chiggers.

PubMed

Myint, K S; Linthicum, K J; Tanskul, P; Lerdthusnee, K; Vaughn, D W; Manomuth, C; Mongkolsirichaikul, D; Hansukjariya, P; Hastriter, M W

1998-07-01

Immunocytochemical methods were developed and tested for their ability to detect the distribution of Orientia tsutsugamushi in paraffin sections of adult chiggers (Leptotrombidium imphalum Vercammen-Grandjean & Langston). Rickettsial antigen was detected by application of a simple direct or amplified immunocytochemistry procedure and an indirect immunofluorescent procedure. In the direct procedure alkaline phosphatase conjugation to the mouse polyclonal antibody to the Karp strain was followed by the HistoMark Red test system to detect rickettsial antigen. The amplification procedure used a similar method but used an unlabeled primary antibody followed by secondary biotinylated antimouse IgG, streptavidin-alkaline phosphatase, and the HistoMark Red test system. The immunofluorescent procedure included a biotinylated secondary antibody followed by addition of a streptavidin-FITC conjugate. Specific tissue tropisms in infected chiggers were observed in the salivary glands, nervous tissue, and ovaries of adult female mites in all procedures; however, nonspecific fluorescence of the chigger limited definitive identification of tissue tropisms with the indirect immunofluorescent procedure.

Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis.

PubMed

Nagalingam, Mohandoss; Thirumalesh, Sushma Rahim Assadi; Kalleshamurthy, Triveni; Niharika, Nakkala; Balamurugan, Vinayagamurthy; Shome, Rajeswari; Sengupta, Pinaki Prasad; Shome, Bibek Ranjan; Prabhudas, Krishnamsetty; Rahman, Habibur

2015-10-01

This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.

Cross-reactions in IgM ELISA tests to Legionella pneumophila sg1 and Bordetella pertussis among children suspected of legionellosis; potential impact of vaccination against pertussis?

PubMed

Pancer, Katarzyna Wanda

2015-01-01

The objective of this study was preliminary evaluation of IgM cross-reaction in sera collected from children hospitalized because of suspected legionellosis. Sera with positive IgM results to L. pneumophila sgs1-7, B. pertussis or with simultaneous detection of IgM antibodies to L. pneumophila sgs1-7 and B. pertussis, or IgM to L. pneumophila sgs1-7 and M. pneumoniae in routine tests, were selected. In total, an adapted pre-absorption test was used for the serological confirmation of legionellosis in the sera of 19 children suspected of legionellosis, and also in 3 adult persons with confirmed Legionnaires' disease. Sera were pre-absorbed with antigens of L. pneumophila sg1, B. pertussis or both, and tested by ELISA tests. The reduction of IgM antibody level by pre-absorption with antigen/antigens was determined. Reduction of anti-Lpsgs1-7 IgM by pre-absorption with L.pneumophila sg1 antigen ranged from 1.5 to 80, and reduction of anti-Bp IgM by pre-absorption with B. pertussis ranged from 2.0 to 23.8. Reduction by both antigens varied depending on the age of the patients: among children <4 yrs.old, the reduction of anti-B. pertussis IgM by both antigens was higher than for B. pertussis antigen alone. Based on the high difference (≥ 2 times) between reduction by L.pneumophila sg1 and by B. pertussis antigen, legionellosis was confirmed in 8/19 children. The majority of them also indicated IgM positive/borderline results for B. pertussis or M.pneumoniae in routine ELISA tests. As a preliminary, we posed a hypothesis of a potential impact of an anti-pertussis vaccination on the results obtained in anti-L. pneumophila ELISA IgM tests among young children.

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.

Serological aspects of rat tumour xenograft growth in athymic nude mice.

PubMed Central

Pimm, M. V.; Baldwin, R. W.

1979-01-01

The serum of athymic nude mice bearing rat tumour xenografts has been examined for tumour-specific antigen. With a sarcoma and a hepatoma, tumour-specific antigen expression continued in xenograft growths, and sera of tumour-bearing mice contained free antigen, assayed by its ability to neutralise reactivity of tumour-immune rat sera against tumour target cells in an indirect membrane-immunofluorescence test. In contrast, no anti-rat antibody was detectable in sera of mice bearing the xenografts, or rejecting cells injected in admixture with BCG. PMID:373782

Detecting decay fungi with antibody-based tests and immunoassays

Treesearch

Carol A. Clausen

2003-01-01

Early detection of wood decay can prolong the service life of wood. Antibodies are the ideal probe for detecting fungi that cause biodeterioration because they are highly specific and can quantitatively determine the fungal antigen concentration from highly complex structures, such as wood. Polyclonal antibodies recognize multiple chemical sites of the targeted...

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.

Reassessing the Detection of B-Virus–Specific Serum Antibodies

PubMed Central

Katz, David; Shi, Wei; Wildes, Martin J; Krug, Peter W; Hilliard, Julia K

2012-01-01

B virus, a natural pathogen of macaques, can cause a fatal zoonotic disease in humans. Serologic screening of macaques by titration ELISA (tELISA, screening test) and by Western blot analysis (WBA, confirmatory test) is one of the principle measures to prevent human infection. Here we slightly modified these 2 tests and reevaluated their correlation. We developed a high-throughput tELISA and used it to screen 278 sera simultaneously against the homologous BV antigen and the heterologous antigens of Papiine herpesvirus 2 and Human herpesvirus 1. More sera (35.6%) were positive by the BV-ELISA than by the HVP2-ELISA (21.6%) or HSV1-ELISA (19.8%). The superiority of the homologous tELISA over the heterologous tELISA was prominent in low-titer sera. WBA confirmed only 21% of the tELISA-positive sera with low or intermediate antibody titers. These sera might have contained antibodies to conformational epitopes that could not be detected by WBA, in which denatured antigens are used, but that could be detected by tELISA, which detects both linear and conformational epitopes. WBA confirmed 82% of the tELISA high-titer sera. However, WBA defined the remaining 18% of sera, which were negative by tELISA, as nonnegative. This finding can be attributed to the difficulties encountered with the subjective interpretation of results by WBA. Together, the current results indicate the inadequacy of WBA as a confirmatory assay for sera with low antibody titers. PMID:23561886

Design and synthesis of multiple antigenic peptides and their application for dengue diagnosis.

PubMed

Rai, Reeta; Dubey, Sameer; Santosh, K V; Biswas, Ashutosh; Mehrotra, Vinit; Rao, D N

2017-09-01

Major difficulty in development of dengue diagnostics is availability of suitable antigens. To overcome this, we made an attempt to develop a peptide based diagnosis which offers significant advantage over other methods. With the help of in silico methods, two epitopes were selected from envelope protein and three from NS1 protein of dengue virus. These were synthesized in combination as three multiple antigenic peptides (MAPs). We have tested 157 dengue positive sera confirmed for NS1 antigen. MAP1 showed 96.81% sera positive for IgM and 68.15% positive for IgG. MAP2 detected 94.90% IgM and 59.23% IgG positive sera. MAP3 also detected 96.17% IgM and 59.87% IgG positive sera. To the best of our knowledge this is the first study describing the use of synthetic multiple antigenic peptides for the diagnosis of dengue infection. This study describes MAPs as a promising tool for the use in serodiagnosis of dengue. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Cross-reactivity profiles of legumes and tree nuts using the xMAP® multiplex food allergen detection assay.

PubMed

Cho, Chung Y; Oles, Carolyn; Nowatzke, William; Oliver, Kerry; Garber, Eric A E

2017-10-01

The homology between proteins in legumes and tree nuts makes it common for individuals with food allergies to be allergic to multiple legumes and tree nuts. This propensity for allergenic and antigenic cross-reactivity means that commonly employed commercial immunodiagnostic assays (e.g., dipsticks) for the detection of food allergens may not always accurately detect, identify, and quantitate legumes and tree nuts unless additional orthogonal analytical methods or secondary measures of analysis are employed. The xMAP ® Multiplex Food Allergen Detection Assay (FADA) was used to determine the cross-reactivity patterns and the utility of multi-antibody antigenic profiling to distinguish between legumes and tree nuts. Pure legumes and tree nuts extracted using buffered detergent displayed a high level of cross-reactivity that decreased upon dilution or by using a buffer (UD buffer) designed to increase the stringency of binding conditions and reduce the occurrence of false positives due to plant-derived lectins. Testing for unexpected food allergens or the screening for multiple food allergens often involves not knowing the identity of the allergen present, its concentration, or the degree of modification during processing. As such, the analytical response measured may represent multiple antigens of varying antigenicity (cross-reactivity). This problem of multiple potential analytes is usually unresolved and the focus becomes the primary analyte, the antigen the antibody was raised against, or quantitative interpretation of the content of the analytical sample problematic. The alternative solution offered here to this problem is the use of an antigenic profile as generated by the xMAP FADA using multiple antibodies (bead sets). By comparing the antigenic profile to standards, the allergen may be identified along with an estimate of the concentration present. Cluster analysis of the xMAP FADA data was also performed and agreed with the known phylogeny of the legumes and tree nuts being analyzed. Graphical abstract The use of cluster analysis to compare the multi-antigen profiles of food allergens.

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.

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.

Semi-Quantitative Scoring of an Immunochromatographic Test for Circulating Filarial Antigen

PubMed Central

Chesnais, Cédric B.; Missamou, François; Pion, Sébastien D. S.; Bopda, Jean; Louya, Frédéric; Majewski, Andrew C.; Weil, Gary J.; Boussinesq, Michel

2013-01-01

The value of a semi-quantitative scoring of the filarial antigen test (Binax Now Filariasis card test, ICT) results was evaluated during a field survey in the Republic of Congo. One hundred and thirty-four (134) of 774 tests (17.3%) were clearly positive and were scored 1, 2, or 3; and 11 (1.4%) had questionable results. Wuchereria bancrofti microfilariae (mf) were detected in 41 of those 133 individuals with an ICT test score ≥ 1 who also had a night blood smear; none of the 11 individuals with questionable ICT results harbored night mf. Cuzick's test showed a significant trend for higher microfilarial densities in groups with higher ICT scores (P < 0.001). The ICT scores were also significantly correlated with blood mf counts. Because filarial antigen levels provide an indication of adult worm infection intensity, our results suggest that semi-quantitative reading of the ICT may be useful for grading the intensity of filarial infections in individuals and populations. PMID:24019435

Humoral and In Vivo Cellular Immunity against the Raw Insect-Derived Recombinant Leishmania infantum Antigens KMPII, TRYP, LACK, and papLe22 in Dogs from an Endemic Area

PubMed Central

Todolí, Felicitat; Solano-Gallego, Laia; de Juan, Rafael; Morell, Pere; del Carmen Núñez, Maria; Lasa, Rodrigo; Gómez-Sebastián, Silvia; Escribano, José M.; Alberola, Jordi; Rodríguez-Cortés, Alhelí

2010-01-01

Leishmania infantum causes visceral leishmaniasis, a severe zoonotic and systemic disease that is fatal if left untreated. Identification of the antigens involved in Leishmania-specific protective immune response is a research priority for the development of effective control measures. For this purpose, we evaluated, in 27 dogs from an enzootic zone, specific humoral and cellular immune response by delayed-type hypersensitivity (DTH) skin test both against total L. infantum antigen and the raw Trichoplusia ni insect-derived kinetoplastid membrane protein-11 (rKMPII), tryparedoxin peroxidase (rTRYP), Leishmania homologue of receptors for activated C kinase (rLACK), and 22-kDa potentially aggravating protein of Leishmania (rpapLe22) antigens from this parasite. rTRYP induced the highest number of positive DTH responses (55% of leishmanin skin test [LST]-positive dogs), showing that TRYP antigen is an important T cell immunogen, and it could be a promising vaccine candidate against this disease. When TRYP-DTH and KMPII-DTH tests were evaluated in parallel, 82% of LST-positive dogs were detected, suggesting that both antigens could be considered as components of a standardized DTH immunodiagnostic tool for dogs. PMID:21118936

Detection of BVDV persistently infected animals in Belgium: evaluation of the strategy implemented.

PubMed

Letellier, C; De Meulemeester, L; Lomba, M; Mijten, E; Kerkhofs, P

2005-11-15

Until now, no official bovine virus diarrhea virus (BVDV) control program has been implemented in Belgium. The only legislation dealing with the detection of BVDV-infected animals concerns the purchase of animals. A strategy of control, based on the identification and elimination of persistently infected (PI) animals and the vaccination of cows before insemination has been designed in both the Northern and the Southern part of the country. The strategy of detection of PI animals relies on PCR testing of pools of blood. Individual blood samples corresponding to the positive pools are then tested by BVDV-antigen ELISA. A first evaluation of the measures already applied in Belgium is presented. Data obtained in 2003 are presented and discussed regarding the validation of the laboratory strategy, the prevalence of positive herds, the genotype of circulating viruses, the outcome of antigen positive animals and the need for improvement of the current legislation.

DETECTION OF Leptospira spp. AND Brucella abortus ANTIBODIES IN FREE-LIVING JAGUARS (Panthera onca) IN TWO PROTECTED AREAS OF NORTHERN PANTANAL, BRAZIL

PubMed Central

ONUMA, Selma Samiko Miyazaki; KANTEK, Daniel Luis Zanella; CRAWSHAW, Peter Gransden; MORATO, Ronaldo Gonçalves; MAY-JÚNIOR, Joares Adenilson; de MORAIS, Zenaide Maria; FERREIRA, José Soares; de AGUIAR, Daniel Moura

2015-01-01

 This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection. PMID:25923900

Detection of Leptospira spp. and Brucella abortus antibodies in free-living jaguars (Panthera onca) in two protected areas of northern Pantanal, Brazil.

PubMed

Onuma, Selma Samiko Miyazaki; Kantek, Daniel Luis Zanella; Crawshaw Júnior, Peter Gransden; Morato, Ronaldo Gonçalves; May-Júnior, Joares Adenilson; Morais, Zenaide Maria de; Ferreira Neto, José Soares; Aguiar, Daniel Moura de

2015-01-01

This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection.

Comparison of Simplexa universal direct PCR with cytotoxicity assay for diagnosis of Clostridium difficile infection: performance, cost, and correlation with disease.

PubMed

Landry, Marie L; Ferguson, David; Topal, Jeffrey

2014-01-01

Simplexa Clostridium difficile universal direct PCR, a real-time PCR assay for the detection of the C. difficile toxin B (tcdB) gene using the 3M integrated cycler, was compared with a two-step algorithm which includes the C. Diff Chek-60 glutamate dehydrogenase (GDH) antigen assay followed by cytotoxin neutralization. Three hundred forty-two liquid or semisolid stools submitted for diagnostic C. difficile testing, 171 GDH antigen positive and 171 GDH antigen negative, were selected for the study. All samples were tested by the C. Diff Chek-60 GDH antigen assay, cytotoxin neutralization, and Simplexa direct PCR. Of 171 GDH-positive samples, 4 were excluded (from patients on therapy or from whom duplicate samples were obtained) and 88 were determined to be true positives for toxigenic C. difficile. Of the 88, 67 (76.1%) were positive by the two-step method and 86 (97.7%) were positive by PCR. Seventy-nine were positive by the GDH antigen assay only. Of 171 GDH antigen-negative samples, none were positive by PCR. One antigen-negative sample positive by the cytotoxin assay only was deemed a false positive based on chart review. Simplexa C. difficile universal direct PCR was significantly more sensitive for detecting toxigenic C. difficile bacteria than cytotoxin neutralization (P = 0.0002). However, most PCR-positive/cytotoxin-negative patients did not have clear C. difficile disease. The estimated cost avoidance provided by a more rapid molecular diagnosis was outweighed by the cost of isolating and treating PCR-positive/cytotoxin-negative patients. The costs, clinical consequences, and impact on nosocomial transmission of treating and/or isolating patients positive for toxigenic C. difficile by PCR but negative for in vivo toxin production merit further study.

Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays.

PubMed

Nakajima, Rie; Escudero, Raquel; Molina, Douglas M; Rodríguez-Vargas, Manuela; Randall, Arlo; Jasinskas, Algis; Pablo, Jozelyn; Felgner, Philip L; AuCoin, David P; Anda, Pedro; Davies, D Huw

2016-07-01

Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays

PubMed Central

Nakajima, Rie; Escudero, Raquel; Molina, Douglas M.; Rodríguez-Vargas, Manuela; Randall, Arlo; Jasinskas, Algis; Pablo, Jozelyn; Felgner, Philip L.; AuCoin, David P.; Anda, Pedro

2016-01-01

Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip. PMID:27098957

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.

Search for Hepatitis A Viruses by New Methods

DTIC Science & Technology

1981-09-01

VII. Detection of HAV and Rotavirus In a Community Water Supply Following an Outbreak of Gastroenteritls and Infectious Hepatitis . . . 1* VUL...22). An "aliquot of each concentrate was further concentrated by ultracentrifugation to assay for HAV antigen and rotavirus . Samples were assayed for... rotavirus using an Indirect "immunofluorescence test (23) and for HAV antigen using a radloirmunoassay (24, 25). Ř. Table 9 shows the concentrations

The First Korean Case Report of Anti-Gerbich

PubMed Central

Jeon, You La; Park, Tae Sung; Cho, Sun Young; Oh, Seung Hwan; Kim, Myeong Hee; Kang, So Young

2012-01-01

In this study, we report the first Korean case of an anti-Gerbich (Ge) alloantibody to a high-incidence antigen that belongs to the Ge blood group system. The alloantibody was detected in a middle-aged Korean woman who did not have a history of transfusion. Her blood type was B+, and findings from the antibody screening test revealed 1+ reactivity in all panels except the autocontrol. The cross-matching test showed incompatible results with all 5 packed red blood cells. Additional blood type antigen and antibody tests confirmed the anti-Ge alloantibody. While rare, cases of hemolytic transfusion reaction or hemolytic disease in newborns due to anti-Ge have been recently reported in the literature. Therefore, additional further studies on alloantibodies to high-incidence antigens, including anti-Ge, are necessary in the future. PMID:23130346

Presence of Human T-Cell Responses to the Mycobacterium leprae 45-Kilodalton Antigen Reflects Infection with or Exposure to M. leprae

PubMed Central

Macfarlane, Anne; Mondragon-Gonzalez, Rafael; Vega-Lopez, Francisco; Wieles, Brigitte; de Pena, Josefina; Rodriguez, Obdulia; Suarez y de la Torre, Raul; de Vries, Rene R. P.; Ottenhoff, Tom H. M.; Dockrell, Hazel M.

2001-01-01

The ability of the 45-kDa serine-rich Mycobacterium leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) proliferation and gamma interferon (IFN-γ) production was measured in leprosy patients, household contacts, and healthy controls from areas of endemicity in Mexico. Almost all the tuberculoid leprosy patients gave strong PBMC proliferation responses to the M. leprae 45-kDa antigen (92.8%; n = 14). Responses were lower in lepromatous leprosy patients (60.6%; n = 34), but some responses to the 45-kDa antigen were detected in patients unresponsive to M. leprae sonicate. The proportion of positive responses to the M. leprae 45-kDa antigen was much higher in leprosy contacts (88%; n = 17) than in controls from areas of endemicity (10%; n = 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the 45-kDa antigen. The 45-kDa antigen induced IFN-γ secretion similar to that induced by the native Mycobacterium tuberculosis 30/31-kDa antigen in tuberculoid leprosy patients and higher responses than those induced by the other recombinant antigens (M. leprae 10- and 65-kDa antigens, thioredoxin, and thioredoxin reductase); in patients with pulmonary tuberculosis it induced lower IFN-γ secretion than the other recombinant antigens. These results suggest that the M. leprae 45-kDa antigen is a potent T-cell antigen which is M. leprae specific in these Mexican donors. This antigen may therefore have diagnostic potential as a new skin test reagent or as an antigen in a simple whole-blood cytokine test. PMID:11329466

Report of data on children with non-typhi Salmonella gastroenteritis in a three-year period.

PubMed

Küçük, Öznur; Biçer, Suat; Ugraş, Meltem; Çöl, Defne; Giray, Tuba; Çiler Erdag, Gülay; Gürol, Yeşim; Yilmaz, Gülden; Yalvaç, Zerrin; Vitrinel, Ayça; Kaspar, Çigdem

2016-09-01

The purpose of this study was to evaluate the clinical and laboratory data of children with acute gastroenteritis caused by non-typhoid Salmonella spp. infections. Clinical (demographic data, symptoms and findings) and laboratory data (stool microscopy, rapid antigen tests, culture, multiplex polymerase chain reaction and blood test results) of children with acute gastroenteritis caused by non-typhoid Salmonella spp. between January 2010 and October 2012 were evaluated. Differences between the groups for categorical variables were estimated with a chi-square or Fisher exact test; for continuous variables with two independent samples a t test was used. P values < 0.05 were considered statistically significant. Sixty-seven children, 39 (58.2%) males and 28 (41.8%) females aged between 1 - 16 years (mean ± SD: 4.64 ± 2.91), were diagnosed with acute bacterial gastroenteritis caused by non-typhoid Salmonella spp. The main serotypes are Salmonella enteritidis (85%) and Salmonella typhimurium (7.5%). The presenting symptoms were diarrhoea (95.5%), fever (61.1%), vomiting (34.3%), abdominal pain (32.8%), loss of appetite (7.4%) and malaise (7.4%). Fever and dehydration (moderate and/or severe) were detected in 11 (16.4%) patients. The mean leukocyte count was 10.930/μL [95% confidence interval (CI), SD: ± 5.710/μL], neutrophil count was 7.880/μL (95% CI, SD: ± 4.960/μL), CRP was 64.16 mg/L (95% CI, SD: ± 76.24 mg/L), and erythrocyte sedimentation rate was 34.72 mm/hour (95% CI, SD: ± 13.64 mm/h). Stool microscopy was positive for leukocytes in 18 patients (26.8%). The definitive diagnosis was made with positive stool culture (n = 65) and/or PCR test (n = 4). Viral antigen positivity was detected in 10 patients (14.9%), evaluated as viral co-infection and false positive results. Antibiotic therapy and hospitalization were required in 26 (38.8%) and 23 (34.3%) patients, respectively. Salmonella carriage was detected in one patient (1.5%). Bloody diarrhoea, leukocytes in stool with an increased erythrocyte sedimentation rate and a CRP level without overt leukocytosis may indicate Salmonella infection. Viral antigens may cause false positive results in fast antigen tests in cases where clinical and laboratory findings indicate bacterial aetiology. Stool culture is a reference method in diagnosis whereas some agents may be detected via molecular techniques (polymerase chain reaction) in spite of negative culture. Multiplex polymerase chain reaction may be used to detect Salmonella spp. and may reveal false positivity for viruses as well as the detection of other bacteria.

Effect of specimen storage, antibiotics, and feminine hygiene products on the detection of group B Streptococcus by culture and the STREP B OIA test.

PubMed

Ostroff, R M; Steaffens, J W

1995-07-01

Agar culture from vaginal swabs is the routine method for diagnosis of maternal Group B Streptococcus (GBS) colonization. Swab specimens are often transported to a clinical laboratory for processing. In these studies, specimen transport was simulated by inoculating swabs with GBS and storing them at selected temperatures and with or without transport medium. The recovery of viable GBS was assessed by agar culture. GBS antigen was detected immunologically with an Optical ImmunoAssay (OIA) method. Swabs that were stored with transport medium harbored viable but rapidly declining numbers of GBS. In contrast, a strong OIA signal was maintained. Recovery of viable GBS organisms declined more quickly when swabs were stored in the absence of transport medium, whereas detection of GBS antigen remained consistent. Both methods were tested for interference from either antibiotics or feminine hygiene products. These compounds inhibited the detection of GBS by culture but had no detrimental effect on the OIA result.

DETECTION OF CANINE PARVOVIRUS ANTIGEN IN DOGS IN KUMASI, GHANA

PubMed Central

Folitse, R. D; Kodie, D.O; Amemor, E.; Dei, D.; Tasiame, W.; Burimuah, V.; Emikpe, B.O

2018-01-01

Background: Canine Parvovirus (CPV) in dogs has been documented in many countries. However, evidence of the infection is scanty in Ghana. This study was conducted to detect canine parvovirus antigen in dogs presented with diarrhoea to the Government Veterinary Clinic in Kumasi, Ghana. Materials and Methods: Faecal samples from 72 dogs presented with diarrhoea were tested for the presence of canine parvovirus antigen using commercially available rapid test kit (BIT® Rapid Colour Canine Parvovirus Ag Test Kit, BIOINDIST Co. Ltd, Korea) based on the principle of immunochromatography. Influence of breed, sex, age, vaccination history and the nature of diarrhoea were assessed. Data obtained was analysed with SPSS and subjected to the chi-square test. Significance was at α0.05 Results: We found 61.11% tested positive (44/72) for CPV. Based on sex, 61.54% of males (20/33) and 60.61% of females tested positive (24/39). A total of 65.67% of samples from puppies below 6 months were positive. 56.25% of CPV vaccinated dogs and 70.83% of unvaccinated dogs were positive respectively. 69.05% of samples from haemorrhagic diarrhoeic dogs and 50.00% from non-haemorrhagic diarrhoeic dogs were positive of CPV. Conclusion: The study is the first documented evidence of the existence of CPV in Ghana. It also revealed that absence of bloody diarrhoea does not necessarily rule out CPV infection. PMID:29302647

DETECTION OF CANINE PARVOVIRUS ANTIGEN IN DOGS IN KUMASI, GHANA.

PubMed

Folitse, R D; Kodie, D O; Amemor, E; Dei, D; Tasiame, W; Burimuah, V; Emikpe, B O

2018-01-01

Canine Parvovirus (CPV) in dogs has been documented in many countries. However, evidence of the infection is scanty in Ghana. This study was conducted to detect canine parvovirus antigen in dogs presented with diarrhoea to the Government Veterinary Clinic in Kumasi, Ghana. Faecal samples from 72 dogs presented with diarrhoea were tested for the presence of canine parvovirus antigen using commercially available rapid test kit (BIT ® Rapid Colour Canine Parvovirus Ag Test Kit, BIOINDIST Co. Ltd, Korea) based on the principle of immunochromatography. Influence of breed, sex, age, vaccination history and the nature of diarrhoea were assessed. Data obtained was analysed with SPSS and subjected to the chi-square test. Significance was at α 0.05 . We found 61.11% tested positive (44/72) for CPV. Based on sex, 61.54% of males (20/33) and 60.61% of females tested positive (24/39). A total of 65.67% of samples from puppies below 6 months were positive. 56.25% of CPV vaccinated dogs and 70.83% of unvaccinated dogs were positive respectively. 69.05% of samples from haemorrhagic diarrhoeic dogs and 50.00% from non-haemorrhagic diarrhoeic dogs were positive of CPV. The study is the first documented evidence of the existence of CPV in Ghana. It also revealed that absence of bloody diarrhoea does not necessarily rule out CPV infection.

Prostate-specific antigen screening impacts on biochemical recurrence in patients with clinically localized prostate cancer.

PubMed

Hashimoto, Takeshi; Ohori, Makoto; Shimodaira, Kenji; Kaburaki, Naoto; Hirasawa, Yosuke; Satake, Naoya; Gondo, Tatsuo; Nakagami, Yoshihiro; Namiki, Kazunori; Ohno, Yoshio

2018-06-01

To clarify the impact of prostate-specific antigen screening on surgical outcomes of prostate cancer. Patients who underwent radical prostatectomy were divided into two groups according to prostate-specific antigen testing opportunity (group 1, prostate-specific antigen screening; group 2, non-prostate-specific antigen screening). Perioperative clinical characteristics were compared using the Wilcoxon rank-sum and χ 2 -tests. Cox proportional hazards models were used to identify independent predictors of postoperative biochemical recurrence-free survival. In total, 798 patients (63.2%) and 464 patients (36.8%) were categorized into groups 1 and 2, respectively. Group 2 patients were more likely to have a higher prostate-specific antigen level and age at diagnosis and larger prostate volume. Clinical T stage, percentage of positive cores and pathological Gleason score did not differ between the groups. The 5-year biochemical recurrence-free survival rate was 83.9% for group 1 and 71.0% for group 2 (P < 0.001). On multivariate analysis, prostate-specific antigen testing opportunity (hazard ratio 2.530; P < 0.001) was an independent predictive factor for biochemical recurrence after surgery, as well as pathological T stage, pathological Gleason score, positive surgical margin and lymphovascular invasion. Additional analyses showed that prostate-specific antigen screening had a greater impact on biochemical recurrence in a younger patients, patients with a high prostate-specific antigen level, large prostate volume and D'Amico high risk, and patients meeting the exclusion criteria of the Prostate Cancer Research International Active Surveillance study. Detection by screening results in favorable outcomes after surgery. Prostate-specific antigen screening might contribute to reducing biochemical recurrence in patients with localized prostate cancer. © 2018 The Japanese Urological Association.

Optical fiber immunosensor based on a poly(pyrrole-benzophenone) film for the detection of antibodies to viral antigen.

PubMed

Konry, T; Novoa, A; Shemer-Avni, Y; Hanuka, N; Cosnier, S; Lepellec, Arielle; Marks, R S

2005-03-15

We describe herein a newly developed optical microbiosensor for the diagnosis of hepatitis C virus (HCV) by using a novel photoimmobilization methodology based on a photoactivable electrogenerated polymer film deposited upon surface-conductive fiber optics, which are then used to link a biological receptor to the fiber tip through light mediation. This fiber-optic electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide on the silica surface of the fiber optics. Monomers are then electropolymerized onto the conductive metal oxide surface; thereafter, the fibers are immersed in a solution containing HCV-E2 envelope protein antigen and illuminated with UV light (wavelength approximately 345 nm). As a result of the photochemical reaction, a thin layer of the antigen becomes covalently bound to the benzophenone-modified surface. The photochemically modified fiber optics were tested as immunosensors for the detection of anti-E2 protein antibody analyte that was measured through chemiluminescence reaction. The biosensor was tested for sensitivity, specificity, and overall practicality. Our results suggest that the detection of anti-E2 antibodies with this microbiosensor may enhance significantly HCV serological standard testing especially among patients during dialysis, which were diagnosed as HCV negative, by standard immunological tests, but were known to carry the virus. If transformed into an easy to use procedure, this assay might be used in the future as an important clinical tool for HCV screening in blood banks.

Rabies in southeast Brazil: a change in the epidemiological pattern.

PubMed

Queiroz, Luzia Helena; Favoretto, Silvana Regina; Cunha, Elenice Maria S; Campos, Angélica Cristine A; Lopes, Marissol Cardoso; de Carvalho, Cristiano; Iamamoto, Keila; Araújo, Danielle Bastos; Venditti, Leandro Lima R; Ribeiro, Erica S; Pedro, Wagner André; Durigon, Edison Luiz

2012-01-01

This epidemiological study was conducted using antigenic and genetic characterisation of rabies virus isolates obtained from different animal species in the southeast of Brazil from 1993 to 2007. An alteration in the epidemiological profile was observed. One hundred two samples were tested using a panel of eight monoclonal antibodies, and 94 were genetically characterised by sequencing the nucleoprotein gene. From 1993 to 1997, antigenic variant 2 (AgV-2), related to a rabies virus maintained in dog populations, was responsible for rabies cases in dogs, cats, cattle and horses. Antigenic variant 3 (AgV-3), associated with Desmodus rotundus, was detected in a few cattle samples from rural areas. From 1998 to 2007, rabies virus was detected in bats and urban pets, and four distinct variants were identified. A nucleotide similarity analysis resulted in two primary groups comprising the dog and bat antigenic variants and showing the distinct endemic cycles maintained in the different animal species in this region.

AN ENVIRONMENTAL TECHNOLOGY VERIFICATION (ETV) TESTING OF THREE IMMUNOASSAY TEST KITS FOR ANTHRAX, BOTULINUM TOXIN AND RICIN

EPA Science Inventory

Immunoassay test kits are based on immunoassay methods, where specific antibodies are used to detect and measure the contaminants of interest. Immunoassay test kits rely on the reaction of a contaminant or antigen with a selective antibody to give a product that can be measures....

[Rapid laboratory detection of antigens of infective agents of infections and technical means for their realization].

PubMed

Kal'noĭ, S M

2003-01-01

A system of new accelerated and rapid methods for the detection of the antigens of the infective agents of plague, cholera, tularemia and brucellosis were developed on the basis of solid phase immunosuspension tests: the passive hemagglutination (PHA) test and the latex agglutination (LA) test. The immunological and physico-chemical properties of suspensions in the PHA and LA tests made it possible to use extraneous sources of energy (centrifugal acceleration and the electric field) to accelerate these tests. The results of the PHA and LA tests were registered with the use of a densitometer, model Ultrascan 2202, and a tester, model C 34014.2. To apply centrifugal acceleration and the electric field, a laboratory centrifuge and an electrophoretic microchamber were designed. Densitometry was carried out on modified plates and conductometry, with the use of modified electrodes. The time of obtaining the results of the PHA and LA tests was 15-30 minutes with the use of centrifugation and 2-5 minutes in the electric field, which made it possible to regard these tests as rapid.

Detection of avian influenza antigens in proximity fiber, droplet, and optical waveguide microfluidics

NASA Astrophysics Data System (ADS)

Yoon, Jeong-Yeol; Heinze, Brian C.; Gamboa, Jessica; You, David J.

2009-05-01

Virus antigens of avian influenza subtype H3N2 were detected on two different microfluidic platforms: microchannel and droplet. Latex immunoagglutination assays were performed using 920-nm highly carboxylated polystyrene beads that are conjugated with antibody to avian influenza virus. The bead suspension was merged with the solutions of avian influenza virus antigens in a Y-junction of a microchannel made by polydimethylsiloxane soft lithography. The resulting latex immunoagglutinations were measured with two optical fibers in proximity setup to detect 45° forward light scattering. Alternatively, 10 μL droplets of a bead suspension and an antigen solution were merged on a superhydrophobic surface (water contact angle = 155°), whose movement was guided by a metal wire, and 180° back light scattering is measured with a backscattering optical probe. Detection limits were 0.1 pg mL-1 for both microchannel with proximity fibers and droplet microfluidics, thanks to the use of micro-positioning stages to help generate reproducible optical signals. Additionally, optical waveguide was tested by constructing optical waveguide channels (filled with mineral oil) within a microfluidic device to detect the same light scattering. Detection limit was 0.1 ng mL-1 for an optical waveguide device, with a strong potential of improvement in the near future. The use of optical waveguide enabled smaller device setup, easier operation, smaller standard deviations and broader linear range of assay than proximity fiber microchannel and droplet microfluidics. Total assay time was less than 10 min.

[THE DEVELOPMENT OF IMMUNE ENZYME AND IMMUNE CHROMATOGRAPHIC MONOCLONAL TEST-SYSTEM FOR DETECTING TULAREMIA AGENT].

PubMed

Eremkin, A V; Elagin, G D; Petchenkin, D V; Fomenkov, O O; Bogatcheva, N V; Kitmanov, A A; Kuklina, G V; Tikhvinskaya, O V

2016-03-01

The immune enzyme and immunochromatographic test-systems for detecting tularemia agent were developed on the basis of selected set of monoclonal antibodies having immunochemical activity to antigens Francisella tularensis. The evaluation of sensitivity and specificity of developed test-systems demonstrated that samples provided detection of strains of F. tularensis in concentration from 5.0 x 105 mkxcm-3 to 1.0 x 106 mkxcm-3 and gave no false positive results in analysis of heterologous microorganisms in concentration of 1.0 x 108 mkxcm-3.

Utility of serum Aspergillus-galactomannan antigen to evaluate the risk of severe acute exacerbation in chronic obstructive pulmonary disease

PubMed Central

Yoshimura, Katsuhiro; Inoue, Yusuke; Nishimoto, Koji; Karayama, Masato; Furuhashi, Kazuki; Enomoto, Noriyuki; Nakamura, Yutaro; Inui, Naoki; Yokomura, Koushi; Imokawa, Shiro; Suda, Takafumi

2018-01-01

Background Recent studies have shown that the microbiome, namely Aspergillus species, play a previously unrecognized role in both stable and exacerbated chronic obstructive pulmonary disease (COPD). Galactomannan is a major component of the Aspergillus cell wall that has been widely used as a diagnostic marker. Objectives To explore whether serum levels of Aspergillus-galactomannan antigen could be used to evaluate the risk of severe acute exacerbation of COPD (AE-COPD). Methods We measured the Aspergillus-galactomannan antigen levels of 191 patients with stable COPD, and examined its clinical relevance including AE-COPD. Results There were 77 (40.3%) patients who were positive for serum Aspergillus-galactomannan antigen (≥0.5). High Aspergillus-galactomannan antigen level (≥0.7) was associated with older age and presence of bronchiectasis and cysts on computed tomography images. Compared to patients with low Aspergillus-galactomannan antigen level (<0.7), patients with high Aspergillus-galactomannan antigen level had significantly higher incidence of severe AE-COPD (P = 0.0039, Gray’s test) and respiratory-related mortality (P = 0.0176, log-rank test). Multivariate analysis showed that high Aspergillus-galactomannan antigen level was independently associated with severe AE-COPD (hazard ratio, 2.162; 95% confidence interval, 1.267−3.692; P = 0.005). Conclusion Serum Aspergillus-galactomannan antigen was detected in patients with COPD, and elevated serum Aspergillus-galactomannan antigen was associated with severe AE-COPD. PMID:29870550

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.

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.

Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye

NASA Astrophysics Data System (ADS)

de La Rica, Roberto; Stevens, Molly M.

2012-12-01

In resource-constrained countries, affordable methodologies for the detection of disease biomarkers at ultralow concentrations can potentially improve the standard of living. However, current strategies for ultrasensitive detection often require sophisticated instruments that may not be available in laboratories with fewer resources. Here, we circumvent this problem by introducing a signal generation mechanism for biosensing that enables the detection of a few molecules of analyte with the naked eye. The enzyme label of an enzyme-linked immunosorbent assay (ELISA) controls the growth of gold nanoparticles and generates coloured solutions with distinct tonality when the analyte is present. Prostate specific antigen (PSA) and HIV-1 capsid antigen p24 were detected in whole serum at the ultralow concentration of 1 × 10-18 g ml-1. p24 was also detected with the naked eye in the sera of HIV-infected patients showing viral loads undetectable by a gold standard nucleic acid-based test.

[Evaluation of the new ImmunoCard STAT!® CGE test for the diagnosis of Amebiasis].

PubMed

Formenti, F; Perandin, F; Bonafini, S; Degani, M; Bisoffi, Z

2015-08-01

For many years, microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of intestinal parasites although the Polymerase Chain Reaction (PCR) analysis is increasingly utilized due to its high accuracy. Recently, PCR has been approved by the World Health Organization as the current method of choice for the diagnosis of Entamoeba histolytica infection. In this study we evaluated a novel immunochromatographic antigen detection rapid test, ImmunoCardSTAT CGE (Meridian Bioscence, Milan, Italy), which has been proposed for the diagnosis of infections caused by Cryptosporidium parvum-Giardia intestinalis-Entamoeba histolytica. There is another rapid test with a similar name, the ImmunoCard STAT! Crypto/Giardia, but it is just for Cryptosporidium and Giardia. We aimed to compare E. histolytica results obtained from the rapid test with those of a rt-PCR for the detection of E. histolytica / E. dispar DNA. The new ImmunoCard rapid antigen detection test exhibited 88% sensitivity and 92% specificity (if assessed on rt-PCR negative samples) but showed a high proportion of cross-reaction between the pathogenic E. histolytica and the non pathogenic E. dispar.

Experimental Genital Infection of Male Guinea Pigs with the Agent of Guinea Pig Inclusion Conjunctivitis and Transmission to Females

PubMed Central

Mount, David T.; Bigazzi, Pierluigi E.; Barron, Almen L.

1973-01-01

Male guinea pigs were inoculated intraurethrally with the agent of guinea pig inclusion conjunctivitis (Gp-ic). Cytoplasmic inclusions were found in superficial epithelial cells of the urethra in smears and stained sections. Gp-ic antigen(s) was detected by immunofluorescent staining of sections. There was no marked urethral exudate, but many animals developed bullous lesions on the glans and the body of the penis and a severe inflammatory lesion of the hind leg. All males demonstrated an antibody response and most of them showed a positive skin test reaction. Venereal transmission to females of Gp-ic infection was shown to occur as determined by detection of inclusions in vaginal smears, antibody response, and positive skin tests. Images PMID:4594119

Ultrasensitive immunochromatographic assay for the simultaneous detection of five chemicals in drinking water.

PubMed

Xing, Changrui; Liu, Liqiang; Song, Shanshan; Feng, Min; Kuang, Hua; Xu, Chuanlai

2015-04-15

In this paper, we describe the development of a multicomponent lateral-flow assay based on an antibody-antigen reaction for the rapid and simultaneous detection of trace contaminants in water, including a heavy metal, algal toxin, antibiotic, hormone, and pesticide. The representative analytes chosen for the study were lead (Pb(II), microcystin-leucine-arginine (MC-LR), chloramphenicol (CAP), testosterone (T), and chlorothalonil (CTN). Five different antigens were immobilized separately in five test lines on a nitrocellulose membrane. The monoclonal antibodies specifically recognized the corresponding antigens, and there was no cross-reactivity between the antibodies in the detection assay. Samples or standards containing the five analytes were preincubated with the freeze-dried colloidal-gold-labeled monoclonal antibody conjugates to improve the sensitivity of the assay. The results were obtained within 20min with a paper-based sensor. The cut-off values for the strip test were 4ng/mL for Pb(II), 1ng/mL for MC-LR, 0.1ng/mL for CAP, 5ng/mL for T, and 5ng/mL for CTN. The assay was evaluated using spiked water samples, and the accuracy and reproducibility of the results were good. In summary, this lateral-flow device provides an effective and rapid method for the onsite detection of multiple contaminants in water samples, with no treatment or devices required. Copyright © 2014 Elsevier B.V. All rights reserved.

Platelet transfusion refractoriness attributable to HLA antibodies produced by donor-derived cells after allogeneic bone marrow transplantation from one HLA-antigen-mismatched mother.

PubMed

Hatakeyama, Naoki; Hori, Tsukasa; Yamamoto, Masaki; Inazawa, Natsuko; Iesato, Kotoe; Miyazaki, Toru; Ikeda, Hisami; Tsutsumi, Hiroyuki; Suzuki, Nobuhiro

2011-12-01

PTR is a serious problem in patients being treated for hematologic disorders. Two patients with acute leukemia developed PTR after allogeneic BMT from one HLA-antigen-mismatched mother attributable to HLA antibodies, which could not be detected in their serum before BMT. HLA antibodies, whose specificity resembled that of each patient, were detected in each donor's serum. Each donor had probably been immunized during pregnancy by their partner's HLA antigens expressed by the fetus, consequently, transplanted donor-derived cells provoked HLA antibodies in each recipient early after BMT, and those HLA antibodies induced PTR. If the mothers are selected as donors for their children, they should be tested for the presence of HLA antibodies. © 2010 John Wiley & Sons A/S.

Detection of new HIV infections in a multicentre HIV antiretroviral pre-exposure prophylaxis trial.

PubMed

Fransen, Katrien; de Baetselier, Irith; Rammutla, Elizabeth; Ahmed, Khatija; Owino, Frederick; Agingu, Walter; Venter, Gustav; Deese, Jen; Van Damme, Lut; Crucitti, Tania

2017-08-01

Monthly specimens collected from FEM-PrEP-a Phase III trial [1] were investigated for the detection of acute HIV (AHI) infection. To evaluate the efficiency of the study-specific HIV algorithm in detecting AHI, and the performance of each of the serological and molecular tests used in diagnosing new infections, and their contribution to narrowing the window period. A total of 83 pre-seroconversion specimens from 61 seroconverters from the FEM-PrEP trial were further analyzed in a sub-study. During the trial, HIV seroconversion was diagnosed on site using a testing algorithm with simple/rapid tests (SRTs) and confirmed with a gold standard testing algorithm (see short communication: Fig. 1). The infection date was determined more accurately by the use of standard ELISAs and Nucleic Acid Amplification Tests (NAAT) in a look-back procedure. For this sub-study, the international central laboratory repeated the study algorithm using SRTs. A total of 83 pre-seroconversions specimens from 61 seroconverters were analyzed in a look-back procedure. RNA was detected in 35/61 seroconverters at the visit before the seroconversion visit as determined at the study sites. Four seroconversion dates were inaccurate at one study site as the international central laboratory detected the HIV infection one visit earlier using the same test algorithm. Using the gold standard, an additional seroconversion was detected at an earlier visit. The combined antigen/antibody and the single antigen test had a higher sensitivity compared to the SRTs in detecting acute infections. In the FEM-PrEP trial, the international central laboratory detected a small number of seroconversions one month earlier than the study sites using the same study algorithm. Standard tests are still the most sensitive tests in detecting pre-seroconversion or acute HIV infection, but they are costly, time consuming and not recommended for use on-site in a clinical trial. Copyright © 2017 Elsevier B.V. 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.

Ethnic and socio-cultural specificities in Tunisia have no impact on the prevalence of anti-Saccharomyces cerevisiae antibodies in Crohn's disease patients, their relatives or associated clinical factors.

PubMed

Hadrich, Ines; Vandewalle, Peggy; Cheikhrouhou, Fatma; Makni, Fattouma; Krichen, Mohamed Salah; Sendid, Boualem; Standaert-Vitse, Annie; Ayadi, Ali; Poulain, Daniel

2007-06-01

In Western Europe and the USA, the presence of anti-Saccharomyces cerevisiae antibodies (ASCAs) in Crohn's disease (CD) patients and their healthy relatives suggests that ASCAs may be influenced by genetic and/or environmental factors. To assess the prevalence of ASCAs in Tunisian patients with CD or ulcerative colitis (UC), and unaffected family members, in relation to clinical phenotype. Patients and methods. Seventy-seven patients (39 CD, 38 UC), 66 healthy relatives of CD patients, 16 relatives of UC patients and 70 healthy controls were studied. ASCAs were quantified with a new isotype-specific ELISA test involving an antigenic extract from S. cerevisiae strain W303 and by the original test which detects total immunoglobulins against S. cerevisiae Su1 mannan. The specificity of the two tests was identical (91%). The isotype-specific ASCA W303 test was more sensitive than the ASCA Su1 test for immunoglobulin detection, but some CD patients were positive only with this latter test. A high percentage of patients with CD (72%) and their unaffected family members (35%) were ASCA-positive in contrast to UC patients (16%) and their relatives (0%) and controls (8.6%). ASCAs were shown to be independent of rural or urban living, disease activity, but were associated with ileal location. The antigen of S. cerevisiae strain W303 discriminated patients depending on age at onset or location of the disease. This study confirms the antigenic heterogeneity of S. cerevisiae strains in their ability to detect ASCA. It suggests that ASCAs are markers of immunoregulatory disturbance in CD, independently of ethnic/cultural differences between Europe, the USA and North Africa.

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.

Long-lived immunity to canine core vaccine antigens in UK dogs as assessed by an in-practice test kit.

PubMed

Killey, R; Mynors, C; Pearce, R; Nell, A; Prentis, A; Day, M J

2018-01-01

To determine the utility of an in-practice test kit to detect protective serum antibody against canine distemper virus, canine adenovirus and canine parvovirus type 2 in a sample of the UK dog population. Serum samples from 486 dogs, last vaccinated between less than 1 month and 124 months previously, were tested with the VacciCheck™ test kit for protective antibodies against distemper, adenovirus and parvovirus type 2. A high proportion of the dogs tested (93·6%) had protective antibody against all three of the core vaccine antigens: 95·7% of the dogs were seropositive against canine distemper virus, 97·3% against canine adenovirus and 98·5% against canine parvovirus type 2. The small number of dogs that were seronegative for one or more of the antigens (n = 31) may have had waning of previous serum antibody or may have been rare genetic non-responders to that specific antigen. UK veterinarians can be reassured that triennial revaccination of adult dogs with core vaccines provides long-lived protective immunity. In-practice serological test kits are a valuable tool for informing decision-making about canine core revaccination. © 2017 British Small Animal Veterinary Association.

[Evaluation of an ELISA test with Fasciola hepatica metabolic antigen for diagnosis of human fascioliasis in Cajamarca, Peru].

PubMed

Cornejo, Hernán; Oblitas, Fátima; Cruzado, Sandro; Quispe, William

2010-01-01

Metabolic (excretion/secretion) antigen was obtained from sheep infected with Fasciola hepatica, with a 1005 μg/μL of protein concentration, composed principally by proteins of molecular weight between 1.2 and 170 KDa. Bands of 170, 150, 31, 24, 18-14 and 10 kDa were detected. With this antigen an ELISA test was developed and the cut off was determined in 0.140. We evaluated 33 serums of patient with fascioliasis confirmed by visualization of eggs in feces, 177 serums of persons without fascioliasis from endemic rural areas of Cajamarca and 88 serums of patients with others parasitic and bacterial infections. We found a 97.0% of sensitivity, 96.6 specificity, 78.1% predictive positive value, 99.6 % predictive negative value. In 9/88 serums was found cross reactions. We recommended the implementation and use of this test for the fascioliasis diagnosis.

RELATION BETWEEN EPSTEIN-BARR VIRAL AND CELL MEMBRANE IMMUNOFLUORESCENCE OF BURKITT TUMOR CELLS

PubMed Central

Klein, G.; Pearson, G.; Nadkarni, J. S.; Nadkarni, J. J.; Klein, E.; Henle, G.; Henle, W.; Clifford, P.

1968-01-01

A comparison was made of the immunofluorescence tests for detection of cell membrane and Epstein-Barr virus antigens in cells from Burkitt tumor biopsies or continuous cultures derived therefrom. On the whole, cell membrane fluorescence in established lines appeared to depend not only upon the presence of EBV but to a considerable degree also upon the extent of the persistent viral infection. There was no constant relationship, however, between the results of the two tests and exceptions to the rule were noted. These observations indicate that different antigens are involved in the two tests. Biopsy cells in general and young cultures may reveal strong MIF activity but few, if any, EBV-positive cells. The reverse, the presence of relatively large numbers of EBV antigen-containing cells in the absence of significant MIF reactions, was also noted on occasion in a few established cultures. The possible interpretations of these findings have been discussed. PMID:4878906

Comparative examination and validation of ELISA test systems for Salmonella typhimurium diagnosis of slaughtering pigs.

PubMed

Szabó, I; Scherer, K; Roesler, U; Appel, B; Nöckler, K; Hensel, A

2008-05-10

The most frequently isolated Salmonella serotype from pork in Germany is S. typhimurium, especially phagetype DT 104. The monitoring programs on Salmonella in swine are based on enzyme-linked immunoadsorbent assay (ELISA) detecting antibodies in serum or meat juice. These serological results are used to classify swine herds in three categories to assess the hygienic status of farm regarding Salmonella infection in pigs. The object of this study was the comparative evaluation of four indirect Salmonella ELISA tests approved in Germany to detect Salmonella typhimurium infection of swine. Three tests (A-C) are based on LPS-antigen and directed against specific IgG-antibodies. The fourth test (D) bases on a whole-cell-lysate antigen and discriminates between Salmonella specific IgA-, IgM- and IgG-antibodies. In a longitudinal study sixteen 6 weeks old weaning pigs were orally infected with S. typhimurium DT 104. During an observation period of 138d clinical and bacteriological parameters were monitored and serum samples obtained at regular intervals as well as meat juice samples taken at slaughter were examined by the respective ELISA systems. Study results reveal that all tested ELISA systems are able to detect S. typhimurium infection in pigs in both sample matrices, blood serum and meat juice whereas test D showed the highest sensitivity to detect Salmonella antibodies in pigs. The sensitivity to detect Salmonella antibodies varied between tests A and C according to the used cut-off (test specific cut-off vs. recommended surveillance cut-off) resulting in a change of seroprevalence and hence may influence the Salmonella status of the farm.

Genotyping Applications for Transplantation and Transfusion Management: The Emory Experience.

PubMed

Fasano, Ross M; Sullivan, Harold Cliff; Bray, Robert A; Gebel, Howard M; Meyer, Erin K; Winkler, Annie M; Josephson, Cassandra D; Stowell, Sean R; Sandy Duncan, Alexander; Roback, John D

2017-03-01

Current genotyping methodologies for transplantation and transfusion management employ multiplex systems that allow for simultaneous detection of multiple HLA antigens, human platelet antigens, and red blood cell (RBC) antigens. The development of high-resolution, molecular HLA typing has led to improved outcomes in unrelated hematopoietic stem cell transplants by better identifying compatible alleles of the HLA-A, B, C, DRB1, and DQB1 antigens. In solid organ transplantation, the combination of high-resolution HLA typing with solid-phase antibody identification has proven of value for highly sensitized patients and has significantly reduced incompatible crossmatches at the time of organ allocation. This database-driven, combined HLA antigen/antibody testing has enabled routine implementation of "virtual crossmatching" and may even obviate the need for physical crossmatching. In addition, DNA-based testing for RBC antigens provides an alternative typing method that mitigates many of the limitations of hemagglutination-based phenotyping. Although RBC genotyping has utility in various transfusion settings, it has arguably been most useful for minimizing alloimmunization in the management of transfusion-dependent patients with sickle cell disease or thalassemia. The availability of high-throughput RBC genotyping for both individuals and large populations of donors, along with coordinated informatics systems to compare patients' antigen profiles with available antigen-negative and/or rare blood-typed donors, holds promise for improving the efficiency, reliability, and extent of RBC matching for this population.

Rapid classification of biological components

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

Thompson, Vicki S.; Barrett, Karen B.; Key, Diane E.

A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens of the surface of a solid support in a preselected pattern to form an array wherein the 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 antigens in the array,more » thereby forming immune complexes; washing away antibodies that do not form immune complexes; and detecting the immune complexes, thereby forming 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 a subject's identity.« less

Rapid classification of biological components

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

Thompson, Vicki S.; Barrett, Karen B.; Key, Diane E.

A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein the 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 antigens in the array,more » thereby forming immune complexes; washing away antibodies that do not form immune complexes; and detecting the immune complexes, thereby forming 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 a subject's identity.« less

Antibody profiling sensitivity through increased reporter antibody layering

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

Apel, William A.; Thompson, Vicki S

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 immunemore » 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.« less

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.

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.

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.

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.

Immunity to Intracellular Salmonella Depends on Surface-associated Antigens

PubMed Central

Claudi, Beatrice; Mazé, Alain; Schemmer, Anne K.; Kirchhoff, Dennis; Schmidt, Alexander; Burton, Neil; Bumann, Dirk

2012-01-01

Invasive Salmonella infection is an important health problem that is worsening because of rising antimicrobial resistance and changing Salmonella serovar spectrum. Novel vaccines with broad serovar coverage are needed, but suitable protective antigens remain largely unknown. Here, we tested 37 broadly conserved Salmonella antigens in a mouse typhoid fever model, and identified antigen candidates that conferred partial protection against lethal disease. Antigen properties such as high in vivo abundance or immunodominance in convalescent individuals were not required for protectivity, but all promising antigen candidates were associated with the Salmonella surface. Surprisingly, this was not due to superior immunogenicity of surface antigens compared to internal antigens as had been suggested by previous studies and novel findings for CD4 T cell responses to model antigens. Confocal microscopy of infected tissues revealed that many live Salmonella resided alone in infected host macrophages with no damaged Salmonella releasing internal antigens in their vicinity. In the absence of accessible internal antigens, detection of these infected cells might require CD4 T cell recognition of Salmonella surface-associated antigens that could be processed and presented even from intact Salmonella. In conclusion, our findings might pave the way for development of an efficacious Salmonella vaccine with broad serovar coverage, and suggest a similar crucial role of surface antigens for immunity to both extracellular and intracellular pathogens. PMID:23093937

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

Competitive and blocking enzyme-linked immunoassay for detection of fetal bovine serum antibodies to bovine viral diarrhea virus.

PubMed

Katz, J B; Hanson, S K

1987-02-01

A competitive blocking enzyme-linked immunoassay (CELIA) was developed to detect bovine viral diarrhea virus (BVDV) antibodies in undiluted fetal bovine serum (FBS). The CELIA was based on competition of serum BVDV antibodies with biotin-labelled anti-BVDV immunoglobulins (Ig) for a limited quantity of solid-phase BVDV antigen. Antigen preparation was simple, FBS could be tested undiluted, and detergent-containing washes were unnecessary. A series of dilutions of postnatal bovine BVDV antiserum prepared in FBS and a set of 147 undiluted abbatoir FBS samples were tested by both CELIA and serum neutralization tests (SNT). CELIA results on both sets of specimens correlated positively with SNT titers (r = 0.99 and r = 0.85). Relative to the SNT, CELIA sensitivity was 100%; specificity was 76%. CELIA detected a level of BVDV antibody below the 1:2-titer threshold detectable with the SNT. Advantages, limitations, and theoretical differences between the CELIA and SNT are discussed. A similar comparison of CELIA with non-competitive enzyme-linked immunoassay approaches to BVDV serodiagnosis is made. It is concluded that the CELIA is valuable in selecting only BVDV-seronegative FBS for use in virologic cell culture media.

Commercial Molecular Tests for Fungal Diagnosis from a Practical Point of View.

PubMed

Lackner, Michaela; Lass-Flörl, Cornelia

2017-01-01

The increasing interest in molecular diagnostics is a result of tremendously improved knowledge on fungal infections in the past 20 years and the rapid development of new methods, in particular polymerase chain reaction. High expectations have been placed on molecular diagnostics, and the number of laboratories now using the relevant technology is rapidly increasing-resulting in an obvious need for standardization and definition of laboratory organization. In the past 10 years, multiple new molecular tools were marketed for the detection of DNA, antibodies, cell wall components, or other antigens. In contrast to classical culture methods, molecular methods do not detect a viable organisms, but only molecules which indicate its presence; this can be nucleic acids, cell components (antigens), or antibodies (Fig. 1). In this chapter, an overview is provided on commercially available detection tools, their strength and how to use them. A main focus is laid on providing tips and tricks that make daily life easier. We try to focus and mention methodical details which are not highlighted in the manufacturer's instructions of these test kits, but are based on our personal experience in the laboratory. Important to keep in mind is that molecular tools cannot replace culture, microscopy, or a critical view on patients' clinical history, signs, and symptoms, but provide a valuable add on tool. Diagnosis should not be based solely on a molecular test, but molecular tools might deliver an important piece of information that helps matching the diagnostic puzzle to a diagnosis, in particular as few tests are in vitro diagnostic tests (IVD) or only part of the whole test carries the IVD certificate (e.g., DNA extraction is often not included). Please be aware that the authors do not claim to provide a complete overview on all commercially available diagnostic assays being currently marketed for fungal detection, as those are subject to constant change. A main focus is put on commonly used panfungal assays and pathogen-specific assays, including Aspergillus-specific, Candida-specific, Cryptococcus specific, Histoplasma-specific, and Pneumocystis-specific assays. Assays are categorized according to their underlying principle in either antigen-detecting or antibody-detecting or DNA-detecting (Fig. 1). Other non-DNA-detecting nucleic acid methods such as FISH and PNA FISH are not summarized in this chapter and an overview on test performance, common false positives, and the clinical evaluation of commercial tests in studies is provided already in a previous book series by Javier Yugueros Marcos and David H. Pincus (Marcos and Pincus, Methods Mol Biol 968:25-54, 2013).

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.

77 FR 7109 - Establishment of User Fees for Filovirus Testing of Nonhuman Primate Liver Samples

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-10

... assay (ELISA) or other appropriate methodology. Each specimen will be held for six months. After six... loss of the only commercially available antigen-detection ELISA filovirus testing facility. Currently... current methodology (ELISA) used to test NHP liver samples. This cost determines the amount of the user...

A novel chemiluminescent immunoassay based on original acridinium ester labels as better solution for diagnosis of human toxoplasmosis than conventional ELISA test.

PubMed

Holec-Gąsior, Lucyna; Ferra, Bartłomiej; Czechowska, Justyna; Serdiuk, Illia E; Krzymiński, Karol

2018-05-01

Toxoplasma gondii infection is one of the most common human zoonosis. Laboratory diagnosis of this disease is mainly based on the results of serological methods detecting specific antibodies in the patient's sera. In this study we aimed to evaluate the performance of a chemiluminescence immunoassay (CLIA) based on the use of a novel immunochemical reagent in the form of the conjugate of original acridinium label (AL) attached to secondary antibody (IgG-AL) and SAG2-GRA1-ROP1 L chimeric antigen for T. gondii specific antibodies detection. The CLIA test was compared with conventional ELISA, which was based on the same recombinant antigen and differed only in terms of the detection methodology of immune complexes. The new CLIA assay proved to be more sensitive and better differentiated sera of patients with T. gondii infection from sera of healthy individuals, being a promising alternative to more labor, cost-demanding and less versatile ELISA as screening test in toxoplasmosis diagnostics. Copyright © 2018 Elsevier Inc. All rights reserved.

Detection of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens

PubMed Central

Vojdani, Aristo

2009-01-01

Background Despite the first documented case of food allergy to cooked food in 1921 by Prausnitz and Kustner, all commercial food antigens are prepared from raw food. Furthermore, all IgE and IgG antibodies against dietary proteins offered by many clinical laboratories are measured against raw food antigens. Methods We developed an enzyme-linked immunosorbent assay for the measurement of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens. Sera with low or high reactivity to modified food antigens were subjected to myelin basic protein, oxidized low density lipoprotein, and advanced glycation end products (AGE) such as AGE-human serum albumin and AGE-hemoglobin. Results Compared to raw food antigens, IgE antibodies showed a 3–8-fold increase against processed food antigens in 31% of the patients. Similarly, IgG, IgA and IgM antibodies against modified food antigens overall were found at much higher levels than antibody reactions against raw food antigens. Almost every tested serum with high levels of antibodies against modified food antigens showed very high levels of antibodies against myelin basic protein, oxidized low density lipoprotein, AGE-human serum albumin and AGE-hemoglobin. Conclusion We conclude that the determination of food allergy, intolerance and sensitivity would be improved by testing IgE, IgG, IgA and IgM antibodies against both raw and processed food antigens. Antibodies against modified food antigens, by reacting with AGEs and tissue proteins, may cause perturbation in degenerative and autoimmune diseases such as diabetes, atherosclerosis, inflammation, autoimmunity, neurodegeneration and neuroautoimmunity. PMID:19435515

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.

Immunoassay procedures for fiber optic sensors

NASA Astrophysics Data System (ADS)

Ligler, Frances S.

1988-04-01

There is an increasing need for the development of an ultrasensitive immunoassay for use with fiber optic sensors. These detection systems can be used for such applications as disease diagnosis, detection of chemical and biological warfare agents or drugs of abuse, pollution control, therapeutic monitoring, and explosive detection. This specific program is designed to produce generic chemistries for use with existing fiber optic-based sensors to detect pathogens of particular threat to Army personnel as determined by USAMRIID. The detection system under development involves the attachment of antibodies to an optical fiber at high density. In addition, the immobilization must be achieved in a way which retains the antibody's ability to bind antigen. The functionality of the antibody will be tested through the binding of a labelled antigen. In the future, this assay could incorporate the antibodies developed by the Army for pathogens of particularly military concern.

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

Diagnostic value of detecting specific IgA and IgM with recombinant Trypanosoma cruzi antigens in congenital Chagas' disease.

PubMed

Lorca, M; Veloso, C; Munoz, P; Bahamonde, M I; Garcia, A

1995-06-01

The present study compares the early diagnosis of congenital Chagas' disease with a DOT assay using recombinant antigens with immunofluorescence antibody testing (IFAT) and an enzyme-linked immunosorbent assay (ELISA). The studies were performed using cord blood and sera of 12 infected newborns (group I) and 12 uninfected ones born to Trypanosoma cruzi-infected mothers (group II). Conventional IFAT and ELISA showed positive results for IgG at high titers, in infants and mothers of both groups; IgA antibodies were detected by ELISA in four of the infected infants and IgM was detected in two of them. All sera of the uninfected infants were negative for IgA and IgM in the ELISA. Application of a DOT assay using eight recombinant T. cruzi antigens allowed detection of specific IgA in the cord blood of six of the infected cases and IgM in eight of them. Repetition of these serologic tests in samples obtained during a monthly follow-up gave positive results for IgA in two of the initially negative infants of group I and for IgM in four of them. This means that diagnosis of congenital T. cruzi infection was confirmed, through demonstration of specific IgM, in all infected infants, and of IgA in eight of them. The importance of late detection of IgM in siblings born of infected mothers is discussed. The detection of IgM and IgA in sera obtained after birth is believed to be due to a congenital transmission of the parasite that occurred late in pregnancy. No IgA or IgM antibodies could be detected by the DOT assay in the sera of the negative controls.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Analytical Validation of the ReEBOV Antigen Rapid Test for Point-of-Care Diagnosis of Ebola Virus Infection

PubMed Central

Cross, Robert W.; Boisen, Matthew L.; Millett, Molly M.; Nelson, Diana S.; Oottamasathien, Darin; Hartnett, Jessica N.; Jones, Abigal B.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Bornholdt, Zachary A.; Fusco, Marnie L.; Abelson, Dafna M.; Oda, Shunichiro; Brown, Bethany L.; Pham, Ha; Rowland, Megan M.; Agans, Krystle N.; Geisbert, Joan B.; Heinrich, Megan L.; Kulakosky, Peter C.; Shaffer, Jeffrey G.; Schieffelin, John S.; Kargbo, Brima; Gbetuwa, Momoh; Gevao, Sahr M.; Wilson, Russell B.; Saphire, Erica Ollmann; Pitts, Kelly R.; Khan, Sheik Humarr; Grant, Donald S.; Geisbert, Thomas W.; Branco, Luis M.; Garry, Robert F.

2016-01-01

Background. Ebola virus disease (EVD) is a severe viral illness caused by Ebola virus (EBOV). The 2013–2016 EVD outbreak in West Africa is the largest recorded, with >11 000 deaths. Development of the ReEBOV Antigen Rapid Test (ReEBOV RDT) was expedited to provide a point-of-care test for suspected EVD cases. Methods. Recombinant EBOV viral protein 40 antigen was used to derive polyclonal antibodies for RDT and enzyme-linked immunosorbent assay development. ReEBOV RDT limits of detection (LOD), specificity, and interference were analytically validated on the basis of Food and Drug Administration (FDA) guidance. Results. The ReEBOV RDT specificity estimate was 95% for donor serum panels and 97% for donor whole-blood specimens. The RDT demonstrated sensitivity to 3 species of Ebolavirus (Zaire ebolavirus, Sudan ebolavirus, and Bundibugyo ebolavirus) associated with human disease, with no cross-reactivity by pathogens associated with non-EBOV febrile illness, including malaria parasites. Interference testing exhibited no reactivity by medications in common use. The LOD for antigen was 4.7 ng/test in serum and 9.4 ng/test in whole blood. Quantitative reverse transcription–polymerase chain reaction testing of nonhuman primate samples determined the range to be equivalent to 3.0 × 105–9.0 × 108 genomes/mL. Conclusions. The analytical validation presented here contributed to the ReEBOV RDT being the first antigen-based assay to receive FDA and World Health Organization emergency use authorization for this EVD outbreak, in February 2015. PMID:27587634

A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool.

PubMed

Schiller, Annemarie; Zhang, Ting; Li, Ruliang; Duechting, Andrea; Sundararaman, Srividya; Przybyla, Anna; Kuerten, Stefanie; Lehmann, Paul V

2017-12-07

Testing of peripheral blood mononuclear cells (PBMC) for immune monitoring purposes requires verification of their functionality. This is of particular concern when the PBMC have been shipped or stored for prolonged periods of time. While the CEF (Cytomegalo-, Epstein-Barr and Flu-virus) peptide pool has become the gold standard for testing CD8 cell functionality, a positive control for CD4 cells is so far lacking. The latter ideally consists of proteins so as to control for the functionality of the antigen processing and presentation compartments, as well. Aiming to generate a positive control for CD4 cells, we first selected 12 protein antigens from infectious/environmental organisms that are ubiquitous: Varicella, Influenza, Parainfluenza, Mumps, Cytomegalovirus, Streptococcus , Mycoplasma , Lactobacillus , Neisseria , Candida , Rubella, and Measles. Of these antigens, three were found to elicited interferon (IFN)-γ-producing CD4 cells in the majority of human test subjects: inactivated cytomegalo-, parainfluenza-, and influenza virions (CPI). While individually none of these three antigens triggered a recall response in all donors, the pool of the three (the 'CPI pool'), did. One hundred percent of 245 human donors tested were found to be CPI positive, including Caucasians, Asians, and African-Americans. Therefore, the CPI pool appears to be suitable to serve as universal positive control for verifying the functionality of CD4 and of antigen presenting cells.

Analysis of HTLV-1 proviral load (PVL) and antibody detected with various kinds of tests in Japanese blood donors to understand the relationship between PVL and antibody level and to gain insights toward better antibody testing.

PubMed

Matsumoto, Chieko; Sagara, Yasuko; Sobata, Rieko; Inoue, Yukiko; Morita, Maiko; Uchida, Shigeharu; Kiyokawa, Hiroyuki; Satake, Masahiro; Tadokoro, Kenji

2017-08-01

Adult T-cell leukemia/lymphoma (ATL) occurs in approximately 5% of individuals infected with human T-cell leukemia virus type 1 (HTLV-1). A high proviral load (PVL; more than four copies per 100 peripheral blood mononuclear cells (PBMCs) or 1.6 copies per 100 blood leukocytes) and being male are risk factors for ATL development. Whether anti-HTLV-1 antibody level is related to such risk is unknown. Here, PVL and antibody levels were examined using real-time PCR and other tests in 600 HTLV-1 positive screened Japanese blood donors to understand the relationship between PVL and antibody level in asymptomatic carriers and to gain insights toward better antibody testing for HTLV-1 infection. The 430 donors in whom proviral DNA was detected were considered as true positives for HTLV-1 infection. Among donors aged 40 years or older, more males than females had a PVL corresponding to more than 1.6% infected leukocytes, and an antibody titer below the median (P = 0.0018). In antibody tests using an HTLV-1 positive cell line or Env antigens there was a large discrepancy in antibody titer among 13 provirus-positive samples, probably suggesting that antibody-based screening tests should incorporate multiple HTLV-1 antigens, such as Gag and Env antigens. © 2017 Wiley Periodicals, Inc.

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.

Use of a Chagas Urine Nanoparticle Test (Chunap) to Correlate with Parasitemia Levels in T. cruzi/HIV Co-infected Patients.

PubMed

Castro-Sesquen, Yagahira E; Gilman, Robert H; Mejia, Carolina; Clark, Daniel E; Choi, Jeong; Reimer-McAtee, Melissa J; Castro, Rosario; Valencia-Ayala, Edward; Flores, Jorge; Bowman, Natalie; Castillo-Neyra, Ricardo; Torrico, Faustino; Liotta, Lance; Bern, Caryn; Luchini, Alessandra

2016-02-01

Early diagnosis of reactivated Chagas disease in HIV patients could be lifesaving. In Latin America, the diagnosis is made by microscopical detection of the T. cruzi parasite in the blood; a diagnostic test that lacks sensitivity. This study evaluates if levels of T. cruzi antigens in urine, determined by Chunap (Chagas urine nanoparticle test), are correlated with parasitemia levels in T. cruzi/HIV co-infected patients. T. cruzi antigens in urine of HIV patients (N = 55: 31 T. cruzi infected and 24 T. cruzi serology negative) were concentrated using hydrogel particles and quantified by Western Blot and a calibration curve. Reactivation of Chagas disease was defined by the observation of parasites in blood by microscopy. Parasitemia levels in patients with serology positive for Chagas disease were classified as follows: High parasitemia or reactivation of Chagas disease (detectable parasitemia by microscopy), moderate parasitemia (undetectable by microscopy but detectable by qPCR), and negative parasitemia (undetectable by microscopy and qPCR). The percentage of positive results detected by Chunap was: 100% (7/7) in cases of reactivation, 91.7% (11/12) in cases of moderate parasitemia, and 41.7% (5/12) in cases of negative parasitemia. Chunap specificity was found to be 91.7%. Linear regression analysis demonstrated a direct relationship between parasitemia levels and urine T. cruzi antigen concentrations (p<0.001). A cut-off of > 105 pg was chosen to determine patients with reactivation of Chagas disease (7/7). Antigenuria levels were 36.08 times (95% CI: 7.28 to 64.88) higher in patients with CD4+ lymphocyte counts below 200/mL (p = 0.016). No significant differences were found in HIV loads and CD8+ lymphocyte counts. Chunap shows potential for early detection of Chagas reactivation. With appropriate adaptation, this diagnostic test can be used to monitor Chagas disease status in T. cruzi/HIV co-infected patients.

Use of a Chagas Urine Nanoparticle Test (Chunap) to Correlate with Parasitemia Levels in T. cruzi/HIV Co-infected Patients

PubMed Central

Castro-Sesquen, Yagahira E.; Gilman, Robert H.; Mejia, Carolina; Clark, Daniel E.; Choi, Jeong; Reimer-McAtee, Melissa J.; Castro, Rosario; Valencia-Ayala, Edward; Flores, Jorge; Bowman, Natalie; Castillo-Neyra, Ricardo; Torrico, Faustino; Liotta, Lance; Bern, Caryn; Luchini, Alessandra

2016-01-01

Background Early diagnosis of reactivated Chagas disease in HIV patients could be lifesaving. In Latin America, the diagnosis is made by microscopical detection of the T. cruzi parasite in the blood; a diagnostic test that lacks sensitivity. This study evaluates if levels of T. cruzi antigens in urine, determined by Chunap (Chagas urine nanoparticle test), are correlated with parasitemia levels in T. cruzi/HIV co-infected patients. Methodology/Principal Findings T. cruzi antigens in urine of HIV patients (N = 55: 31 T. cruzi infected and 24 T. cruzi serology negative) were concentrated using hydrogel particles and quantified by Western Blot and a calibration curve. Reactivation of Chagas disease was defined by the observation of parasites in blood by microscopy. Parasitemia levels in patients with serology positive for Chagas disease were classified as follows: High parasitemia or reactivation of Chagas disease (detectable parasitemia by microscopy), moderate parasitemia (undetectable by microscopy but detectable by qPCR), and negative parasitemia (undetectable by microscopy and qPCR). The percentage of positive results detected by Chunap was: 100% (7/7) in cases of reactivation, 91.7% (11/12) in cases of moderate parasitemia, and 41.7% (5/12) in cases of negative parasitemia. Chunap specificity was found to be 91.7%. Linear regression analysis demonstrated a direct relationship between parasitemia levels and urine T. cruzi antigen concentrations (p<0.001). A cut-off of > 105 pg was chosen to determine patients with reactivation of Chagas disease (7/7). Antigenuria levels were 36.08 times (95% CI: 7.28 to 64.88) higher in patients with CD4+ lymphocyte counts below 200/mL (p = 0.016). No significant differences were found in HIV loads and CD8+ lymphocyte counts. Conclusion Chunap shows potential for early detection of Chagas reactivation. With appropriate adaptation, this diagnostic test can be used to monitor Chagas disease status in T. cruzi/HIV co-infected patients. PMID:26919324

Comparison between two commercially available serological tests and polymerase chain reaction in the diagnosis of Cryptosporidium in animals and diarrhoeic children.

PubMed

Helmy, Yosra A; Krücken, Jürgen; Nöckler, Karsten; von Samson-Himmelstjerna, Georg; Zessin, Karl-H

2014-01-01

For the detection of Cryptosporidium species in 804 animals and 165 diarrhoeic children (<10 years) in Egypt, two copro-antigen tests, the RIDASCREEN® Cryptosporidium test [enzyme immunoassay (EIA)] and the RIDA®QUICK Cryptosporidium/Giardia Combi [immuno-chromatographic test (ICT)] as well as polymerase chain reaction (PCR) were used. Prevalence of Cryptosporidium was 15.0, 19.5 and 32.3% in animals and 2.4, 6.7 and 49.1% in children using EIA, ICT and PCR, respectively.Using PCR as reference method, animal samples sensitivity (Se) of the EIA was 46.5% when questionable samples were considered positive, whereas specificity (Sp) was 100%. Se of the ICT was 60.4% while Sp was 100%. Positive predictive values (PPVs) for both EIA and ICT test were 100%, and negative predictive values (NPVs) for EIA were 79.7 and 84.1% for ICT. For the children samples, the Se of EIA was 5%, Sp was 100%, PPV was 100% and NPV was 52.2%, while the Se of ICT was 13.6%, Sp was 100%, PPV was 100% and NPV was 54.6%.The Kappa score of agreement between PCR and ICT was 67.4%, 54.1% between PCR and EIA and 84.4% between ICT and EIA. Until the second serial dilution of the EIA and ICT test, 9 × 10(3) oocysts/μl of Cryptosporidia was detected, whereas in PCR, they were detected until the sixth serial dilution. Copro-antigen tests were easy to perform and less time-consuming but less sensitive compared to PCR. They obviously are best applicable for screening and epidemiological studies of large numbers of subjects, for batch specimen processing and in isolated or rural areas where reliable tests like PCR are unfeasible. When in children, a single stool sample is used for the diagnosis of clinical cases; better results can be obtained when non-standardized PCR due low specificity is coupled with copro-antigen tests.

Detection of rabies antigen in the saliva and brains of apparently healthy dogs slaughtered for human consumption and its public health implications in abia state, Nigeria.

PubMed

Mshelbwala, P P; Ogunkoya, A B; Maikai, B V

2013-01-01

The study was carried out in eight dogs slaughtering outlets within four Local Government Areas of the State for the determination of rabies antigen in the saliva and brain of apparently healthy dogs slaughtered for human consumption. A total of one hundred (100) samples each of saliva and brain were collected before and after slaughter, respectively, between April to June, 2013, in the selected areas. The saliva was subjected to rapid immune-chromatographic test (RICT) while direct fluorescent antibody test (DFAT) was carried out on the brain samples. Structured questionnaire was administered to nineteen (19) dog meat processors comprising 18 males and 1 female in the selected areas. Sixty four percent of the samples tested were from female dogs while 36% were from males, 5% tested positive for rabies antigen with the use of both tests; there was no statistical association between sex and rabies status of the dogs sampled (P > 0.05). Butchers bitten during the course of slaughtering were 94.7% out of which 72.8% utilized traditional method of treatment and only 27.8% reported to the hospital for proper medical attention. This study has established the presence of rabies antigen in apparently healthy dogs in the study area.

The implementation of nucleic acid amplification technology testing for living tissue donors.

PubMed

Westby, J; Lomas, R J; Kearney, J N

2010-05-01

There is a significant requirement within the United Kingdom for tissue grafts from living donors. To ensure safety, blood samples from these donors are tested for pathogens at donation, and at 180 days post donation. Nucleic acid amplification technology (NAT) permits more sensitive detection of pathogens in blood samples than serum antigen testing. NAT testing can be applied to samples from living tissue donors to eliminate the need to re-test these donors 180 days post-donation before grafts can be implanted. This has major financial and operational advantages for a tissue bank, and this manuscript describes how NAT testing was assessed and implemented by NHSBT Tissue Services. When compared to traditional serum antigen testing, NAT testing was more cost effective, more convenient for donors and resulted in a greater proportion of donated grafts being made available for transplant.

Streamlined, PCR-based testing for pfhrp2- and pfhrp3-negative Plasmodium falciparum.

PubMed

Parr, Jonathan B; Anderson, Olivia; Juliano, Jonathan J; Meshnick, Steven R

2018-04-02

Rapid diagnostic tests (RDTs) that detect histidine-rich protein 2 (PfHRP2) are used throughout Africa for the diagnosis of Plasmodium falciparum malaria. However, recent reports indicate that parasites lacking the pfhrp2 and/or histidine-rich protein 3 (pfhrp3) genes, which produce antigens detected by these RDTs, are common in select regions of South America, Asia, and Africa. Proving the absence of a gene is challenging, and multiple PCR assays targeting these genes have been described. A detailed characterization and comparison of published assays is needed to facilitate robust and streamlined testing approaches. Among six pfhrp2 and pfhrp3 PCR assays tested, the lower limit of detection ranged from 0.01 pg/µL to 0.1 ng/µL of P. falciparum 3D7 strain DNA, or approximately 0.4-4000 parasite genomes/µL. By lowering the elongation temperature to 60 °C, a tenfold improvement in the limit of detection and/or darker bands for all exon 1 targets and for the first-round reaction of a single exon 2 target was achieved. Additionally, assays targeting exon 1 of either gene yielded spurious amplification of the paralogous gene. Using these data, an optimized testing algorithm for the detection of pfhrp2- and pfhrp3-negative P. falciparum is proposed. Surveillance of pfhrp2- and pfhrp3-negative P. falciparum requires careful laboratory workflows. PCR-based testing methods coupled with microscopy and/or antigen testing serve as useful tools to support policy development. Standardized approaches to the detection of pfhrp2- and pfhrp3-negative P. falciparum should inform efforts to define the impact of these parasites.

A recombinant Toscana virus nucleoprotein in a diagnostic immunoblot test system.

PubMed

Schwarz, T F; Gilch, S; Schätzl, H M

1998-01-01

Sandfly fever, a vector-borne disease endemic in the Mediterranean region, is caused by Toscana virus (TOS). The disease is increasingly important as a travel-related infection. Serological diagnosis is currently dependent on viral antigens derived from TOS-infected cell cultures. In this study, we report the cloning and expression of the TOS nucleoprotein (N) in Escherichia coli and evaluation of the recombinant (r) TOS N protein as an antigen for immunoblot assays. The TOS N gene was amplified by reverse-transcriptase polymerase chain reaction and cloned into the bacterial expression vector pTrcHis-A. Sera with known TOS antibody status were used to evaluate the immunoblot assay. The expressed rTOS N protein was purified and used as antigen for immunoblots. By recombinant immunoblot, the TOS antibody status (IgM and/or IgG) of the test panel was correctly identified. No cross-reactivity was detected. The rTOS N protein is useful as an antigen for immunoblot assays, and will enable more laboratories to perform TOS antibody diagnosis.

Comparing validation of four ELISA-systems for detection of Salmonella derby- and Salmonella infantis-infected pigs.

PubMed

Roesler, Uwe; Szabo, Istvan; Matthies, Claudia; Albrecht, Kerstin; Leffler, Martin; Scherer, Kathrin; Nöckler, Karsten; Lehmann, Jörg; Methner, Ulrich; Hensel, Andreas; Truyen, Uwe

2011-01-01

The objective of this study was the comparative evaluation of four indirect Salmonella ELISA tests at study time approved in Germany to detect Salmonella infection in pigs.Three tests are based on a LPS-antigen mix and directed against specific IgG antibodies. The fourth test is based on a purified S. Typhimurium whole-cell lysate antigen and discriminates between Salmonella-specific IgM-, IgA-, and IgG- antibodies. In a longitudinal study, two groups of six weeks old hybrid piglets were orally infected with a porcine S. Infantis or S. Derby strain. Clinical and bacteriological parameters were monitored weekly during an observation period of 130 days after infection and serum samples were investigated in parallel with the respective ELISAs. Apparently, the LPS-based ELISA systems used in this study failed to recognize S. Infantis-infected pigs although those animals shed the pathogen in high amounts throughout the study until day 81 post infection (p. i.). In contrast, the isotype-specific Salmonella Typhimurium whole-cell-lysate based ELISA was capable of detecting Salmonella-infected pigs from day ten p. i. at all tested serotypes and revealed the highest sensitivity in detection of S. Infantis-infected pigs. Furthermore, it became apparent that the often used surveillance cut-off value of 40 OD% is not appropriate for intra-vitam detection of S. Infantis- and S. Derby-infected pigs. In contrast, the cut-off values of the ELISAs given by the suppliers result in considerable higher detection rates.

Detection of soluble antigens of Toxoplasma gondii by a four-layer modification of an enzyme immunoassay.

PubMed Central

Turunen, H J

1983-01-01

A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of the antigen assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the antigen assay was found to be at least 10 ng of antigen protein per ml. The suitability of the method for detecting antigens of T. gondii in different specimens was studied by experimental toxoplasma infection in mice. Antigenic components of T. gondii could be detected in different tissue specimens from infected animals from the first day after infection onwards. Toxoplasma antigen in serum and urine samples from infected mice reached detectable levels on day 2 after infection followed by a linear increase in antigen concentration in succeeding samples. This method might offer a valuable aid for a rapid etiological diagnosis also in human cases of acute toxoplasmosis. PMID:6345574

A case of hyper-reactive malarial splenomegaly. The role of rapid antigen-detecting and PCR-based tests.

PubMed

Mothe, B; Lopez-Contreras, J; Torres, O H; Muñoz, C; Domingo, P; Gurgui, M

2008-03-01

Hyper-reactive malarial splenomegaly (HMS) - originally referred to as tropical splenomegaly syndrome - is characterized by a massive splenomegaly, high titres of anti-malarial antibodies and polyclonal IgM hypergammaglobulinemia. It is believed to be a consequence of an aberrant immunological response to prolonged exposure to malarial parasites. Although it is a frequent disease in the tropics, it is infrequent in western countries and is only seen in long-term residents from endemic areas. We describe the case of a 67-year-old Spanish man, a missionary in Cameroon for 30 years, who presented with a clinical history that fulfilled the diagnosis of HMS. We discuss the role and importance of PCR-based techniques in demonstrating lowgrade malarial parasitemia and the usefulness of new rapid antigen-detecting dipstick tests.

Testing Eurasian wild boar piglets for serum antibodies against Mycobacterium bovis.

PubMed

Che' Amat, A; González-Barrio, D; Ortiz, J A; Díez-Delgado, I; Boadella, M; Barasona, J A; Bezos, J; Romero, B; Armenteros, J A; Lyashchenko, K P; Venteo, A; Rueda, P; Gortázar, C

2015-09-01

Animal tuberculosis (TB) caused by infection with Mycobacterium bovis and closely related members of the M. tuberculosis complex (MTC), is often reported in the Eurasian wild boar (Sus scrofa). Tests detecting antibodies against MTC antigens are valuable tools for TB monitoring and control in suids. However, only limited knowledge exists on serology test performance in 2-6 month-old piglets. In this age-class, recent infections might cause lower antibody levels and lower test sensitivity. We examined 126 wild boar piglets from a TB-endemic site using 6 antibody detection tests in order to assess test performance. Bacterial culture (n=53) yielded a M. bovis infection prevalence of 33.9%, while serum antibody prevalence estimated by different tests ranged from 19% to 38%, reaching sensitivities between 15.4% and 46.2% for plate ELISAs and between 61.5% and 69.2% for rapid immunochromatographic tests based on dual path platform (DPP) technology. The Cohen kappa coefficient of agreement between DPP WTB (Wildlife TB) assay and culture results was moderate (0.45) and all other serological tests used had poor to fair agreements. This survey revealed the ability of several tests for detecting serum antibodies against the MTC antigens in 2-6 month-old naturally infected wild boar piglets. The best performance was demonstrated for DPP tests. The results confirmed our initial hypothesis of a lower sensitivity of serology for detecting M. bovis-infected piglets, as compared to older wild boar. Certain tests, notably the rapid animal-side tests, can contribute to TB control strategies by enabling the setup of test and cull schemes or improving pre-movement testing. However, sub-optimal test performance in piglets as compared to that in older wild boar should be taken into account. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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

Evaluation of Fluorescence Microsphere Immunoassay for the Detection of Antibodies to Rift Valley Fever Nucleocapsid Protein and Glycoproteins

USDA-ARS?s Scientific Manuscript database

Rift Valley Fever virus (RVFV) is a zoonotic virus that infects ruminants including cattle, sheep, goats, camels and buffalo. Multiplexing diagnostic assays that can simultaneously detect antibodies against multiple RVFV antigens offer a high throughput test for disease surveillance and vaccine eva...

[Pestivirus as causative agent of abortion and perinatal mortality in cattle and sheep in Switzerland].

PubMed

Thür, B; Caplazi, P; Hilbe, M; Zlinszky, K; Strasser, M; Corboz, L; Ehrensperger, F

1998-04-01

The causal involvement of bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) infection in bovine and ovine abortion and perinatal mortality remain unclear. From 1992 until 1994, 213 bovine and 31 ovine foetuses as well as 36 calves and 25 lambs which had died perinatally were investigated. Tissue samples were tested for the presence of pestiviruses and serum or fluid from the body cavities were analysed for the presence of pestivirus antibodies. Detection of pestiviruses was performed by (i) cell culture isolation, (ii) antigen ELISA and (iii) immunohistochemical staining for viral antigen. For antibody-testing an indirect ELISA was used. In nine bovine foetuses and in two calves BVDV was isolated. Pestiviruses, most likely BDV were detected in one ovine foetus and three lambs. In 6% of the bovine and 11% of the ovine foetuses anti-pestivirus antibodies were detected. However, clinical features and history of bovine cases did not show a correlation with the diagnostic results, In contrast, the presence of central nervous system signs in neonatal lambs and the detection of BDV was correlated.

Rapid viral diagnosis of acute respiratory infections: comparison of enzyme-linked immunosorbent assay and the immunofluorescence technique for detection of viral antigens in nasopharyngeal secretions.

PubMed Central

Grandien, M; Pettersson, C A; Gardner, P S; Linde, A; Stanton, A

1985-01-01

Nasopharyngeal secretions from adults and children were obtained in Stockholm, Sweden, for routine diagnosis of influenza A virus, influenza B virus, respiratory syncytial (RS) virus, parainfluenza type 3 virus, and adenovirus infections by demonstration of viral antigens directly in the specimens. The cells in nasopharyngeal secretions were pelleted by centrifugation for preparation of cell deposits for diagnosis by the immunofluorescence technique (IF) in London, England, and in Stockholm, whereas the supernatants were used to diagnose infection by the enzyme-linked immunosorbent assay (ELISA) in Stockholm. Titrations of the various purified viruses showed that ELISA could detect viral antigens in amounts corresponding to 1 to 10 ng of virus protein per test well. In a series of 73 specimens tested for influenza A, RS, and parainfluenza type 3 viruses by IF in London and by ELISA in Stockholm, 15 of 18 RS, 14 of 15 influenza A, and 2 of 2 parainfluenza type 3 viral infections were diagnosed by ELISA as compared with IF, giving sensitivities for RS and influenza A viral diagnosis of 83 and 93%, respectively, and a specificity of 100%. In another series of specimens from 35 patients tested for influenza B virus and adenovirus, five influenza B virus and four adenovirus infections were diagnosed by both methods; one additional influenza B infection was detected only by IF and another only by ELISA. Comparisons of diagnostic results between the two methods performed in Stockholm gave nonagreement of results for 37 of 1,593 tests (2.5%) for the five viruses. The conclusion reached was that the described ELISA, although a satisfactory test, had somewhat less sensitivity than did IF for the detection of respiratory viral infections. This could possibly be explained by unnecessary dilutions of specimens at the time of collection; transportation, processing, and storage of specimens were less complicated than for IF. PMID:2997270

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

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

Occurrence of nonspecific reactions among stool specimens tested by the Abbott TestPack rotavirus enzyme immunoassay.

PubMed Central

Lipson, S M; Leonardi, G P; Salo, R J; Schutzbank, T E; Kaplan, M H

1990-01-01

Sixty-five stool specimens obtained from children suffering from gastroenteritis were tested for the presence of antigen to rotavirus by the Abbott TestPack Rotavirus (TestPack) enzyme immunoassay kit. The Kallestad Pathfinder enzyme immunoassay, polyacrylamide gel electrophoresis, immune electron microscopy, and virus isolation were utilized as reference assays. Fifty-four specimens were in accord by TestPack and Kallestad Pathfinder. Among 11 discordant specimens positive with TestPack but negative by Kallestad Pathfinder, rotavirus was not identified by polyacrylamide gel electrophoresis, immune electron microscopy, or isolation in primary African green monkey kidney cell cultures. TestPack displayed a performance specificity of 83%. The inordinately high number of stool specimens reported as false-positive by TestPack precludes the incorporation of this antigen detection kit into our routine regimen of diagnostic virologic testing. Images PMID:2166074

Blocking effect of rheumatoid factor and cold agglutinins on complement fixation tests for histoplasmosis.

PubMed Central

Johnson, J E; Roberts, G D

1976-01-01

The blocking effect of rheumatoid factor (RF) and cold agglutinins (CA) on the detection of complement-fixing (CF) antibodies for Histoplasma capsulatum using a mycelial (histoplasmin) and a yeast antigen was studied. Sera from 213 patients serologically positive for histoplasmosis were screened for the presence of RF or CA. CF antibodies to H. capsulatum in sera containing RF or CA were studied before and after removal of these factors (RF and CA) by treatment with dithiothreitol. Results suggest that RF or CA may interfere with the CF reaction to the yeast antigen of H. capsulatum but not to the mycelial antigen (histoplasmin). PMID:1254713

Antigenic Determinants of Alpha-Like Proteins of Streptococcus agalactiae

PubMed Central

Maeland, Johan A.; Bevanger, Lars; Lyng, Randi Valsoe

2004-01-01

The majority of group B streptococcus (GBS) isolates express one or more of a family of surface-anchored proteins that vary by strain and that form ladder-like patterns on Western blotting due to large repeat units. These proteins, which are important as GBS serotype markers and as inducers of protective antibodies, include the alpha C (Cα) and R4 proteins and the recently described alpha-like protein 2 (Alp2), encoded by alp2, and Alp3, encoded by alp3. In this study, we examined antigenic determinants possessed by Alp2 and Alp3 by testing of antibodies raised in rabbits, mainly by using enzyme-linked immunosorbent assays (ELISA) and an ELISA absorption test. The results showed that Alp2 and Alp3 shared an antigenic determinant, which may be a unique immunological marker of the Alp variants of GBS proteins. Alp2, in addition, possessed an antigenic determinant which showed specificity for Alp2 and a third determinant which showed serological cross-reactivity with Cα. Alp3, in addition to the determinant common to Alp2 and Alp3, harbored an antigenic site which also was present in the R4 protein, whereas no Alp3-specific antigenic site was detected. These ELISA-based results were confirmed by Western blotting and a fluorescent-antibody test. The results are consistent with highly complex antigenic structures of the alpha-like proteins in a fashion which is in agreement with the recently described structural mosaicism of the alp2 and alp3 genes. The results are expected to influence GBS serotyping, immunoprotection studies, and GBS vaccine developments. PMID:15539502

Antigenic typing of canine parvovirus using differential PCR.

PubMed

Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Sharma, N S

2014-12-01

Canine parvovirus (CPV) is an enteric pathogen causing hemorrhagic enteritis in pups of 3-6 months of age and is mainly transmitted via feco-oral route. In the present study, a total of 85 animals rectal swabs suspected of CPV were tested using a PCR, nested PCR and a newly designed differential PCR. Using PCR 7 (8.23 %) animals were positive whereas 39 (45.88 %) were positive by using nested PCR and 40 (47.05 %) were positive for either one or more than one antigenic types of CPV using differential PCR. Using differential PCR it was found that CPV-2a and CPV-2b were the most prevailing antigenic types. Also it was found that dogs that were vaccinated too yielded positive CPV indicating a possible presence of additional CPV antigenic types. Thus, the primers used in differential PCR can be used in a single PCR reaction to detect various antigenic types of CPV.

Prognostic Significance of Digital Rectal Examination and Prostate Specific Antigen in the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Arm.

PubMed

Halpern, Joshua A; Shoag, Jonathan E; Mittal, Sameer; Oromendia, Clara; Ballman, Karla V; Hershman, Dawn L; Wright, Jason D; Shih, Ya-Chen Tina; Nguyen, Paul L; Hu, Jim C

2017-02-01

The absence of definitive data or explicit guidelines regarding the use of digital rectal examination for prostate cancer screening may lead to confusion for physicians and patients alike. We evaluated the prognostic value of abnormal digital rectal examination and prostate specific antigen following the widespread dissemination of prostate specific antigen testing in the U.S. Collectively, men comprising the screening arm of the PLCO cancer screening trial who underwent digital rectal examination screening (35,350) were followed for 314,033 person-years. Adjusted analyses with competing risks regression were performed to assess the association of suspicious (nodularity, induration, asymmetry) digital rectal examination and abnormal prostate specific antigen (4 ng/ml or greater) with the detection of clinically significant prostate cancer, prostate cancer specific mortality and overall mortality. Among all screening encounters with a suspicious digital rectal examination only 15.4% had a concurrently abnormal prostate specific antigen (McNemar's test p <0.001). During followup there were 1,612 clinically significant prostate cancers detected, 64 prostate cancer specific deaths and 4,600 deaths. On multivariable analysis suspicious digital rectal examination and abnormal prostate specific antigen were associated with a greater risk of clinically significant prostate cancer (HR 2.21, 95% CI 1.99-2.44 vs HR 5.48, 95% CI 5.05-5.96, p <0.001 and p <0.001) and prostate cancer specific mortality (HR 2.54, 95% CI 1.41-4.58 vs HR 5.23, 95% CI 3.08-8.88, p=0.002 and p <0.001), respectively. In a secondary analysis of a contemporary U.S. cohort, suspicious digital rectal examination and abnormal prostate specific antigen on routine screening were independently associated with clinically significant prostate cancer and prostate cancer specific mortality. However, additional research is needed to optimize screening protocols. Copyright © 2017 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Early Detection of Breast Cancer Using Autoantibody Markers — EDRN Public Portal

Cancer.gov

To identify large numbers of antigens that can be used to recognize the presence of cancer by detecting antibodies to tumor proteins in the serum of the test subjects. Our technology will provide an early detection test for breast cancer in asymptomatic women. We will use bioinformatics techniques to analyze these protein microarray-immunoassays to discriminate between cancer patients and healthy subjects so as to detect disease prior to standard diagnoses as well as discriminate patients with benign conditions or other cancers that might be a false positive in less specific assays.

Development of a blocking latex agglutination test for the detection of antibodies to chicken anemia virus.

PubMed

Trinh, Dai Quang; Ogawa, Haruko; Bui, Vuong Nghia; Nguyen, Tham Thi Hong; Gronsang, Dulyatad; Baatartsogt, Tugsbaatar; Kizito, Mugimba Kahoza; AboElkhair, Mohammed; Yamaguchi, Shigeo; Nguyen, Viet Khong; Imai, Kunitoshi

2015-09-01

A blocking latex agglutination test (b-LAT) developed in this study was evaluated for the detection of antibodies against chicken anemia virus (CAV) in chickens. Polystyrene latex beads were coupled with a neutralizing monoclonal antibody (mAb) to CAV (mAb-beads). When mAb-beads were mixed with antigens prepared from the lysate of MDCC-MSB1 cells infected with CAV, agglutination occurred. A short pre-incubation of CAV antigens with CAV-specific antiserum inhibited the agglutination of mAb-beads. The test results were obtained within 5min. The specificity of b-LAT was evaluated using sera from specific pathogen-free chickens and sera containing antibodies to avian influenza virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus; nonspecific agglutination and cross-reactivity with antibodies to unrelated viruses were not observed. The examination of 94 serum samples collected from commercial breeder chickens of various ages (17-63 weeks) revealed good agreement (93.6%, Kappa value=0.82) between b-LAT and a virus neutralization test, known to be most sensitive and specific in the detection of antibodies to CAV. These results indicate that b-LAT, a simple and rapid test, is a useful and reliable tool in CAV serology. Copyright © 2015 Elsevier B.V. All rights reserved.

Enzymatic signal amplification for sensitive detection of intracellular antigens by flow cytometry.

PubMed

Karkmann, U; Radbruch, A; Hölzel, V; Scheffold, A

1999-11-19

Flow cytometry is the method of choice for the analysis of single cells with respect to the expression of specific antigens. Antigens can be detected with specific antibodies either on the cell surface or within the cells, after fixation and permeabilization of the cell membrane. Using conventional fluorochrome-labeled antibodies several thousand antigens are required for clear-cut separation of positive and negative cells. More sensitive reagents, e.g., magnetofluorescent liposomes conjugated to specific antibodies permit the detection of less than 200 molecules per cell but cannot be used for the detection of intracellular antigens. Here, we describe an enzymatic amplification technique (intracellular tyramine-based signal amplification, ITSA) for the sensitive cytometric analysis of intracellular cytokines by immunofluorescence. This approach results in a 10 to 15-fold improvement of the signal-to-noise ratio compared to conventional fluorochrome labeled antibodies and permits the detection of as few as 300-400 intracellular antigens per cell.

Optoacoustic sensing of ocular bacterial antigen using targeted gold nanorods

NASA Astrophysics Data System (ADS)

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

2008-02-01

Bacterial contamination can be detected using a minimally invasive optical method, based on laser-induced optoacoustic spectroscopy, to probe for specific antigens associated with a specific infectious agent. As a model system, we have used a surface antigen (Ag), isolated from Chlamydia trachomatis, and a complementary antibody (Ab). A preparation of 0.2 mg/ml of monoclonal Ab specific to the C. trachomatis surface Ag was conjugated to gold nanorods using standard commercial reagents, in order to produce a targeted contrast agent with a strong optoacoustic signal. The C. trachomatis Ag was absorbed in standard plastic microwells, and the binding of the complementary Ab-nanorod conjugate was tested in an immunoaffinity assay. Optoacoustic signals were elicited from the bound nanorods, using an optical parametric oscillator (OPO) laser system as the optical pump. The wavelength tuneability of the OPO optimized the spectroscopic measurement by exciting the nanorods at their optical absorption maxima. Optoacoustic responses were measured in the microwells using a probe beam deflection technique. Immunoaffinity assays were performed on several dilutions of purified C. trachomatis antigen ranging from 50 μg/ml to 1 pg/ml, in order to determine the detection limit for the optoacoustic-based assay. Only when the antigen was present, and the complementary Ab-NR reagent was introduced into the microwell, was an enhanced optoacoustic signal obtained, which indicated specific binding of the Ab-NR complex. The limit of detection with the current system design is between 1 and 5 pg/ml of bacterial Ag.

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.

[Detection of stool antigens of Echinococcus granulosus in dogs belonging to slaughterhouse workers and offal merchants in Metropolitan Lima].

PubMed

Merino, Veronika; Falcón, Néstor; Morel, Noelia; González, Gualberto

2017-04-20

To demonstrate the presence of Echinoccocus granulosus in the definitive host in the city of Lima, Perú, by detecting parasite antigens in the stool of dogs belonging to offal handlers and merchants in authorized slaughterhouses in Metropolitan Lima. Stool samples were collected from 58 dogs and examined using the coproELISA technique for the detection of secretory/excretory antigens of E. granulosus. A survey was conducted to obtain information on pet feeding and handling practices. Positivity to E. granulosus was detected in 13.8% (8/58) of the dogs. In 27.8% (5/18) of the homes, at least one animal showed positivity, and in families that had more than four dogs the chances of finding positivity in at least one dog were higher (P < 0.05). In all homes where at least one dog tested positive the pets were fed on offal. Of study participants, 94.4% (17) knew nothing about the routes of transmission of hydatid disease. Results show the presence of definitive hosts in the urban area of Lima and underscore the need to more widely disseminate practices for the prevention of parasite transmission.

Leprosy (Hansen's Disease)

MedlinePlus

... See all Leprosy (Hansen's Disease) related NIAID Now posts Basic Research NIAID-funded investigators are developing the armadillo as a research animal model for human leprosy and developing improved skin test antigens to detect leprosy. Read more about leprosy ...

Single-step colony assay for screening antibody libraries.

PubMed

Kato, Mieko; Hanyu, Yoshiro

2017-08-10

We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3×10 3 scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries. Copyright © 2017 Elsevier B.V. All rights reserved.

Malaria rapid diagnostic tests in endemic settings.

PubMed

Maltha, J; Gillet, P; Jacobs, J

2013-05-01

Malaria rapid diagnostic tests (RDTs) are instrument-free tests that provide results within 20 min and can be used by community health workers. RDTs detect antigens produced by the Plasmodium parasite such as Plasmodium falciparum histidine-rich protein-2 (PfHPR2) and Plasmodium lactate dehydrogenase (pLDH). The accuracy of RDTs for the diagnosis of uncomplicated P. falciparum infection is equal or superior to routine microscopy (but inferior to expert microscopy). Sensitivity for Plasmodium vivax is 75-100%; for Plasmodium ovale and Plasmodium malariae, diagnostic performance is poor. Design limitations of RDTs include poor sensitivity at low parasite densities, susceptibility to the prozone effect (PfHRP2-detecting RDTs), false-negative results due to PfHRP2 deficiency in the case of pfhrp2 gene deletions (PfHRP2-detecting RDTs), cross-reactions between Plasmodium antigens and detection antibodies, false-positive results by other infections and susceptibility to heat and humidity. End-user's errors relate to safety, procedure (delayed reading, incorrect sample and buffer volumes) and interpretation (not recognizing invalid test results, disregarding faint test lines). Withholding antimalarial treatment in the case of negative RDT results tends to be infrequent and tendencies towards over-prescription of antibiotics have been noted. Numerous shortcomings in RDT kits' labelling, instructions for use (correctness and readability) and contents have been observed. The World Health Organization and partners actively address quality assurance of RDTs by comparative testing of RDTs, inspections of manufacturing sites, lot testing and training tools but no formal external quality assessment programme of end-user performance exists. Elimination of malaria requires RDTs with lower detection limits, for which nucleic acid amplification tests are under development. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

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.

Performance of the fourth-generation Bio-Rad GS HIV Combo Ag/Ab enzyme immunoassay for diagnosis of HIV infection in Southern Africa

PubMed Central

Piwowar-Manning, Estelle; Fogel, Jessica M.; Richardson, Paul; Wolf, Shauna; Clarke, William; Marzinke, Mark A.; Fiamma, Agnès; Donnell, Deborah; Kulich, Michal; Mbwambo, Jessie K.K.; Richter, Linda; Gray, Glenda; Sweat, Michael; Coates, Thomas J.; Eshleman, Susan H.

2015-01-01

Background Fourth-generation HIV assays detect both antigen and antibody, facilitating detection of acute/early HIV infection. The Bio-Rad GS HIV Combo Ag/Ab assay (Bio-Rad Combo) is an enzyme immunoassay that simultaneously detects HIV p24 antigen and antibodies to HIV-1 and HIV-2 in serum or plasma. Objective To evaluate the performance of the Bio-Rad Combo assay for detection of HIV infection in adults from Southern Africa. Study design Samples were obtained from adults in Soweto and Vulindlela, South Africa and Dar es Salaam, Tanzania (300 HIV-positive samples; 300 HIV-negative samples; 12 samples from individuals previously classified as having acute/early HIV infection). The samples were tested with the Bio-Rad Combo assay. Additional testing was performed to characterize the 12 acute/early samples. Results All 300 HIV-positive samples were reactive using the Bio-Rad Combo assay; false positive test results were obtained for 10 (3.3%) of the HIV-negative samples (sensitivity: 100%, 95% confidence interval [CI]: 98.8–100%); specificity: 96.7%, 95% CI: 94.0–98.4%). The assay detected 10 of the 12 infections classified as acute/early. The two infections that were not detected had viral loads < 400 copies/mL; one of those samples contained antiretroviral drugs consistent with antiretroviral therapy. Conclusions The Bio-Rad Combo assay correctly classified the majority of study specimens. The specificity reported here may be higher than that seen in other settings, since HIV-negative samples were pre-screened using a different fourth-generation test. The assay also had high sensitivity for detection of acute/early infection. False-negative test results may be obtained in individuals who are virally suppressed. PMID:25542477

Design, fabrication, and characterization of polymeric bioMEMS for the detection of feline immunodeficiency virus (FIV)

NASA Astrophysics Data System (ADS)

Cohen, Brian; Gadre, Anand; Kaloyeros, Alain E.

2007-02-01

This project comprises the development of a novel polymeric BioMEMS device capable of rapidly detecting FIV in a minimally invasive manner. FIV severely inhibits the infected feline from mounting an immune response, and causes susceptibility to other types of diseases. Vaccines against FIV do exist, but have some strong limitations to their effectiveness; so early detection is the best method to combat the spread of the disease. Current testing methods look for antibodies to the FIV protein p24 in feline blood using established Enzyme Linked ImmunoSorbent Assay (ELISA) protocols. The focus of this research is to design and construct a device that can detect antibodies to p24 in a salivary sample by non-intrusive electrochemical means. The device is constructed upon a silicon substrate with gold microelectrodes coated with polypyrrole (PPy), an electrically conducting and biocompatible polymer. In the current phase of the research, the PPy deposition process has been optimized with regards to film thickness, uniformity and conductivity. Microfluidic channels have been fabricated using SU-8, an epoxy based polymer that enables the test sample and other solutions to pass freely through the device. The PPy will be coated with anti-FIV p24 antibodies that can capture FIV p24 antigens present in a salivary sample. Future research will involve the analysis of PPy/antibody interaction and its effect on functionality. The capture of such antigens will interfere with a reduction-oxidation (redox) reaction in a subsequently added ionic solution. This interference will change the characteristic resistance of the solution yielding a qualitative test for the presence of the viral antigens in the sample and hence determining the occurrence of infection.

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 Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites

PubMed Central

Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

2016-01-01

Background Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Methodology/Principal Findings Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Conclusions/Significance Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species. PMID:26986566

Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

PubMed

Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

2016-03-01

Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

Comparison of Xpert Flu rapid nucleic acid testing with rapid antigen testing for the diagnosis of influenza A and B.

PubMed

DiMaio, Michael A; Sahoo, Malaya K; Waggoner, Jesse; Pinsky, Benjamin A

2012-12-01

Influenza infections are associated with thousands of hospital admissions and deaths each year. Rapid detection of influenza is important for prompt initiation of antiviral therapy and appropriate patient triage. In this study the Cepheid Xpert Flu assay was compared with two rapid antigen tests, BinaxNOW Influenza A & B and BD Directigen EZ Flu A+B, as well as direct fluorescent antibody testing for the rapid detection of influenza A and B. Using real-time, hydrolysis probe-based, reverse transcriptase PCR as the reference method, influenza A sensitivity was 97.3% for Xpert Flu, 95.9% for direct fluorescent antibody testing, 62.2% for BinaxNOW, and 71.6% for BD Directigen. Influenza B sensitivity was 100% for Xpert Flu and direct fluorescent antibody testing, 54.5% for BinaxNOW, and 48.5% for BD Directigen. Specificity for influenza A was 100% for Xpert Flu, BinaxNOW, and BD Directigen, and 99.2% for direct fluorescent antibody testing. All methods demonstrated 100% specificity for influenza B. These findings support the use of the Xpert Flu assay in settings requiring urgent diagnosis of influenza A and B. Copyright © 2012 Elsevier B.V. All rights reserved.

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

[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.

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.

Antigenemia in Patients with Paracoccidioidomycosis: Detection of the 87-Kilodalton Determinant during and after Antifungal Therapy

PubMed Central

Gómez, B. L.; Figueroa, J. I.; Hamilton, A. J.; Diez, S.; Rojas, M.; Tobón, A. M.; Hay, R. J.; Restrepo, A.

1998-01-01

Serological diagnosis and follow-up of paracoccidioidomycosis (PCM) patients have relied mainly on the detection of antibody responses by using techniques such as complement fixation (CF) and immunodiffusion. We recently described a novel inhibition enzyme-linked immunosorbent assay (inh-ELISA) which proved to be useful in the diagnosis of PCM via the detection of an 87-kDa determinant in patient sera (B. L. Gomez, J. I. Figueroa, A. J. Hamilton, B. Ortiz, M. A. Robledo, R. J. Hay, and A. Restrepo, J. Clin. Microbiol. 35:3278–3283, 1997). This test has now been assessed as a means of following up PCM patients. A total of 24 PCM patients, classified according to their clinical presentation (6 with the acute form of the disease, of whom two had AIDS, 12 with the multifocal form of the disease, and 6 with the unifocal form of the disease), were studied. The four human immunodeficiency virus-negative patients with acute PCM showed a statistically significant decrease in circulating antigen levels after the start of antifungal therapy. Antigen levels in this group became negative by our criteria (≤2.3 μg/ml) before week 20 and remained so in three of four of these patients. In contrast, the two AIDS patients who also presented with the acute form of PCM showed no statistically significant decrease in circulating antigen levels even after 68 weeks of therapy. Taken together as a group, the patients with the multifocal form showed a statistically significant decrease in antigenemia after 28 weeks of therapy. In addition, five of six patients with the unifocal form became antigen negative by week 40. Antigen level decrease mirrored clinical cure in the majority of patients in all clinical groups; in contrast, measurement of anti-PCM antibodies via the CF test showed wide fluctuations in titers during the follow-up period. The inh-ELISA for the detection of the 87-kDa Paracoccidioides brasiliensis determinant would appear to be a valuable additional tool in the follow-up of PCM patients. PMID:9774584

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.

Analytical Validation of the ReEBOV Antigen Rapid Test for Point-of-Care Diagnosis of Ebola Virus Infection.

PubMed

Cross, Robert W; Boisen, Matthew L; Millett, Molly M; Nelson, Diana S; Oottamasathien, Darin; Hartnett, Jessica N; Jones, Abigal B; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Bornholdt, Zachary A; Fusco, Marnie L; Abelson, Dafna M; Oda, Shunichiro; Brown, Bethany L; Pham, Ha; Rowland, Megan M; Agans, Krystle N; Geisbert, Joan B; Heinrich, Megan L; Kulakosky, Peter C; Shaffer, Jeffrey G; Schieffelin, John S; Kargbo, Brima; Gbetuwa, Momoh; Gevao, Sahr M; Wilson, Russell B; Saphire, Erica Ollmann; Pitts, Kelly R; Khan, Sheik Humarr; Grant, Donald S; Geisbert, Thomas W; Branco, Luis M; Garry, Robert F

2016-10-15

Ebola virus disease (EVD) is a severe viral illness caused by Ebola virus (EBOV). The 2013-2016 EVD outbreak in West Africa is the largest recorded, with >11 000 deaths. Development of the ReEBOV Antigen Rapid Test (ReEBOV RDT) was expedited to provide a point-of-care test for suspected EVD cases. Recombinant EBOV viral protein 40 antigen was used to derive polyclonal antibodies for RDT and enzyme-linked immunosorbent assay development. ReEBOV RDT limits of detection (LOD), specificity, and interference were analytically validated on the basis of Food and Drug Administration (FDA) guidance. The ReEBOV RDT specificity estimate was 95% for donor serum panels and 97% for donor whole-blood specimens. The RDT demonstrated sensitivity to 3 species of Ebolavirus (Zaire ebolavirus, Sudan ebolavirus, and Bundibugyo ebolavirus) associated with human disease, with no cross-reactivity by pathogens associated with non-EBOV febrile illness, including malaria parasites. Interference testing exhibited no reactivity by medications in common use. The LOD for antigen was 4.7 ng/test in serum and 9.4 ng/test in whole blood. Quantitative reverse transcription-polymerase chain reaction testing of nonhuman primate samples determined the range to be equivalent to 3.0 × 10 5 -9.0 × 10 8 genomes/mL. The analytical validation presented here contributed to the ReEBOV RDT being the first antigen-based assay to receive FDA and World Health Organization emergency use authorization for this EVD outbreak, in February 2015. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

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

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.

Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

PubMed

Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

2016-08-01

Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Expression of myeloid differentiation antigens on normal and malignant myeloid cells.

PubMed Central

Griffin, J D; Ritz, J; Nadler, L M; Schlossman, S F

1981-01-01

A series of monoclonal antibodies have been characterized that define four surface antigens (MY3, MY4, MY7, and MY8) of human myeloid cells. They were derived from a fusion of the NS-1 plasmacytoma cell line with splenocytes from a mouse immunized with human acute myelomonocytic leukemia cells. MY3 and MY4 are expressed by normal monocytes and by greater than 90% of patients with acute monocytic leukemia or acute myelomonocytic leukemia, but are detected much less often on other types of myeloid leukemia. MY7 is expressed by granulocytes, monocytes, and 5% of normal bone marrow cells. 79% of all acute myeloblastic leukemia (AML) patients tested (72 patients) express MY7 without preferential expression by any AML subtype. MY8 is expressed by normal monocytes, granulocytes, all peroxidase-positive bone marrow cells, and 50% of AML patients. MY3, MY4, and MY8 define myeloid differentiation antigens in that they are not detected on myeloid precursor cells and appear at discrete stages of differentiation. These antigens are not expressed by lymphocytes, erythrocytes, platelets, or lymphoid malignancies. The monoclonal antisera defining these antigens have been used to study differentiation of normal myeloid cells and malignant cell lines. Images PMID:6945311

An optimized immunohistochemistry protocol for detecting the guidance cue Netrin-1 in neural tissue.

PubMed

Salameh, Samer; Nouel, Dominique; Flores, Cecilia; Hoops, Daniel

2018-01-01

Netrin-1, an axon guidance protein, is difficult to detect using immunohistochemistry. We performed a multi-step, blinded, and controlled protocol optimization procedure to establish an efficient and effective fluorescent immunohistochemistry protocol for characterizing Netrin-1 expression. Coronal mouse brain sections were used to test numerous antigen retrieval methods and combinations thereof in order to optimize the stain quality of a commercially available Netrin-1 antibody. Stain quality was evaluated by experienced neuroanatomists for two criteria: signal intensity and signal-to-noise ratio. After five rounds of testing protocol variants, we established a modified immunohistochemistry protocol that produced a Netrin-1 signal with good signal intensity and a high signal-to-noise ratio. The key protocol modifications are as follows: •Use phosphate buffer (PB) as the blocking solution solvent.•Use 1% sodium dodecyl sulfate (SDS) treatment for antigen retrieval. The original protocol was optimized for use with the Netrin-1 antibody produced by Novus Biologicals. However, we subsequently further modified the protocol to work with the antibody produced by Abcam. The Abcam protocol uses PBS as the blocking solution solvent and adds a citrate buffer antigen retrieval step.

A New Intrusion Detection Method Based on Antibody Concentration

NASA Astrophysics Data System (ADS)

Zeng, Jie; Li, Tao; Li, Guiyang; Li, Haibo

Antibody is one kind of protein that fights against the harmful antigen in human immune system. In modern medical examination, the health status of a human body can be diagnosed by detecting the intrusion intensity of a specific antigen and the concentration indicator of corresponding antibody from human body’s serum. In this paper, inspired by the principle of antigen-antibody reactions, we present a New Intrusion Detection Method Based on Antibody Concentration (NIDMBAC) to reduce false alarm rate without affecting detection rate. In our proposed method, the basic definitions of self, nonself, antigen and detector in the intrusion detection domain are given. Then, according to the antigen intrusion intensity, the change of antibody number is recorded from the process of clone proliferation for detectors based on the antigen classified recognition. Finally, building upon the above works, a probabilistic calculation method for the intrusion alarm production, which is based on the correlation between the antigen intrusion intensity and the antibody concen-tration, is proposed. Our theoretical analysis and experimental results show that our proposed method has a better performance than traditional methods.

SNAP Assay Technology.

PubMed

O'Connor, Thomas P

2015-12-01

The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Validation of a urine circulating cathodic antigen cassette test for detection of Schistosoma haematobiumin uMkhanyakude district of South Africa.

PubMed

Rubaba, O; Chimbari, M J; Soko, W; Manyangadze, T; Mukaratirwa, S

2018-06-01

Circulating cathodic antigen (CCA) tests for schistosomiasis are fast and less complicated allowing making them good candidates for routine qualitative screening for schistosomiasis at point of care. The urine-CCA has been evaluated for detection of S. mansoni with promising results. Its specificity and consistency in detecting S. haematobium infection in different endemic regions has been variable. This study validated a rapid urine-CCA cassette test for qualitative detection of S. haematobium infection in an S. haematobium endemic area with low S. mansoni prevalence. Microscopic examination for the standard urine filtration technique was used to validate the commercially available urine-CCA cassette test (rapid medical diagnostics ® ). The validation was done in a sample of primary school pupils (n = 420) aged 10-15 years in schools in the Jozini Municipality, KZN. There was a relationship between infection intensity and a positive urine-CCA test. Using the urine filtration method as the gold standard, the prevalence for S. haematobium was 40%, the accuracy of the CCA kit was 54.8%, sensitivity was 68.1% while the specificity was 45.8%. The positive predictive value was 45.82% while the negative predictive value was 68.05%. Both the urine filtration and the urine-CCA methods detected heavy (≥50 eggs/10 mL urine) and light infections at statistically significant levels. The overall accuracy, sensitivity and specificity of the urine-CCA cassette test were low. The urine-CCA cassette test performed much better for heavy infections than low infections (p < 0.05) implying that the kit may not be suitable for low endemic areas. Copyright © 2018 Elsevier B.V. All rights reserved.

Recombinant blood group proteins for use in antibody screening and identification tests.

PubMed

Seltsam, Axel; Blasczyk, Rainer

2009-11-01

The present review elucidates the potentials of recombinant blood group proteins (BGPs) for red blood cell (RBC) antibody detection and identification in pretransfusion testing and the achievements in this field so far. Many BGPs have been eukaryotically and prokaryotically expressed in sufficient quantity and quality for RBC antibody testing. Recombinant BGPs can be incorporated in soluble protein reagents or solid-phase assays such as ELISA, color-coded microsphere and protein microarray chip-based techniques. Because novel recombinant protein-based assays use single antigens, a positive reaction of a serum with the recombinant protein directly indicates the presence and specificity of the target antibody. Inversely, conventional RBC-based assays use panels of human RBCs carrying a huge number of blood group antigens at the same time and require negative reactions of samples with antigen-negative cells for indirect determination of antibody specificity. Because of their capacity for single-step, direct RBC antibody determination, recombinant protein-based assays may greatly facilitate and accelerate the identification of common and rare RBC antibodies.

Operational accuracy and comparative persistent antigenicity of HRP2 rapid diagnostic tests for Plasmodium falciparum malaria in a hyperendemic region of Uganda.

PubMed

Kyabayinze, Daniel J; Tibenderana, James K; Odong, George W; Rwakimari, John B; Counihan, Helen

2008-10-29

Parasite-based diagnosis of malaria by microscopy requires laboratory skills that are generally unavailable at peripheral health facilities. Rapid diagnostic tests (RDTs) require less expertise, but accuracy under operational conditions has not been fully evaluated in Uganda. There are also concerns about RDTs that use the antigen histidine-rich protein 2 (HRP2) to detect Plasmodium falciparum, because this antigen can persist after effective treatment, giving false positive test results in the absence of infection. An assessment of the accuracy of Malaria Pf immuno-chromatographic test (ICT) and description of persistent antigenicity of HRP2 RDTs was undertaken in a hyperendemic area of Uganda. Using a cross-sectional design, a total of 357 febrile patients of all ages were tested using ICT, and compared to microscopy as the gold standard reference. Two independent RDT readings were used to assess accuracy and inter-observer reliability. With a longitudinal design to describe persistent antigenicity of ICT and Paracheck, 224 children aged 6-59 months were followed up at 7-day intervals until the HRP2 antigens where undetectable by the RDTs. Of the 357 patients tested during the cross-sectional component, 40% (139) had positive blood smears for asexual forms of P. falciparum. ICT had an overall sensitivity of 98%, a specificity of 72%, a negative predictive value (NPV) of 98% and a positive predictive value (PPV) of 69%. ICT showed a high inter-observer reliability under operational conditions, with 95% of readings having assigned the same results (kappa statistics 0.921, p < 0.001). In children followed up after successful antimalaria treatment, the mean duration of persistent antigenicity was 32 days, and this duration varied significantly depending on pre-treatment parasitaemia. In patients with parasite density >50,000/microl, the mean duration of persistent antigenicity was 37 days compared to 26 days for parasitaemia less than 1,000/microl (log rank 21.9, p < 0.001). ICT is an accurate and appropriate test for operational use as a diagnostic tool where microscopy is unavailable. However, persistent antigenicity reduces the accuracy of this and other HRP2-based RDTs. The low specificity continues to be of concern, especially in children below five years of age. These pose limitations that need consideration, such as their use for diagnosis of patients returning with symptoms within two to four weeks of treatment. Good clinical skills are essential to interpret test results.

Operational accuracy and comparative persistent antigenicity of HRP2 rapid diagnostic tests for Plasmodium falciparum malaria in a hyperendemic region of Uganda

PubMed Central

Kyabayinze, Daniel J; Tibenderana, James K; Odong, George W; Rwakimari, John B; Counihan, Helen

2008-01-01

Background Parasite-based diagnosis of malaria by microscopy requires laboratory skills that are generally unavailable at peripheral health facilities. Rapid diagnostic tests (RDTs) require less expertise, but accuracy under operational conditions has not been fully evaluated in Uganda. There are also concerns about RDTs that use the antigen histidine-rich protein 2 (HRP2) to detect Plasmodium falciparum, because this antigen can persist after effective treatment, giving false positive test results in the absence of infection. An assessment of the accuracy of Malaria Pf™ immuno-chromatographic test (ICT) and description of persistent antigenicity of HRP2 RDTs was undertaken in a hyperendemic area of Uganda. Methods Using a cross-sectional design, a total of 357 febrile patients of all ages were tested using ICT, and compared to microscopy as the gold standard reference. Two independent RDT readings were used to assess accuracy and inter-observer reliability. With a longitudinal design to describe persistent antigenicity of ICT and Paracheck, 224 children aged 6–59 months were followed up at 7-day intervals until the HRP2 antigens where undetectable by the RDTs. Results Of the 357 patients tested during the cross-sectional component, 40% (139) had positive blood smears for asexual forms of P. falciparum. ICT had an overall sensitivity of 98%, a specificity of 72%, a negative predictive value (NPV) of 98% and a positive predictive value (PPV) of 69%. ICT showed a high inter-observer reliability under operational conditions, with 95% of readings having assigned the same results (kappa statistics 0.921, p < 0.001). In children followed up after successful antimalaria treatment, the mean duration of persistent antigenicity was 32 days, and this duration varied significantly depending on pre-treatment parasitaemia. In patients with parasite density >50,000/μl, the mean duration of persistent antigenicity was 37 days compared to 26 days for parasitaemia less than 1,000/μl (log rank 21.9, p < 0.001). Conclusion ICT is an accurate and appropriate test for operational use as a diagnostic tool where microscopy is unavailable. However, persistent antigenicity reduces the accuracy of this and other HRP2-based RDTs. The low specificity continues to be of concern, especially in children below five years of age. These pose limitations that need consideration, such as their use for diagnosis of patients returning with symptoms within two to four weeks of treatment. Good clinical skills are essential to interpret test results. PMID:18959777

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.

How should a district general hospital immunology service screen for anti-nuclear antibodies? An ‘in-the-field’ audit

PubMed Central

Hira-Kazal, R; Shea-Simonds, P; Peacock, J L; Maher, J

2015-01-01

Anti-nuclear antibody (ANA) testing assists in the diagnosis of several immune-mediated disorders. The gold standard method for detection of these antibodies is by indirect immunofluorescence testing on human epidermoid laryngeal carcinoma (HEp-2) cells. However, many laboratories test for these antibodies using solid-phase assays such as enzyme-linked immunosorbent assay (ELISA), which allows for higher throughput testing at reduced cost. In this study, we have audited the performance of a previously established ELISA assay to screen for ANA, making comparison with the gold standard HEp-2 immunofluorescence test. A prospective and unselected sample of 89 consecutive ANA test requests by consultant rheumatologists were evaluated in parallel over a period of 10 months using both tests. ELISA and HEp-2 screening assays yielded 40 (45%) and 72 (81%) positive test results, respectively, demonstrating lack of concordance between test methods. Using standard and clinical samples, it was demonstrated that the ELISA method did not detect several ANA with nucleolar, homogeneous and speckled immunofluorescence patterns. None of these ELISANEG HEp-2POS ANA were reactive with a panel of six extractable nuclear antigens or with double-stranded DNA. Nonetheless, 13 of these samples (15%) originated from patients with recognized ANA-associated disease (n = 7) or Raynaud's phenomenon (n = 6). We conclude that ELISA screening may fail to detect clinically relevant ANA that lack defined specificity for antigen. PMID:25412573

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

A comparative study of the diagnostic methods for Group A streptococcal sore throat in two reference hospitals in Yaounde, Cameroon

PubMed Central

Gonsu, Hortense Kamga; Bomki, Cynthia Mbimenyuy; Djomou, François; Toukam, Michel; Ndze, Valantine Ngum; Lyonga, Emilia Enjema; Mbakop, Calixte Didier; Koulla-Shiro, Sinata

2015-01-01

Introduction Sore throat is a common complaint in general practice which is more frequent in children. The most frequent pathogenic bacteria associated with this infection is Streptococcus pyogenes. Rapid Antigen Diagnostic Test (RADT) facilitates the rapid identification and consequently prompt treatment of patients, prevents complications, and also reduces the risk of spread of Group A Streptococcus (GAS). The main objective of this study was to assess the diagnostic value of a rapid streptococcal antigen detection test in patients with sore throat. Methods A cross-sectional descriptive study was carried out from January to April 2011 on patients aged 3 to 72 years consulting for pharyngitis or sore throat at the paediatric and Ear, Nose and Throat units of the University Teaching Hospital Yaounde and the Central Hospital Yaounde. Two throat swabs were collected per patient. One was used for the rapid test and the other for standard bacteriological analysis. Results The prevalence of GAS in the study population was 22.5%. Out of the 71 samples collected, the RADT detected group A streptococcal antigens in 12 of 16 positive cultures giving a sensitivity of 75%. The specificity of the rapid test was 96%, with positive predictive value of 85.7%, and negative predictive value of 93% respectively. Conclusion Rapid test may have an additional value in the management of patients with high risk of having GAS infection. However, tests with a higher sensitivity are needed for accurate and reliable results for early diagnosis of patients with sore throat caused by GAS. PMID:27386017

A comparative study of the diagnostic methods for Group A streptococcal sore throat in two reference hospitals in Yaounde, Cameroon.

PubMed

Gonsu, Hortense Kamga; Bomki, Cynthia Mbimenyuy; Djomou, François; Toukam, Michel; Ndze, Valantine Ngum; Lyonga, Emilia Enjema; Mbakop, Calixte Didier; Koulla-Shiro, Sinata

2015-01-01

Sore throat is a common complaint in general practice which is more frequent in children. The most frequent pathogenic bacteria associated with this infection is Streptococcus pyogenes. Rapid Antigen Diagnostic Test (RADT) facilitates the rapid identification and consequently prompt treatment of patients, prevents complications, and also reduces the risk of spread of Group A Streptococcus (GAS). The main objective of this study was to assess the diagnostic value of a rapid streptococcal antigen detection test in patients with sore throat. A cross-sectional descriptive study was carried out from January to April 2011 on patients aged 3 to 72 years consulting for pharyngitis or sore throat at the paediatric and Ear, Nose and Throat units of the University Teaching Hospital Yaounde and the Central Hospital Yaounde. Two throat swabs were collected per patient. One was used for the rapid test and the other for standard bacteriological analysis. The prevalence of GAS in the study population was 22.5%. Out of the 71 samples collected, the RADT detected group A streptococcal antigens in 12 of 16 positive cultures giving a sensitivity of 75%. The specificity of the rapid test was 96%, with positive predictive value of 85.7%, and negative predictive value of 93% respectively. Rapid test may have an additional value in the management of patients with high risk of having GAS infection. However, tests with a higher sensitivity are needed for accurate and reliable results for early diagnosis of patients with sore throat caused by GAS.

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.

Physiological and Antigenic Characteristics of Virulent and Attenuated Strains of Legionella pneumophila (Philadelphia 3)

DTIC Science & Technology

1981-07-01

serogroups of Legionnaires ’ disease bacteria defined by immunofluorescence. Ann. Intern. Med. 90:621-624. 17. Ristroph, J. D., K. W. Hedlund, and S ...major antigens isolated from the Legionnaires ’ disease bacterium. Ann. Intern. Med. 90:634-638. 21. Wong, M. C., E. P. Ewing, Jr., C. S . Callaway, and W...J. C. Feeley. 1978. A microagglutination test for detecting antibodies, p. 163-168. In C. L. Jones and G. A. Hebert (ed.), " Legionnaires " the disease

9 CFR 145.15 - Diagnostic surveillance program for low pathogenic avian influenza.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Diagnostic surveillance program for low pathogenic avian influenza. 145.15 Section 145.15 Animals and Animal Products ANIMAL AND PLANT... antigen detection test. Memoranda of understanding or other means must be used to establish testing and...

9 CFR 145.15 - Diagnostic surveillance program for low pathogenic avian influenza.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Diagnostic surveillance program for low pathogenic avian influenza. 145.15 Section 145.15 Animals and Animal Products ANIMAL AND PLANT... antigen detection test. Memoranda of understanding or other means must be used to establish testing and...

9 CFR 145.15 - Diagnostic surveillance program for low pathogenic avian influenza.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Diagnostic surveillance program for low pathogenic avian influenza. 145.15 Section 145.15 Animals and Animal Products ANIMAL AND PLANT... antigen detection test. Memoranda of understanding or other means must be used to establish testing and...

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.

External quality assessment of dengue and chikungunya diagnostics in the Asia Pacific region, 2015

PubMed Central

Soh, Li Ting; Squires, Raynal C; Tan, Li Kiang; Pok, Kwoon Yong; Yang, HuiTing; Liew, Christina; Shah, Aparna Singh; Aaskov, John; Abubakar, Sazaly; Hasabe, Futoshi; Ng, Lee Ching

2016-01-01

Objective To conduct an external quality assessment (EQA) of dengue and chikungunya diagnostics among national-level public health laboratories in the Asia Pacific region following the first round of EQA for dengue diagnostics in 2013. Methods Twenty-four national-level public health laboratories performed routine diagnostic assays on a proficiency testing panel consisting of two modules. Module A contained serum samples spiked with cultured dengue virus (DENV) or chikungunya virus (CHIKV) for the detection of nucleic acid and DENV non-structural protein 1 (NS1) antigen. Module B contained human serum samples for the detection of anti-DENV antibodies. Results Among 20 laboratories testing Module A, 17 (85%) correctly detected DENV RNA by reverse transcription polymerase chain reaction (RT–PCR), 18 (90%) correctly determined serotype and 19 (95%) correctly identified CHIKV by RT–PCR. Ten of 15 (66.7%) laboratories performing NS1 antigen assays obtained the correct results. In Module B, 18/23 (78.3%) and 20/20 (100%) of laboratories correctly detected anti-DENV IgM and IgG, respectively. Detection of acute/recent DENV infection by both molecular (RT–PCR) and serological methods (IgM) was available in 19/24 (79.2%) participating laboratories. Discussion Accurate laboratory testing is a critical component of dengue and chikungunya surveillance and control. This second round of EQA reveals good proficiency in molecular and serological diagnostics of these diseases in the Asia Pacific region. Further comprehensive diagnostic testing, including testing for Zika virus, should comprise future iterations of the EQA. PMID:27508088

Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization.

PubMed

Okamoto-Uchida, Yoshimi; Nakamura, Ryosuke; Matsuzawa, Yumiko; Soma, Megumi; Kawakami, Hiroshi; Ishii-Watabe, Akiko; Nishimaki-Mogami, Tomoko; Teshima, Reiko; Saito, Yoshiro

2016-01-01

The physicochemical nature of allergen molecules differ from the liquid phase to the solid phase. However, conventional allergy tests are based on the detection of immunoglobulin (Ig)E binding to immobilized allergens. We recently developed an in vitro allergy testing method using a luciferase-reporting humanized rat mast cell line to detect IgE crosslinking-induced luciferase expression (EXiLE test). The aim of the present study was to evaluate the effects of antigen immobilization on the results of different in vitro allergy tests using two anti-ovalbumin (OVA) antibodies (Abs), E-C1 and E-G5, with different properties in the OVA-induced allergic reaction. Both Abs showed clear binding to OVA with an enzyme-linked immunosorbent assay and by BIAcore analysis. However, only E-C1 potentiated EXiLE response for the liquid-phase OVA. On the other hand, OVA immobilized on solid-phase induced EXiLE responses in both E-C1 Ab- and E-G5 Ab-sensitized mast cells. Western blotting of OVA indicated that E-C1 Ab binds both to OVA monomers and dimers, unlike E-G5 Ab, which probably binds only to the OVA dimer. These results suggest that antigen immobilization enhanced IgE crosslinking ability through multimerization of allergen molecules in the solid phase, resulting in an increase in false positives in IgE binding-based conventional in vitro allergy tests. These findings shed light on the physicochemical nature of antigens as an important factor for the development and evaluation of in vitro allergy tests and suggest that mast cell activation-based allergy testing with liquid-phase allergens is a promising strategy to evaluate the physiological interactions of IgE and allergens.

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

PubMed

Carrio, Roberto; Zhang, Ge; Drake, Donald R; Schanen, Brian C

2018-05-07

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4 + T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4 + T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4 + T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4 + T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

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.

Detection of antibodies to both M. leprae PGL-I and MMP-II to recognize leprosy patients at an early stage of disease progression.

PubMed

Wang, Hongsheng; Liu, Weijing; Jin, Yali; Yu, Meiwen; Jiang, Haiqin; Tamura, Toshiki; Maeda, Yumi; Makino, Masahiko

2015-11-01

Antibodies to phenolic glycolipid (PGL)-I and major membrane protein (MMP)-II were evaluated for serodiagnosis of leprosy in Southwest China, and the role in predicting the occurrence of the disease in household contacts (HHCs) of leprosy was examined. Using PGL-I (natural disaccharide-octyl-bovine serum albumin) antigen-based diagnosis (IgM antibodies), we could detect 94.9% of multibacillary (MB) leprosy and 38.9% paucibacillary (PB) leprosy patients, whereas using MMP-II (IgG antibody), 88.1% of MB and 61.1% of PB patients were positive. By combining the 2 tests and considering either test positive as positive, 100% of MB patients and 72.2% of PB patients were found to test positive. Of the HHCs of leprosy, 28.3% and 30% had positive levels of PGL-I and MMP-II Abs, respectively. Seven out of 21 HHCs, who had high Ab titer to either antigen, developed leprosy during the follow-up period of 3 years. These data suggest that the measurement of both anti-PGL-I as well as anti-MMP-II antibodies could facilitate early detection of leprosy. Copyright © 2015 Elsevier Inc. All rights reserved.

Detection of intracellular canine distemper virus antigen in mink inoculated with an attenuated or a virulent strain of canine distemper virus.

PubMed

Blixenkrone-Møller, M

1989-09-01

Using an indirect immunofluorescence technique, the distribution of viral antigen in various tissues and blood mononuclear leukocytes was studied in wild mink, either vaccinated with an attenuated vaccine strain of canine distemper virus (CDV) or experimentally inoculated with the virulent Snyder-Hill strain of CDV. Viral antigen was detected in cells of the lymphoid system 6 to 12 days after vaccination. From 2 to 3 days after inoculation with the virulent strain, CDV antigen was demonstrated in cells of the lymphoid system and, during the incubation period, the antigen had spread to the epithelia and brain at days 6 and 12, respectively. In clinical cases of acute fatal canine distemper, the viral antigen was detected in a wide variety of tissues, including the cells of the lymphoid system, epithelial cells of skin, mucous membranes, lung, kidney, and cells of the CNS. The diagnostic importance of CDV antigen detection is discussed on the basis of these findings.

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.

Peste des petits ruminants infection in domestic ruminants in Sudan.

PubMed

Intisar, K S; Ali, Y H; Haj, M A; Sahar, M A T; Shaza, M M; Baraa, A M; Ishag, O M; Nouri, Y M; Taha, K M; Nada, E M; Ahmed, A M; Khalafalla, A I; Libeau, G; Diallo, A

2017-04-01

The existence of peste des petits ruminants (PPR) in domestic ruminants and camels in Sudan during 2008-2012 was investigated. Lung tissues and serum samples were randomly collected from sheep, goats, cattle, and camels at different areas of Sudan. A total of 12,384 serum samples were collected from clinically healthy 7413 sheep, 1988 camels, 1501 cattle, 1459 goats, and 23 gazelles at different areas in the Sudan. They were examined for PPR antibodies using competitive ELISA (cELISA). The overall detected seroprevalence of PPR in tested sera was 49.4%; seroprevalence values within species were 67.1, 48.2, 25.8, 2.1, and 21.7% in sheep, goat, cattle, camels, and gazelles, respectively. The highest seroprevalence (68.1%) was observed in sera collected from Darfur states, then the central states (54.3%). A total of 1276 lung tissue samples (623 sheep, 324 cattle, 220 camels, and 109 goats) were collected. The majority of lung samples were collected from clinically healthy animals that showed lesions on PM in slaughterhouses (95%) and during PPR outbreaks; samples were tested for PPR antigen using immunocapture ELISA (IcELISA). PPR antigen was detected in 233 out of the 1276 tested samples (18.3%). Positive results were observed in samples collected from clinically healthy and diseased animals. The observed prevalence values in each species were 33.6, 21.1, 15.4, and 12.3% in camel, goat, sheep, and cattle, respectively. PPR antigen was detected in samples from different areas; however, the highest prevalence (63.9%) was found in samples collected from the eastern states, then Khartoum state (28%). Trials for virus isolation were done in different cell cultures. Out of 30 IcELISA-positive samples inoculated in primary bovine and ovine kidney cells, Vero cells, the PPR virus was successfully isolated from 15 (eight sheep, five camels, and two goats) samples in the three cell culture types. Using RT-PCR, PPRV nucleic acid was detected in all 25 IcELISA-positive tested samples.

Clinical significance and diagnostic capacity of serum TK1, CEA, CA 19-9 and CA 72-4 levels in gastric and colorectal cancer patients.

PubMed

Ning, Shufang; Wei, Wene; Li, Jilin; Hou, Bingbing; Zhong, Jianhong; Xie, Yuxuan; Liu, Haizhou; Mo, Xianwei; Chen, Jiansi; Zhang, Litu

2018-01-01

Despite extensive progress in treatment for cancer in recent decades, the early diagnosis for gastric cancer (GC) and colorectal cancer (CRC) remains poor. In this study, we explore the diagnostic value of joint detection of thymidine kinase 1 (TK1), carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA 19-9) and carbohydrate antigen 72-4 (CA 72-4) in the diagnosis of GC and CRC, and to evaluated the relationship between TK1 expression and clinical pathological characteristics in the patients. Serum TK1, CA 19-9, CA 72-4 and CEA levels were measured in 169 patients with GC, 344 patients with CRC and 75 healthy controls using electro-chemiluminescence. The TK1 concentration was significantly higher in patients with cancer than in healthy controls and patients with clinical stage Ⅲ+Ⅳ had higher TK1 levels than clinical stage Ⅰ+Ⅱ ( P <0.05). The levels of TK1 is significantly associated with tumor stage, lymph node metastasis, distant metastasis, tumor differentiation and age ( P <0.05). When the tumor markers (TK1, CA 19-9 and CA 72-4) were detected respectively, the area under receiver operating characteristics curve (AUC) of TK1 for three cancers was the highest (0.823-0.895). However, the combination of AUC was higher than that for each tumor marker detected respectively (0.934-0.953), and the Hosmer-Lemeshow test showed an adequate model of calibration (P>0.05). Moreover, the AUCs varied significantly between the combination tests and single biomarker tests (Z test, P <0.01). In conclusion, serum TK1 may be an independent tumor marker for GC and CRC patients, and the combination of TK1, CA 19-9 and CA 72-4 and CEA performed even better. This study suggests that combination detection of four tumor markers may prove to be useful for the diagnosis of GC and CRC.

Mapping of epitopes and structural analysis of antigenic sites in the nucleoprotein of rabies virus.

PubMed

Goto, H; Minamoto, N; Ito, H; Ito, N; Sugiyama, M; Kinjo, T; Kawai, A

2000-01-01

Linear epitopes on the rabies virus nucleoprotein (N) recognized by six MAbs raised against antigenic sites I (MAbs 6-4, 12-2 and 13-27) and IV (MAbs 6-9, 7-12 and 8-1) were investigated. Based on our previous studies on sites I and IV, 24 consecutively overlapping octapeptides and N- and C-terminal-deleted mutant N proteins were prepared. Results showed that all three site I epitopes studied and two site IV epitopes (for MAbs 8-1 and 6-9) mapped to aa 358-367, and that the other site IV epitope of MAb 7-12 mapped to aa 375-383. Tests using chimeric and truncated proteins showed that MAb 8-1 also requires the N-terminal sequence of the N protein to recognize its binding region more efficiently. Immunofluorescence studies demonstrated that all three site I-specific MAbs and one site IV-specific MAb (7-12) stained the N antigen that was diffusely distributed in the whole cytoplasm; the other two site IV-specific MAbs (6-9 and 8-1) detected only the N antigen in the cytoplasmic inclusion bodies (CIB). An antigenic site II-specific MAb (6-17) also detected CIB-associated N antigen alone. Furthermore, the level of diffuse N antigens decreased after treatment of infected cells with cycloheximide. These results suggest that epitopes at site I are expressed on the immature form of the N protein, but epitope structures of site IV MAbs 6-9 and 8-1 are created and/or exposed only after maturation of the N protein.

Serology of Typhoid Fever in an Area of Endemicity and Its Relevance to Diagnosis

PubMed Central

House, Deborah; Wain, John; Ho, Vo A.; Diep, To S.; Chinh, Nguyen T.; Bay, Phan V.; Vinh, Ha; Duc, Minh; Parry, Christopher M.; Dougan, Gordon; White, Nicholas J.; Hien, Tran Tinh; Farrar, Jeremy J.

2001-01-01

Currently, the laboratory diagnosis of typhoid fever is dependent upon either the isolation of Salmonella enterica subsp. enterica serotype Typhi from a clinical sample or the detection of raised titers of agglutinating serum antibodies against the lipopolysaccharide (LPS) (O) or flagellum (H) antigens of serotype Typhi (the Widal test). In this study, the serum antibody responses to the LPS and flagellum antigens of serotype Typhi were investigated with individuals from a region of Vietnam in which typhoid is endemic, and their usefulness for the diagnosis of typhoid fever was evaluated. The antibody responses to both antigens were highly variable among individuals infected with serotype Typhi, and elevated antibody titers were also detected in a high proportion of serum samples from healthy subjects from the community. In-house enzyme-linked immunosorbent assays (ELISAs) for the detection of specific classes of anti-LPS and antiflagellum antibodies were compared with other serologically based tests for the diagnosis of typhoid fever (Widal TO and TH, anti-serotype Typhi immunoglobulin M [IgM] dipstick, and IDeaL TUBEX). At a specificity of ≥0.93, the sensitivities of the different tests were 0.75, 0.55, and 0.52 for the anti-LPS IgM, IgG, and IgA ELISAs, respectively; 0.28 for the antiflagellum IgG ELISA; 0.47 and 0.32 for the Widal TO and TH tests, respectively; and 0.77 for the anti-serotype Typhi IgM dipstick assay. The specificity of the IDeaL TUBEX was below 0.90 (sensitivity, 0.87; specificity, 0.76). The serological assays based on the detection of IgM antibodies against either serotype Typhi LPS (ELISA) or whole bacteria (dipstick) had a significantly higher sensitivity than the Widal TO test when used with a single acute-phase serum sample (P ≤ 0.007). These tests could be of use for the diagnosis of typhoid fever in patients who have clinical typhoid fever but are culture negative or in regions where bacterial culturing facilities are not available. PMID:11230418

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

PubMed

Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

2012-11-01

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

Method for detection of antibodies for metallic elements

DOEpatents

Barrick, C.W.; Clarke, S.M.; Nordin, C.W.

1993-11-30

An apparatus and method for detecting antibodies specific to non-protein antigens. The apparatus is an immunological plate containing a plurality of plastic projections coated with a non-protein material. Assays utilizing the plate are capable of stabilizing the non-protein antigens with detection levels for antibodies specific to the antigens on a nanogram level. A screening assay with the apparatus allows for early detection of exposure to non-protein materials. Specifically metallic elements are detected. 10 figures.

[Laboratory diagnosis of genital herpes--direct immunofluorescence method].

PubMed

Majewska, Anna; Romejko-Wolniewicz, Ewa; Zareba-Szczudlik, Julia; Kilijańczyk, Marek; Gajewska, Małgorzata; Młynarczyk, Grazyna

2013-07-01

Aim of the study was to determine clinical usefulness of direct immunofluorescence method in the laboratory diagnosis of genital herpes in women. Overall 187 anogenital swabs were collected from 120 women. Using a dacron-tipped applicator 83 swabs were collected from women suspected of genital herpes and 104 from patients with no signs of genital infection. All samples were tested using cell culture (Vero cell line) and then direct immunofluorescence method (DIF) for the identification of antigens of herpes simplex viruses: HSV-1 and HSV-2. Characteristic cytopathic effect (CPE), indicative of alphaherpesvirus infection, was observed in 43.4% of cultures with clinical specimens collected from women with suspected genital herpes and in 29.8% of cultures of clinical specimens taken from patients with no clinical symptoms of genital herpes. Herpes simplex viruses were determined in 73 samples by direct immunofluorescence method after amplification of the virus in cell culture. The DIF test confirmed the diagnosis based on the microscopic CPE observation in 85%. In 15% of samples (taken from pregnant women without clinical signs of infection) we reported positive immunofluorescence in the absence of CPE. The frequency of antigen detection was statistically significantly higher in samples that were positive by culture study (chi-square test with Yates's correction, p < 0.01). This method proved to be highly sensitive (97%) in women with clinically suspected infection. High negative predictive value (99%) proves the clinical utility of the DIF in these group of patients. In asymptomatic infections, viral antigens were detected most frequently in the swabs from the cervical canal, and in cases of suspected genital herpes in swabs taken from the vestibule of the vagina and the vulva. However, there was no statistically significant difference in the frequency of detection of Herpes Simplex Virus antigens in specimens from different parts of the genital tract in both groups of women (chi-square test, p > 0.05). In our study HHV-1 was the main causative agent of genital herpes. The growing worldwide prevalence of genital herpes, challenges with the clinical diagnosis, and availability of effective antiviral therapy are the main reasons for a growing interest in rapid, proper laboratory diagnosis of infected patients. Optimal testing diagnostic algorithm depends on patient population, clinical circumstances and availability. Our results indicated that combination of laboratory tests may help to establish the diagnosis if genital herpes is suspected but there are no typical signs.

DIRECT AND INDIRECT FLUORESCENT-ANTIBODY TECHNIQUES FOR THE PSITTACOSIS-LYMPHOGRANULOMA VENEREUM-TRACHOMA GROUP OF AGENTS1

PubMed Central

Ross, Martin R.; Borman, Earle K.

1963-01-01

Ross, Martin R. (Connecticut State Department of Health, Hartford) and Earle K. Borman. Direct and indirect fluorescent-antibody techniques for the psittacosis-lymphogranuloma venereum-trachoma group of agents. J. Bacteriol. 85:851–858. 1963.—Direct and indirect fluorescent-antibody (FA) techniques were developed for the detection of group antigen in infected tissue cultures and the titration of group antibody in human antiserum. The growth of the agent of meningopneumonitis (MP) in mouse embryo lung cell monolayers was followed by infectivity and complement-fixing (CF) antigen titrations, and cytological examination of FA stained cultures. Although infectivity and CF antigen reached a peak at 2 days and remained constant for an additional 3 days, only cells tested 2 to 3 days after infection were suitable for FA staining with labeled anti-MP serum because of excessive artifacts in the older cultures. Fluorescein isothiocyanate-labeled rooster and guinea pig anti-MP serums and human antipsittacosis serums were titrated in direct FA and hemagglutination-inhibition (HI) tests. The rooster conjugate showed brighter staining and higher antibody titers than the guinea pig or human conjugates and was more effective in detecting minimal amounts of virus antigen. FA staining reactions with 1 and 2 units of labeled rooster serum were inhibited by unlabeled rooster serum but clear-cut inhibition with human antipsittacosis serum could not be demonstrated. The indirect FA technique was successfully used for the titration of group antibody in human serum. A comparison of the indirect FA, HI, and CF tests showed the indirect FA technique to be intermediate in sensitivity between the HI and CF tests. None of the three tests showed significant cross reactions with human serums reactive for influenza A and B; parainfluenza 1, 2, and 3; respiratory syncytial virus; Q fever; or the primary atypical pneumonia agent. PMID:14044954

Genotyping applications for transplantation and transfusion management: The Emory Experience

PubMed Central

Fasano, Ross M.; Sullivan, Harold Cliff; Bray, Bob; Gebel, Howie; Meyer, Erin K.; Winkler, Annie M.; Josephson, Cassandra D.; Stowell, Sean R.; Duncan, Sandy; Roback, John D.

2018-01-01

Current genotyping methodologies for transplantation and transfusion management employ multiplex systems that allow for the simultaneous detection of multiple human leukocyte antigens (HLA), human platelet antigens (HPA) and red blood cell (RBC) antigens. The development of high resolution molecular HLA typing has led to improved outcomes of unrelated hematopoietic stem cell transplants by better identifying suitable donors typed at the allele level for HLA-A, B, C, DRB1 and DQB1 antigens. In solid organ transplantation, the combination of high resolution HLA typing along with solid-phase antibody identification and the calculated PRA have shown to be of specific benefit to highly sensitized patients, and have resulted in significant reductions of incompatible crossmatches at the time of organ allocation. This database-driven combined HLA antigen/antibody testing has promoted the routine implementation of the virtual crossmatch, in which an electronic crossmatch is performed, and perhaps even obviates the need for a physical crossmatch. Additionally, DNA-based testing for RBC antigens provides as an alternative typing method that mitigates many of the limitations of hemagglutination-based phenotyping. Although there are many applications of RBC genotyping in various transfusion settings, it has arguably been most useful in the management of transfusion-dependent patients with sickle cell disease (SCD) and thalassemia to minimize alloimmunization. The availability of high-throughput RBC genotyping for both patients and large populations of donors, along with coordinated informatics systems to link patients’ antigen needs with available antigen-negative and/or rare blood-typed donors, offer promise toward improving the efficiency, reliability, and extent of RBC matching for this population. PMID:28234571

Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody.

PubMed

He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Yang, Xiqin; Bai, Guanzhong; Ling, Shigan; Zhang, Heqiu

2011-08-01

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

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.

Radioimmunoassays of hidden viral antigens

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

Neurath, A.R.; Strick, N.; Baker, L.

1982-07-01

Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis Bmore » virus DNA were detected. Antibody-bound adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure.« less

Membrane insertion for the detection of lipopolysaccharides: Exploring the dynamics of amphiphile-in-lipid assays

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

Stromberg, Loreen R.; Hengartner, Nicolas W.; Swingle, Kirstie L.

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assaysmore » to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. In addition, we also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.« less

Membrane insertion for the detection of lipopolysaccharides: Exploring the dynamics of amphiphile-in-lipid assays

DOE PAGES

Stromberg, Loreen R.; Hengartner, Nicolas W.; Swingle, Kirstie L.; ...

2016-05-26

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assaysmore » to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. In addition, we also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.« less

Six-week follow-up after HIV-1 exposure: a position statement from the Public Health Agency of Sweden and the Swedish Reference Group for Antiviral Therapy.

PubMed

Gaines, Hans; Albert, Jan; Axelsson, Maria; Berglund, Torsten; Gisslén, Magnus; Sönnerborg, Anders; Blaxhult, Anders; Bogdanovic, Gordana; Brytting, Maria; Carlander, Christina; Flamholc, Leo; Follin, Per; Haggar, Axana; Hagstam, Per; Johansson, Marcus; Navér, Lars; Persson Blom, Jenny; Samuelson, Agneta; Ström, Helena; Sundqvist, Martin; Svedhem Johansson, Veronica; Tegmark Wisell, Karin; Tegnell, Anders; Thorstensson, Rigmor

2016-02-01

In 2014 the Public Health Agency of Sweden and the Swedish Reference Group for Antiviral Therapy (RAV) conducted a review and analysis of the state of knowledge on the duration of follow-up after exposure to human immunodeficiency virus (HIV). Up until then a follow-up of 12 weeks after exposure had been recommended, but improved tests and new information on early diagnosis motivated a re-evaluation of the national recommendations by experts representing infectious diseases and microbiology, county medical officers, the RAV, the Public Health Agency, and other national authorities. Based on the current state of knowledge the Public Health Agency of Sweden and the RAV recommend, starting in April 2015, a follow-up period of 6 weeks after possible HIV-1 exposure, if HIV testing is performed using laboratory-based combination tests detecting both HIV antibody and antigen. If point-of-care rapid HIV tests are used, a follow-up period of 8 weeks is recommended, because currently available rapid tests have insufficient sensitivity for detection of HIV-1 antigen. A follow-up period of 12 weeks is recommended after a possible exposure for HIV-2, since presently used assays do not include HIV-2 antigens and only limited information is available on the development of HIV antibodies during early HIV-2 infection. If pre- or post-exposure prophylaxis is administered, the follow-up period is recommended to begin after completion of prophylaxis. Even if infection cannot be reliably excluded before the end of the recommended follow-up period, HIV testing should be performed at first contact for persons who seek such testing.

Immune response to measles vaccine in Peruvian children.

PubMed Central

Bautista-López, N. L.; Vaisberg, A.; Kanashiro, R.; Hernández, H.; Ward, B. J.

2001-01-01

OBJECTIVE: To evaluate the immune response in Peruvian children following measles vaccination. METHODS: Fifty-five Peruvian children received Schwarz measles vaccine (about 10(3) plaque forming units) at about 9 months of age. Blood samples were taken before vaccination, then twice after vaccination: one sample at between 1 and 4 weeks after vaccination and the final sample 3 months post vaccination for evaluation of immune cell phenotype and lymphoproliferative responses to measles and non-measles antigens. Measles-specific antibodies were measured by plaque reduction neutralization. FINDINGS: The humoral response developed rapidly after vaccination; only 4 of the 55 children (7%) had plaque reduction neutralization titres <200 mlU/ml 3 months after vaccination. However, only 8 out of 35 children tested (23%) had lymphoproliferative responses to measles antigens 3-4 weeks after vaccination. Children with poor lymphoproliferative responses to measles antigens had readily detectable lymphoproliferative responses to other antigens. Flow cytometric analysis of peripheral blood mononuclear cells revealed diffuse immune system activation at the time of vaccination in most children. The capacity to mount a lymphoproliferative response to measles antigens was associated with expression of CD45RO on CD4+ T-cells. CONCLUSION: The 55 Peruvian children had excellent antibody responses after measles vaccination, but only 23% (8 out of 35) generated detectable lymphoproliferative responses to measles antigens (compared with 55-67% in children in the industrialized world). This difference may contribute to the less than uniform success of measles vaccination programmes in the developing world. PMID:11731811

Comparison of Buffered, Acidified Plate Antigen to Standard Serologic Tests for the Detection of Serum Antibodies to Brucella abortus in Elk (Cervus canadensis).

PubMed

Clarke, P Ryan; Edwards, William H; Hennager, Steven G; Block, Jean F; Yates, Angela M; Ebel, Eric; Knopp, Douglas J; Fuentes-Sanchez, Antonio; Jennings-Gaines, Jessica; Kientz, Rebecca L; Simunich, Marilyn

2015-07-01

Brucellosis (caused by the bacterium Brucella abortus) is a zoonotic disease endemic in wild elk (Cervus canadensis) of the Greater Yellowstone Ecosystem, US. Because livestock and humans working with elk or livestock are at risk, validated tests to detect the B. abortus antibody in elk are needed. Using the κ-statistic, we evaluated the buffered, acidified plate antigen (BAPA) assay for agreement with the results of the four serologic tests (card test [card], complement fixation test [CF], rivanol precipitation plate agglutination test [RIV], standard plate agglutination test [SPT]) that are approved by the US Department of Agriculture for the detection of the B. abortus antibody in elk. From 2006 to 2010, serum samples collected from elk within B. abortus-endemic areas (n = 604) and nonendemic areas (n = 707) and from elk culture-positive for B. abortus (n = 36) were split and blind tested by four elk serum diagnostic laboratories. κ-Values showed a high degree of agreement for the card (0.876), RIV (0.84), and CF (0.774) test pairings and moderate agreement for the SPT (0.578). Sensitivities for the BAPA, card, RIV, CF, and SPT were 0.859, 0.839, 0.899, 1.00, and 0.813, whereas specificities were 0.986, 0.993, 0.986, 0.98, and 0.968, respectively. The positive predictive values and the negative predictive values were calculated for 2.6%, 8.8%, and 16.2% prevalence levels. These findings suggest the BAPA test is a suitable screening test for the B. abortus antibodies in elk.

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

The novel Group A Streptococcus antigen SpnA combined with bead-based immunoassay technology improves streptococcal serology for the diagnosis of acute rheumatic fever.

PubMed

Hanson-Manful, Paulina; Whitcombe, Alana L; Young, Paul G; Atatoa Carr, Polly E; Bell, Anita; Didsbury, Alicia; Mitchell, Edwin A; Dunbar, P Rod; Proft, Thomas; Moreland, Nicole J

2018-04-01

Streptococcal serology provides evidence of prior Group A Streptococcus (GAS) exposure, crucial to the diagnosis of acute rheumatic fever (ARF) and post-streptococcal glomerulonephritis. However, current tests, which measure anti-streptolysin-O and anti-DNaseB antibodies, are limited by false positives in GAS endemic settings, and incompatible methodology requiring the two tests to be run in parallel. The objective was to improve streptococcal serology by combining the novel GAS antigen, SpnA, with streptolysin-O and DNaseB in a contemporary, bead-based immunoassay. Recombinant streptolysin-O, DNAseB and SpnA were conjugated to polystyrene beads with unique fluorescence positions so antibody binding to all three antigens could be detected simultaneously by cytometric bead array. Multiplex assays were run on sera collected in three groups: ARF; ethnically matched healthy children; and healthy adults. The ability of the antigens to detect a previous GAS exposure in ARF was assessed using the 80th centile of the healthy children group as cut-off (upper limit of normal). SpnA had the highest sensitivity at 88%, compared with 75% for streptolysin-O and 56% for DNaseB. SpnA has favorable immunokinetics for streptococcal serology, and can be combined with anti-streptolysin-O and anti-DNaseB in a multiplex format to improve efficiency and accuracy. Copyright © 2017 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Designed Ankyrin Repeat Proteins: A New Approach to Mimic Complex Antigens for Diagnostic Purposes?

PubMed Central

Hausammann, Stefanie; Vogel, Monique; Kremer Hovinga, Johanna A.; Lacroix-Desmazes, Sebastien; Stadler, Beda M.; Horn, Michael P.

2013-01-01

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures. PMID:23626669

Mapping the fine specificity of ABO monoclonal reagents with A and B type-specific function-spacer-lipid constructs in kodecytes and inkjet printed on paper.

PubMed

Barr, Katie; Korchagina, Elena; Ryzhov, Ivan; Bovin, Nicolai; Henry, Stephen

2014-10-01

Monoclonal (MoAb) reagents are routinely used and are usually very reliable for the serologic determination of ABO blood types. However, the fine specificity and cross-reactivity of these reagents are often unknown, particularly against synthetic antigens used in some diagnostic assays. If nonserologic assays or very sensitive techniques other than those specifically prescribed by the manufacturer are used, then there is a risk of incorrect interpretation of results. Forty-seven MoAbs and two polyclonal ABO reagents were tested against red blood cell (RBC) kodecytes prepared with A trisaccharide, A Type 1, A Type 2, A Type 3, A Type 4, B trisaccharide, B Type 1, B Type 2, acquired B trisaccharide, and Le(a) trisaccharide function-spacer-lipid (FSL) constructs. Natural RBCs were tested in parallel. In addition these FSL constructs were printed onto paper with a desktop inkjet printer and used in a novel immunoassay that identifies reactivity through the appearance of alphanumeric characters. Mapping of MoAbs with kodecytes and printed FSL constructs revealed a series of broad recognition patterns. All ABO MoAbs tested were reactive with the RBC dominant Type 2 ABO antigens. Unexpectedly some anti-A reagents were reactive against the B Type 1 antigen, while others were poorly reactive with trisaccharide antigens. All ABO MoAbs detect the RBC dominant Type 2 ABO antigens; however, some reagents may show minor reactivity with inappropriate blood group antigens, which needs to be considered when using these reagents in alternative or highly sensitive analytic systems. © 2014 AABB.

Detection of sperm-reactive antibodies in wild sika deer and identification of the sperm antigens.

PubMed

Kawase, Osamu; Jimbo, Mitsuru

2018-03-16

Antisperm antibodies potentially inhibit sperm functions causing the sterility in humans and experimentally treated animals. However, there is no information about antisperm antibodies emerging spontaneously in wildlife. In this study, we searched for the sperm-reactive antibodies, spontaneously produced in wild sika deer (Cervus nippon), and identified the sperm antigens. We collected 529 fecal masses of sika deer in Japanese cities, from which we extracted the mucosal antibodies to test them for reactivities to deer sperm proteins by ELISA. Two of the extracts contained IgAs that were highly reactive to the sperm proteins. The molecular weights of the active IgAs, partially purified by DEAE-sephadex A-50, were estimated at more than 100 kDa, suggesting that the IgAs evaded drastic digestion in the gastrointestinal tract. Two-dimensional electrophoresis and immunoblotting detected three major antigens, and the following LC-MS/MS analysis identified them as alpha-enolase, phosphoglycerate kinase 2 and acrosin-binding protein. The antibodies were cross-reactive to a recombinant human acrosin-binding protein. To our knowledge, this is the first research to find that the sperm-reactive antibodies are produced spontaneously in wildlife and they recognize a common antigen found in humans.

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.

A multiplex immunochromatographic test using gold nanoparticles for the rapid and simultaneous detection of four nitrofuran metabolites in fish samples.

PubMed

Wang, Quan; Liu, Yingchun; Wang, Mingyan; Chen, Yongjun; Jiang, Wei

2018-01-01

There is an urgent need for the rapid and simultaneous detection of multiple analytes present in a sample matrix. Here, a multiplex immunochromatographic test (multi-ICT) was developed that successfully allowed for the rapid and simultaneous detection of four major nitrofuran metabolites, i.e., 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), in fish samples. Four different antigens were separately immobilized in four test lines on a nitrocellulose membrane. Goat anti-mouse immunoglobulin (IgG) was used as a control. Sensitive and specific monoclonal antibodies (mAbs) that recognize the corresponding antigens were selected for the assay, and no cross-reactivity between the antibodies in the detection assay was observed. The free analytes in samples or standards were pre-incubated with freeze-dried mAb-gold conjugates to improve the sensitivity of the detection assay. The multi-ICT detection was accomplished in less than 15 min by the naked eye. The cutoff values for the strip test were 0.5 ng/mL for AOZ and 0.75 ng/mL for AHD, SEM, and AMOZ, which were all below the maximum residue levels set by the European Union and China. A high degree of consistency was observed between the multi-ICT method and commercially available enzyme-linked immunosorbent assay (ELISA) kits using spiked, incurred, and "blind" fish samples, indicating the accuracy, reproducibility, and reliability of the novel test strip. This newly developed multi-ICT strip assay is suitable for the rapid and high-throughput screening of four nitrofuran metabolites in fish samples on-site, with no treatment or devices required. Graphical abstract A multiplex immunochromatographic test (multi-ICT) was developed that successfully allowed for the rapid and simultaneous detection of four major nitrofuran metabolites (AOZ, SEM, AMOZ, and AHD) in fish samples.

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

Type 1 and type 2 diabetes mellitus: are they mutually exclusive?

PubMed

Fatima, Aziz; Khawaja, Khadija Irfan; Burney, Saira; Minhas, Khushroo; Mumtaz, Usman; Masud, Faisal

2013-07-01

With advancement in the understanding of the pathogenesis underlying diabetes mellitus (DM), the boundary between type 1 and type 2 DM (T1DM and T2DM) does not seem to be as clear cut as previously thought. This study was designed to test the possibility of overlap between the spectra of immune-mediated DM and insulin resistance. To test for the possibility of overlap, we looked for autoantibodies typical of T1DM in patients with classical T2DM, and insulin resistance in patients with T1DM. Autoantibodies against islet cell antigen, glutamic acid decarboxylase-65 and insulinoma-associated antigen-2 were tested in 82 patients with T2DM and 27 patients with T1DM. The patients had been diagnosed on clinical criteria using standard laboratory techniques. Clinical parameters of diagnostic importance were noted, and homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using fasting insulin and fasting blood glucose ratio. Autoantibodies against one or more beta cell antigens were detected in 12.19% of patients clinically diagnosed to have T2DM, and insulin resistance (HOMA-IR > 2.5) was diagnosed in 37.03% of patients with T1DM. It was not possible to identify any combination of clinical or biochemical markers that could predict autoantibody positivity in T2DM patients. T1DM patients with insulin resistance had a significantly higher body mass index than their insulin-sensitive counterparts (p = 0.02). Autoantibodies against beta cell antigens are detectable in insulin-resistant T2DM patients, and insulin resistance may be present in relatively overweight T1DM patients. No differentiating clinical features that might predict autoantibody positivity in T2DM patients were found.

Helicobacter pylori HopE and HopV porins present scarce expression among clinical isolates

PubMed Central

Lienlaf, Maritza; Morales, Juan Pablo; Díaz, María Inés; Díaz, Rodrigo; Bruce, Elsa; Siegel, Freddy; León, Gloria; Harris, Paul R; Venegas, Alejandro

2010-01-01

AIM: To evaluate how widely Helicobacter pylori (H. pylori) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile. METHODS: H. pylori hopE and hopV genes derived from strain CHCTX-1 were cloned by polymerase chain reaction (PCR), sequenced and expressed in Escherichia coli AD494 (DE3). Gel-purified porins were used to prepare polyclonal antibodies. The presence of both genes was tested by PCR in a collection of H. pylori clinical isolates and their expression was detected in lysates by immunoblotting. Immune responses against HopE, HopV and other H. pylori antigens in sera from infected and non-infected patients were tested by Western blotting using these sera as first antibody on recombinant H. pylori antigens. RESULTS: PCR and Western blotting assays revealed that 60 and 82 out of 130 Chilean isolates carried hopE and hopV genes, respectively, but only 16 and 9, respectively, expressed these porins. IgG serum immunoreactivity evaluation of 69 H. pylori-infected patients revealed that HopE and HopV were infrequently recognized (8.7% and 10.1% respectively) compared to H. pylori VacA (68.1%) and CagA (59.5%) antigens. Similar values were detected for IgA serum immunoreactivity against HopE (11.6%) and HopV (10.5%) although lower values for VacA (42%) and CagA (17.4%) were obtained when compared to the IgG response. CONCLUSION: A scarce expression of HopE and HopV among Chilean isolates was found, in agreement with the infrequent seroconversion against these antigens when tested in infected Chilean patients. PMID:20082477

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.

[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.

System and method for a parallel immunoassay system

DOEpatents

Stevens, Fred J.

2002-01-01

A method and system for detecting a target antigen using massively parallel immunoassay technology. In this system, high affinity antibodies of the antigen are covalently linked to small beads or particles. The beads are exposed to a solution containing DNA-oligomer-mimics of the antigen. The mimics which are reactive with the covalently attached antibody or antibodies will bind to the appropriate antibody molecule on the bead. The particles or beads are then washed to remove any unbound DNA-oligomer-mimics and are then immobilized or trapped. The bead-antibody complexes are then exposed to a test solution which may contain the targeted antigens. If the antigen is present it will replace the mimic since it has a greater affinity for the respective antibody. The particles are then removed from the solution leaving a residual solution. This residual solution is applied a DNA chip containing many samples of complimentary DNA. If the DNA tag from a mimic binds with its complimentary DNA, it indicates the presence of the target antigen. A flourescent tag can be used to more easily identify the bound DNA tag.

Serological Detection of Viral Infections in Captive Wild Cats from Costa Rica

PubMed Central

Blanco, Kinndle; Peña, Roberto; Hernández, Carmen; Jiménez, Mauricio; Araya, Luis Nazario; Romero, Juan José; Dolz, Gaby

2011-01-01

Serum samples from a total of 44 wildcats, 28 margays (Leopardus wiedii), 10 ocelots (Leopardus pardalis), four jaguaroundis (Herpailurus yaguaroundi), one oncilla (Leopardus tigrina), and one jaguar (Panthera onca) were obtained between January 2001 and August 2002 from the Profelis Centre for rehabilitation of wild felids, located in the northwestern region of Costa Rica. Forty three samples were tested for antibodies against feline immunodeficiency virus (FIV) and p27 antigen of feline leukemia virus (FeLV), 42 samples for antibodies against feline parvovirus (FPV), and 30 for antibodies against feline calicivirus (FCV). None of the samples contained detectable antibodies against FIV or p27 antigen of FeLV, all samples contained antibodies against FPV, and one sample contained antibodies against FCV. PMID:21547230

Serological detection of viral infections in captive wild cats from costa rica.

PubMed

Blanco, Kinndle; Peña, Roberto; Hernández, Carmen; Jiménez, Mauricio; Araya, Luis Nazario; Romero, Juan José; Dolz, Gaby

2011-04-03

Serum samples from a total of 44 wildcats, 28 margays (Leopardus wiedii), 10 ocelots (Leopardus pardalis), four jaguaroundis (Herpailurus yaguaroundi), one oncilla (Leopardus tigrina), and one jaguar (Panthera onca) were obtained between January 2001 and August 2002 from the Profelis Centre for rehabilitation of wild felids, located in the northwestern region of Costa Rica. Forty three samples were tested for antibodies against feline immunodeficiency virus (FIV) and p27 antigen of feline leukemia virus (FeLV), 42 samples for antibodies against feline parvovirus (FPV), and 30 for antibodies against feline calicivirus (FCV). None of the samples contained detectable antibodies against FIV or p27 antigen of FeLV, all samples contained antibodies against FPV, and one sample contained antibodies against FCV.

[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.

Quantitative blood group typing using surface plasmon resonance.

PubMed

Then, Whui Lyn; Aguilar, Marie-Isabel; Garnier, Gil

2015-11-15

The accurate and reliable typing of blood groups is essential prior to blood transfusion. While current blood typing methods are well established, results are subjective and heavily reliant on analysis by trained personnel. Techniques for quantifying blood group antibody-antigen interactions are also very limited. Many biosensing systems rely on surface plasmon resonance (SPR) detection to quantify biomolecular interactions. While SPR has been widely used for characterizing antibody-antigen interactions, measuring antibody interactions with whole cells is significantly less common. Previous studies utilized SPR for blood group antigen detection, however, showed poor regeneration causing loss of functionality after a single use. In this study, a fully regenerable, multi-functional platform for quantitative blood group typing via SPR detection is achieved by immobilizing anti-human IgG antibody to the sensor surface, which binds to the Fc region of human IgG antibodies. The surface becomes an interchangeable platform capable of quantifying the blood group interactions between red blood cells (RBCs) and IgG antibodies. As with indirect antiglobulin tests (IAT), which use IgG antibodies for detection, IgG antibodies are initially incubated with RBCs. This facilitates binding to the immobilized monolayer and allows for quantitative blood group detection. Using the D-antigen as an example, a clear distinction between positive (>500 RU) and negative (<100 RU) RBCs is achieved using anti-D IgG. Complete regeneration of the anti-human IgG surface is also successful, showing negligible degradation of the surface after more than 100 regenerations. This novel approach is validated with human-sourced whole blood samples to demonstrate an interesting alternative for quantitative blood grouping using SPR analysis. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Detection of H5N1 high-pathogenicity avian influenza virus in meat and tracheal samples from experimentally infected chickens.

PubMed

Das, Amaresh; Spackman, Erica; Thomas, Colleen; Swayne, David E; Suarez, David L

2008-03-01

The Asian H5N1 highly pathogenic avian influenza (HPAI) virus causes a systemic disease with high mortality of poultry and is potentially zoonotic. In both chickens and ducks, the virus has been demonstrated to replicate in both cardiac and skeletal muscle cells. Experimentally, H5N1 HPAI virus has been transmitted to chickens through the consumption of raw infected meat. In this study, we investigated virus replication in cardiac and skeletal muscle and in the trachea of chickens after experimental intranasal inoculation with the H5N1 HPAI virus. The virus was detected in tissues by real-time reverse transcription-polymerase chain reaction (RRT-PCR) and virus isolation, and in the trachea by RRT-PCR and a commercial avian influenza (AI) viral antigen detection test. A modified RNA extraction protocol was developed for rapid detection of the virus in tissues by RRT-PCR. The H5N1 HPAI virus was sporadically detected in meat and the tracheas of infected birds without any clinical sign of disease as early as 6 hr postinfection (PI), and was detected in all samples tested at 24 hr PI and later. No differences in sensitivity were seen between virus isolation and RRT-PCR in meat samples. The AI viral antigen detection test on tracheal swabs was a useful method for identifying infected chickens when they were sick or dead, but was less sensitive in detecting infected birds when they were preclinical. This study provides data indicating that preslaughter tracheal swab testing can identify birds infected with HPAI among the daily mortality and prevent infected flocks from being sent to processing plants. In addition, the modified RNA extraction and RRT-PCR test on meat samples provide a rapid and sensitive method of identifying HPAI virus in illegal contraband or domestic meat samples.

Two cases of false-positive dengue non-structural protein 1 (NS1) antigen in patients with hematological malignancies and a review of the literature on the use of NS1 for the detection of Dengue infection.

PubMed

Chung, Shimin J; Krishnan, Prabha U; Leo, Yee Sin

2015-02-01

Early diagnosis of dengue has been made easier in recent years owing to the advancement in diagnostic technologies. The rapid non-structural protein 1 (NS1) test strip is widely used in many developed and developing regions at risk of dengue. Despite the relatively high specificity of this test, we recently encountered two cases of false-positive dengue NS1 antigen in patients with underlying hematological malignancies. We reviewed the literature for causes of false-positive dengue NS1. © The American Society of Tropical Medicine and Hygiene.

[Laboratory diagnosis of bacterial meningitis: usefulness of various tests for the determination of the etiological agent].

PubMed

Carbonnelle, E

2009-01-01

Despite breakthroughs in the diagnosis and treatment of infectious diseases, meningitis still remains an important cause of mortality and morbidity. An accurate and rapid diagnosis of acute bacterial meningitis is essential for a good outcome. The gold-standard test for diagnosis is CSF analysis. Gram staining of CSF reveals bacteria in about 50 to 80 % of cases and cultures are positive in at best 80 % of cases. However, the sensitivity of both tests is less than 50 % in patients who are already on antibiotic treatment. CSF leukocyte count and concentration of protein and glucose lack specificity and sensitivity for the diagnosis of meningitis. Other biological tests are available for the diagnosis. Latex agglutination test were adapted for rapid and direct detection of soluble bacterial antigens in CSF of patients suspected with bacterial meningitis. This test is efficient in detecting antigens of most common central nervous system bateria but lacks sensibility. Furthermore, in the early phases of acute bacterial and viral meningitis, signs and symptoms are often non specific and it is not always possible to make a differential diagnosis. Markers like CRP, procalcitonin, or sTREM-1 may be very useful for the diagnosis and to differentiate between viral and bacterial meningitis. Bacterial meningitis diagnosis and management require various biological tests and a multidisciplinary approach.

K99 antigen-positive enterotoxigenic Escherichia coli from piglets with diarrhea in Sweden.

PubMed Central

Smyth, C J; Olsson, E; Moncalvo, C; Söderlind, O; Orskov, F; Orskov, I

1981-01-01

K88 antigen-negative enterotoxigenic Escherichia coli and non-enterotoxigenic strains isolated from piglets with diarrhea were examined for K99 antigen by agglutination tests after growth on Minca-IsoVitaleX (BBL Microbiology Systems, Cockeysville, Md.) agar medium. Of 64 K88-negative enterotoxigenic strains from as many piglets, 17 were found to be K99 positive. Of these, 10 were Swedish and 7 were of Norwegian origin. All 17 produced heat-stable enterotoxin detectable in the infant mouse assay, but only 2 gave positive ligated loop tests in 3- to 7-week-old piglets. Ligated loop tests in 5- to 12-day-old piglets were positive for each of the 15 K99-positive strains tested. Each of the Swedish K99-positive isolates was from a piglet of lesser than or equal to 7 days of age. One piglet harboring a K99-positive strain also harbored an O141:K88 enterotoxigenic strain producing only heat-stable enterotoxin. Five of the Swedish piglets yielding K99-positive isolates were from dams vaccinated wih a multicomponent bacterial vaccine containing K88 antigen. The K99 strains were O:K:H serotyped. The O serogroups represented were O8, O9, O64, O101, and O140. None of the 101 non-enterotoxigenic porcine isolates, representing 42 serogroups and non-O-groupable and rough strains, was found to be K99 positive. The findings indicate that so-called class 2 or atypical porcine enterotoxigenic E. coli should be routinely examined for the presence of K99 antigen. PMID:7009634

Utility of a stool antigen test to detect the incidence of helicobacter pylori infection and familial and community enviromental risk factors for this infection in pediatric age.

PubMed

Sabbi, T; Dall'Oglio, L; De Angelis, P; Torroni, E; Colistro, F; Azzolina, M; Santoni, A; Di Ciommo, V; Benedetto, M

2012-01-01

Helicobacter pylori (Hp) infection is mainly acquired during childhood; it is recognised as a cause of gastritis and peptic ulcer and it has been classified as a group A carcinogen by World Health Organization. The exact mode of transmission is as yet, not known. Aim of our study has been to identify risk factors associated with Helicobacter pylori infection in a preschool and school population and to confirm if Hp antigen in faeces is useful as screening in epidemiological studies. We interviewed, with questionnaire, 400 children (203 male; age range 3-10 years; mean age 6 years) of 3 different schools and stool samples were collected of all children too. 35 of 400 (8%) children underwent to upper gastrointestinal endoscopy because of a suspect of upper gastrointestinal disease. stool were collected from 400 school children and 35 of them shown positivity of Hp antigen test. A questionnaire about presence of nausea, vomit, recurrent abdominal pain, family size, parent's occupations and education, use of antibiotics, country of birth of child and parents, personal hygiene, breast feeding, presence of the animals was completed. 35 children with positive Hp stool antigen test and a suspicious of upper gastrointestinal disease (recurrent abdominal pain, diurnal or nocturnal abdominal pain, nausea, vomiting, iron deficiency) underwent to esophagogastroduodenoscopy (EGDS) that demonstrated antral gastritis and positive histology and urease rapid test. the results of this study suggest that risk factors for Hp infection are low socioeconomics factors, hygiene and living conditions and that Hp antigen in faeces is useful as screening test.

Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in tissues of postharvest fruits

PubMed Central

2011-01-01

Background Botrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparently healthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitive methods are necessary for its detection and quantification. This article describes the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for quantification of B. cinerea in apple (Red Delicious), table grape (pink Moscatel), and pear (William's) tissues. Results The method was based in the competition for the binding site of monoclonal antibodies between B. cinerea antigens present in fruit tissues and B. cinerea purified antigens immobilized by a crosslinking agent onto the surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity, precision, accuracy and sensibility. The calculated detection limit was 0.97 μg mL-1 B. cinerea antigens. The immobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The fungus was detected and quantified in any of the fruits tested when the rot was not visible yet. Results were compared with a DNA quantification method and these studies showed good correlation. Conclusions The developed method allowed detects the presence of B. cinerea in asymptomatic fruits and provides the advantages of low cost, easy operation, and short analysis time determination for its possible application in the phytosanitary programs of the fruit industry worldwide. PMID:21970317

Evaluation of the LIA-ANA-Profile-17S for the detection of autoantibodies to nuclear antigens.

PubMed

Yi, Ahram; Lee, Chang-Hoon; Moon, Hee-Won; Kim, Hanah; Hur, Mina; Yun, Yeo-Min

2018-05-01

The diagnostic tests for autoimmune disease include screening for autoantibodies for nuclear antigens (ANA) and antibodies against extractable nuclear antigens (ENA). Using the line immunoassay (LIA) method, various kinds of ENA antibodies can be detected simultaneously. We evaluated the performance of the newly launched LIA-ANA-Profile-17S (Shenzhen YHLO Biotech, Shenzhen, China) as compared to a conventional LIA kit. Residual samples were collected from 200 patients who had been tested for ANA using indirect immunofluorescence. The LIA-ANA-Profile-17S was compared to the EuroLine ANA (Euroimmun, Oberlausitz, Germany) for the analysis of 17 different autoantibodies. The concordance rate and agreement between assays were determined. Samples showing discrepancies between the LIA-ANA-Profile-17S and EuroLine tests were further examined through additional analysis. The overall agreement was moderate (kappa = 0.759, 95% CI = 0.712-0.805). Agreement between assays ranged from weak to almost perfect, except for those tests targeting nucleosomes, histones, and PM-Scl. Of the 57 disparate results between LIA-ANA-Profile-17S and EuroLine, 38 (66.7%) samples tested positive under an additional assay, showing variable patterns between types of autoantibodies. The positive rate of each autoantibody between LIA-ANA-Profile-17S and EuroLine did not differ significantly, except for anti-nucleosome and anti-histone assays in samples from patients diagnosed with systemic lupus erythematosus (P = 0.004 and 0.001, respectively). Compared to those from the conventional EuroLine assay, the LIA-ANA-Profile-17S results showed variable agreement in samples showing different prevalence of each autoantibody. The most frequently detected antibodies showed almost perfect agreement. The LIA-ANA-Profile-17S could play a role in the diagnosis of systemic autoimmune disease in ANA-positive samples. Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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

Is HCV core antigen a reliable marker of viral load? An evaluation of HCV core antigen automated immunoassay

PubMed Central

Hadziyannis, Emilia; Minopetrou, Martha; Georgiou, Anastasia; Spanou, Fotini; Koskinas, John

2013-01-01

Background Hepatitis C viral (HCV) load detection and quantification is routinely accomplished by HCV RNA measurement, an expensive but essential test, both for the diagnosis and treatment of chronic hepatitis C (CHC). HCV core antigen (Ag) testing has been suggested as an attractive alternative to molecular diagnostics. The aim of the study was to evaluate an automated chemiluminescent immunoassay (CLIA) for HCV core Ag measurement in comparison to quantitative HCV RNA determination. Methods HCV Ag was measured in 105 anti-HCV positive patients, from which 89 were HCV RNA positive with CHC and 16 HCV RNA negative after spontaneous HCV clearance. Viral load was quantified with branched DNA (bDNA, Versant, Siemens). Sera were stored at -70°C and then tested with the Architect HCV Ag test (Abbott Laboratories), a two-step CLIA assay, with high throughput and minimal handling of the specimens. Statistical analysis was performed on logarithmically transformed values. Results HCV-Ag was detectable and quantifiable in 83/89 and in grey zone in 4/89 HCV RNA positive sera. HCV-Ag was undetectable in all 16 HCV RNA negative samples. The sample with the lowest viral load that tested positive for HCV-Ag contained 1200 IU/mL HCV RNA. There was a positive correlation between HCV RNA and HCV-Ag (r=0.89). The HCV RNA/ HCV Ag ratio varied from 1.5 to 3.25. Conclusion The HCV core Ag is an easy test with comparable sensitivity (>90%) and satisfactory correlation with the HCV RNA bDNA assay. Its role in diagnostics and other clinical applications has to be determined based on cost effectiveness. PMID:24714621

Etiologic predictive value of a rapid immunoassay for the detection of group A Streptococcus antigen from throat swabs in patients presenting with a sore throat.

PubMed

Orda, Ulrich; Gunnarsson, Ronny; Orda, Sabine; Fitzgerald, Mark; Rofe, Geoff; Dargan, Anna

2016-04-01

Clinical reasoning utilizing certain symptoms and scores has not proven to be a reliable decision-making tool to determine whether or not to suspect a group A Streptococcus (GAS) infection in the patient presenting with a sore throat. Culture as the so-called 'gold standard' is impracticable because it takes 1 to 2 days (and even longer in remote locations) for a result, and thus treatment decisions will be made without the result available. Rapid diagnostic antigen tests have demonstrated sufficient sensitivities and specificities in detecting GAS antigens to identify GAS throat infections. Throat swab samples were collected from patients attending the Mount Isa Hospital emergency department for a sore throat; these samples were compared to swab samples collected from healthy controls who did not have a sore throat. Both groups were aged 3-15 years. All swab samples were analyzed with a point-of-care test (Alere Test Pack +Plus with OBC Strep A). The etiologic predictive value (EPV) of the throat swab was calculated. The 95% confidence interval for positive EPV was 88-100% and for negative EPV was 97-99%, depending on assumptions made. This study demonstrates that the point-of-care test Alere Test Pack +Plus Strep A has a high positive predictive value and is able to rule in GAS infection as long as the proportion of carriers is low. Also the negative predictive value for ruling out GAS as the etiologic agent is very high irrespective of the carrier rate. Hence, this test is always useful to rule out GAS infection. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis.

PubMed

Cortina, María E; Balzano, Rodrigo E; Rey Serantes, Diego A; Caillava, Ana J; Elena, Sebastián; Ferreira, A C; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

2016-06-01

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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A review of the basis of the immunological diagnosis of ruminant brucellosis.

PubMed

Ducrotoy, Marie J; Conde-Álvarez, Raquel; Blasco, José María; Moriyón, Ignacio

2016-03-01

Bacteria of the genus Brucella cause brucellosis, the most common bacterial zoonosis worldwide. The diagnosis of Brucella abortus and Brucella melitensis ruminant brucellosis is based on bacteriological and immunological tests, the latter being routinely used in control and eradication and surveillance programs. Infections by smooth and rough Brucella spp., the use of smooth and rough vaccines, and the false-positive serological reactions caused by Yersinia enterocolitica O:9 and other cross-reacting bacteria represent the immunological contexts in which those tests are used. This complex context explains the large number of brucellosis tests that have been developed, and that vary in antigen type, antigen presentation, antibody and conditions for the reaction, the response detected and the sample required. This wealth of information and an imperfect understanding of Brucella antigens and of the peculiarities of the immunoresponse to Brucella has created confusion and led to several misconceptions on the usefulness and limitations of the brucellosis diagnostic tests. In this review, Brucella antigens are examined focusing on cellular topology, supramolecular properties, epitopic structure and lipopolysaccharide and protein cross-reactivity in the various contexts of the immune response in ruminants. Then, the significance of these features in diagnostic tests that use whole bacteria is discussed with respect to the activities of ruminant immunoglobulins, and the effect of pH on unspecific agglutinations, non-agglutinating and blocking antibodies, pseudo-prozones and complement activation. Similarly, the bacterial surface lipopolysaccharides and cognate polysaccharides are discussed with regards to topological effects, epitope exposure, ionic strength and antibody avidity in immunoprecipitation, immunosorbent and fluorescence polarization assays. Finally, the search for immunodominant protein antigens and their use in immunological tests is reviewed. Critical review of the existing information is necessary both to select optimal tests according to the logistical means available and the epidemiological context, and to focus the development of new tests. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of potential target antigens for immunotherapy on primary and metastatic prostate cancers.

PubMed

Zhang, S; Zhang, H S; Reuter, V E; Slovin, S F; Scher, H I; Livingston, P O

1998-02-01

Defining the expression of tumor-associated antigens on primary and metastatic prostate cancer is the crucial first step in selecting appropriate targets for immune attack. In this study, the distribution of the tumor-associated antigens GM2, Tn, sTn, Thompson-Friedenreich antigen (TF), Globo H, Le(y), MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, carcinoembryonic antigen, beta chain of human chorionic gonadotropin (hCG beta), HER2/neu, PSMA, and KSA on primary and metastatic prostate cancer and 16 types of normal tissues was compared by immunohistochemistry, using a panel of well-characterized monoclonal antibodies. Our results show that GM2, KSA, and MUC2 were strongly expressed on 8 or 9 of 9 metastatic prostate cancer biopsy specimens and, with PSMA, hCG beta, TF, Tn, and sTn, on 8 or more of 11 primary prostate cancer specimens. Tn, MUC1, and PSMA were expressed on 4-6 of 9 metastatic specimens. The remaining antigens were expressed on no more than three of nine metastatic specimens. Normal tissues were also tested with all antibodies. With regard to the eight antigens most widely expressed on prostate cancers, PSMA was not expressed significantly on any of the normal tissues except prostate epithelium. Tn, sTn, hCG beta, and MUC2 were detected on up to 3 of 10 types of normal epithelia. GM2, TF, MUC1, and KSA were more broadly distributed on normal epithelia, all primarily at the secretory borders. STn, KSA, and hCG beta were also detected in the testis, and GM2 was expressed on gray matter of brain. From the 30 antigens that we have screened, this study provides the basis for selecting GM2, TF, Tn, sTn, hCG beta, MUC1, MUC2, KSA, and PSMA as target antigens for specific immunotherapy of prostate cancer.

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

Multiplexed immunophenotyping of human antigen-presenting cells in whole blood by polychromatic flow cytometry

PubMed Central

Fung, Erik; Esposito, Laura; Todd, John A.; Wicker, Linda S.

2010-01-01

We describe two modular protocols for immunostaining and multiparameter flow cytometric analysis of major human antigen-presenting cells (dendritic cells, monocytes, B lymphocytes) in minimally manipulated whole blood. Simultaneous detection of up to eight colors is enabled by careful selection and testing of cell-subset-defining monoclonal antibodies (anchor markers) in the appropriate fluorochrome combinations, to demonstrate the quantification of surface expression levels of molecules involved in chemotaxis (e.g. CX3CR1, CCR2), adhesion (e.g. CD11b, CD62L), antigen presentation (e.g. CD83, CD86, CD209) and immune regulation (e.g. CD101) on circulating antigen-presenting cells. Each immunostaining reaction requires as little as 50–100 μl of peripheral whole blood, no density-gradient separation, and the entire procedure from preparation of reagents to flow cytometry can be completed in <5 h. PMID:20134434

Rapid assessment of the antigenic integrity of tetrameric HLA complexes by human monoclonal HLA antibodies.

PubMed

Eijsink, Chantal; Kester, Michel G D; Franke, Marry E I; Franken, Kees L M C; Heemskerk, Mirjam H M; Claas, Frans H J; Mulder, Arend

2006-08-31

The ability of tetrameric major histocompatibility complex (MHC) class I-peptide complexes (tetramers) to detect antigen-specific T lymphocyte responses has yielded significant information about the generation of in vivo immunity in numerous antigenic systems. Here we present a novel method for rapid validation of tetrameric HLA molecules based on the presence of allodeterminants. Human monoclonal antibodies (mAbs) recognizing polymorphic determinants on HLA class I were immobilized on polystyrene microparticles and used to probe the structural integrity of tetrameric HLA class I molecules by flow cytometry. A total of 22 tetramers, based on HLA-A1, A2, A3, A24, B7 and B8 were reactive with their counterpart mAbs, thus confirming their antigenic integrity. A positive outcome of this mAb test ensures that tetrameric HLA class I can be used with greater confidence in subsequent functional assays.

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.

The bovine immune response to Brucella abortus. II. Elimination of some sporadic serological reactions by chelation of divalent cations.

PubMed Central

Nielsen, K; Samagh, B S; Speckmann, G; Stemshorn, B

1979-01-01

The standard agglutination tests for detecting antibody to Brucella abortus were modified by addition of chelating agents (EDTA and EGTA) to the antigens. Approximately 80% of "singleton" agglutination test reactions, negative on the diagnostic complement fixation test, obtained with cattle sera were eliminated while no decrease in titer was apparent when sera from B. abortus infected or vaccinated cattle were tested. PMID:121242

[Detection of antibodies against Lamblia antigens in serum from atopic patients].

PubMed

Akhapkina, I G

2004-04-01

The method of solid-phase immune-enzyme assay was used to test the sera of atopic patients for the purpose of detecting IgG to lamblias. A high degree of protozoal invasion was established especially among pre-school and school children. It was proven as expedient to make the serological examinations of atopic patients in cases the clinical finding is unclear.

Performance evaluation of the Bio-Rad Laboratories GS HIV Combo Ag/Ab EIA, a 4th generation HIV assay for the simultaneous detection of HIV p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in human serum or plasma.

PubMed

Bentsen, Christopher; McLaughlin, Lisa; Mitchell, Elizabeth; Ferrera, Carol; Liska, Sally; Myers, Robert; Peel, Sheila; Swenson, Paul; Gadelle, Stephane; Shriver, M Kathleen

2011-12-01

A multi-center study was conducted to evaluate the Bio-Rad GS HIV Combo Ag/Ab EIA, a 4th generation HIV-1/HIV-2 assay for the simultaneous detection of HIV p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in human serum or plasma in adult and pediatric populations. The objectives of the study were to assess assay performance for the detection of acute HIV infections; sensitivity in known HIV positive samples; percent agreement with HIV status; specificity in low and high risk individuals of unknown HIV status; and to compare assay performance to a 3rd generation HIV assay. The evaluation included testing 9150 samples at four U.S. clinical trial sites, using three kit lots. Unlinked samples were from routine testing, repositories or purchased from vendors. GS HIV Combo Ag/Ab EIA detection in samples from individuals in two separate populations with acute HIV infection was 95.2% (20/21) and 86.4% (38/44). Sensitivity was 100% (1603/1603) in known antibody positive [HIV-1 Groups M and O, and HIV-2] samples. HIV p24 antigen detection was 100% (53/53) in HIV-1 culture supernatants. HIV-1 seroconversion panel detection improved by a range of 0-20 days compared to a 3rd generation HIV test. Specificity was 99.9% (5989/5996) in low risk, 99.9% (959/960) in high risk and 100% (100/100) in pediatric populations. The GS HIV Combo Ag/Ab EIA significantly reduced the diagnostic window when compared to the 3rd generation screening assay, enabling earlier diagnosis of HIV infection. The performance parameters of the Bio-Rad GS HIV Combo Ag/Ab EIA are well suited for use in HIV diagnostic settings. Copyright © 2011 Elsevier B.V. All rights reserved.

Early detection of the growth of Mycobacterium tuberculosis using magnetophoretic immunoassay in liquid culture.

PubMed

Kim, Jeonghyo; Lee, Kil-Soo; Kim, Eun Bee; Paik, Seungwha; Chang, Chulhun L; Park, Tae Jung; Kim, Hwa-Jung; Lee, Jaebeom

2017-10-15

Tuberculosis (TB) is an often neglected, epidemic disease that remains to be controlled by contemporary techniques of medicine and biotechnology. In this study, a nanoscale sensing system, referred to as magnetophoretic immunoassay (MPI) was designed to capture culture filtrate protein (CFP)-10 antigens effectively using two different types of nanoparticles (NPs). Two specific monoclonal antibodies against CFP-10 antigen were used, including gold NPs for signaling and magnetic particles for separation. These results were carefully compared with those obtained using the commercial mycobacteria growth indicator tube (MGIT) test via 2 sequential clinical tests (with ca. 260 clinical samples). The sensing linearity of MPI was shown in the range of pico- to micromoles and the detection limit was 0.3pM. MPI using clinical samples shows robust and reliable sensing while monitoring Mycobacterium tuberculosis (MTB) growth with monitoring time 3-10 days) comparable to that with the MGIT test. Furthermore, MPI distinguished false-positive samples from MGIT-positive samples, probably containing non-tuberculous mycobacteria. Thus, MPI shows promise in early TB diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Autoantibodies and their antigens in autoimmune hepatitis.

PubMed

Bogdanos, Dimitrios P; Mieli-Vergani, Giorgina; Vergani, Diego

2009-08-01

Autoantibody detection assists in the diagnosis and allows differentiation of autoimmune hepatitis (AIH) type 1 (AIH-1), characterized by antinuclear antibody (ANA) and/or smooth muscle antibody (SMA), and type 2 (AIH-2), distinguished by the presence of antibodies to liver-kidney microsome type 1 (anti-LKM1) and/or antibodies to liver cytosol type 1 (anti-LC1). Detection of atypical perinuclear antineutrophil cytoplasmic antibodies (pANCA) and anti-soluble liver antigen (SLA) antibodies can act as an additional pointer toward the diagnosis of AIH, particularly in the absence of the conventional autoantibodies. Routine autoantibody testing by indirect immunofluorescence has been recently complemented by molecular assays based on purified or recombinant antigens. Although the AIH-1-specific ANA and SMA targets need better definition, those of anti-LKM1 and anti-LC1 in AIH-2 have been clearly identified; the fine specificity of antibody reactivity and its clinical relevance to disease pathogenesis are the focus of ongoing investigation. This article critically discusses the current knowledge of the diagnostic and clinical significance of AIH-related autoantibody reactivities, focusing on key issues that the physician needs to be aware of to be able to request the appropriate testing and to interpret correctly the laboratory results within the clinical context of the patient. Copyright Thieme Medical Publishers.

Cost-effective antigen testing for delimitation, monitoring and evaluation in bancroftian filariasis.

PubMed

Das, L K; Pani, S P; Vanamail, P; Vijayalakshmi, G; Debritto, L J

2012-01-31

This study was focussed on identifying a cost-effective method for delimitation, monitoring and evaluation in bancroftian filariasis. Finger prick blood samples were collected between 20.00 and 23.00 hours for the detection of microfilariae (mf) from the available population in a village which was endemic for lymphatic filariasis. Simultaneously, from each individual, four spots of 25-μl blood samples were collected on Whatman number 3 filter paper and air dried. Dried filter paper spots were pooled in quantities of 1, 5, 10, 15, 20 and 25 on unknown and simulated mf and antigen prevalence. Pooled samples were assayed for circulating filarial antigen (CFA) using TropBIO Og4C3 ELISA kits. The community mf and CFA rates were 3.4% and 25.9%, respectively. The pool sizes of 20 and 25 showed CFA positivity in all the above categories tested. The results of the pooled blood spot samples suggest that, in areas with mf and CFA prevalence rates between 1 and 10%, pools of 20 or 25 could be considered as the ideal pool size for the detection of filarial infection in the community. CFA prevalence at the level of 5-6% following desirable rounds of mass drug administration (MDA) indicates that the community mf prevalence is likely to be at the 1% level.

Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

PubMed

Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Leitolis, Amanda; Crestani, Sandra; Foti, Leonardo; de Souza, Wayner Vieira; Gomes, Yara de Miranda; Krieger, Marco Aurélio

2017-10-01

Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi -specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti- T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania , a pathogen with high similarity to T. cruzi , showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD. Copyright © 2017 American Society for Microbiology.

Characterisation of antibody responses in pigs induced by recombinant oncosphere antigens from Taenia solium.

PubMed

Jayashi, César M; Gonzalez, Armando E; Castillo Neyra, Ricardo; Kyngdon, Craig T; Gauci, Charles G; Lightowlers, Marshall W

2012-12-14

Recombinant antigens cloned from the oncosphere life cycle stage of the cestode parasite Taenia solium (T. solium) have been proven to be effective as vaccines for protecting pigs against infections with T. solium. Previous studies have defined three different host protective oncosphere antigens, TSOL18, TSOL16 and TSOL45. In this study, we evaluated the potential for combining the antigens TSOL16 and TSOL18 as a practical vaccine. Firstly, in a laboratory trial, we compared the immunogenicity of the combined antigens (TSOL16/18) versus the immunogenicity of the antigens separately. Secondly, in a field trial, we tested the ability of the TSOL16/18 vaccine to induce detectable antibody responses in animals living under environmental stress and traditionally reared in areas where T. solium cysticercosis is endemic; and finally, we characterised the immune response of the study population. Pigs of 8-16 weeks of age were vaccinated with 200 μg each of TSOL16 and TSOL18, plus 5mg of Quil-A. Specific total IgG, IgG(1) and IgG(2) antibody responses induced by TSOL16 and TSOL18 were determined with ELISA. The immunogenicity of both antigens was retained in the combined TSOL16/18 vaccine. The combined vaccine TSOL16/18 induced detectable specific anti-TSOL18 antibody responses in 100% (113/113) and specific anti-TSOL16 in 99% (112/113) of the vaccinated animals measured at 2 weeks following the booster vaccination. From the two IgG antibody subtypes analysed we found there was stronger response to IgG(2). Copyright © 2012 Elsevier Ltd. All rights reserved.

A study on the prevalence of dog erythrocyte antigen 1.1 and detection of canine Babesia by polymerase chain reaction from apparently healthy dogs in a selected rural community in Zimbabwe.

PubMed

Dhliwayo, Solomon; Makonese, Tariro A; Whittall, Belinda; Chikerema, Silvester M; Pfukenyi, Davies M; Tivapasi, Musavenga T

2016-10-26

A study was carried out to determine the prevalence of blood group antigen dog erythrocyte antigen (DEA) 1.1 in mixed breed dogs in rural Chinamhora, Zimbabwe. DEA 1.1 is clinically the most important canine blood group as it is the most antigenic blood type; hence, DEA 1.1 antibodies are capable of causing acute haemolytic, potentially life-threatening transfusion reactions. In this study, blood samples were collected from 100 dogs in Chinamhora, and blood typing was carried out using standardised DEA 1.1 typing strips with monoclonal anti-DEA 1.1 antibodies (Alvedia® LAB DEA 1.1 test kits). Polymerase chain reaction for detecting Babesia spp. antigen was carried out on 58 of the samples. Of the 100 dogs, 78% were DEA 1.1 positive and 22% were DEA 1.1 negative. A significantly (p = 0.02) higher proportion of females (90.5%) were DEA 1.1 positive than males (69.0%). The probability of sensitisation of recipient dogs following first-time transfusion of untyped or unmatched blood was 17.2%, and an approximately 3% (2.95%) probability of an acute haemolytic reaction following a second incompatible transfusion was found. Babesia spp. antigen was found in 6.9% of the samples. No significant relationship (χ2 = 0.56, p = 0.45) was found between DEA 1.1 positivity and Babesia spp. antigen presence. Despite a low probability of haemolysis after a second incompatibility transfusion, the risk remains present and should not be ignored. Hence, where possible, blood typing for DEA 1.1 is recommended. A survey of DEA 3, 4, 5 and 7 in various breeds is also recommended.

Inaccuracy of Enzyme-Linked Immunosorbent Assay Using Soluble and Recombinant Antigens to Detect Asymptomatic Infection by Leishmania infantum

PubMed Central

Moreno, Elizabeth Castro; Gonçalves, Andréa Vieira; Chaves, Anderson Vieira; Melo, Maria Norma; Lambertucci, José Roberto; Andrade, Antero Silva Ribeiro; Negrão-Corrêa, Deborah; Antunes, Carlos Mauricio de Figueiredo; Carneiro, Mariângela

2009-01-01

Background One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period. Methodology Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve. Findings The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10). Conclusions Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias in population studies. Therefore, studies which have used serological assays to estimate prevalence, to evaluate intervention programs or to identify risk factors for Leishmania infection, may have had their results compromised. PMID:19841736

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

Monoclonal antibodies against LipL32, the major outer membrane protein of pathogenic Leptospira: production, characterization, and testing in diagnostic applications.

PubMed

Fernandes, Cláudia P H; Seixas, Fabiana K; Coutinho, Mariana L; Vasconcellos, Flávia A; Seyffert, Núbia; Croda, Julio; McBride, Alan J; Ko, Albert I; Dellagostin, Odir A; Aleixo, José A G

2007-02-01

Pathogenic serovars of Leptospira have a wide antigenic diversity attributed mainly to the lipopolysaccharide present in the outer membrane. In contrast, antigens conserved among pathogenic serovars are mainly represented by outer membrane proteins. Surface exposure of a major and highly conserved outer membrane lipoprotein (LipL32) was recently demonstrated on pathogenic Leptospira. LipL32 in its recombinant form (rLipL32) was used to immunize BALB/c mice to develop murine monoclonal antibodies (MAbs). Three MAbs against rLipL32 were produced, isotyped, and evaluated for further use in diagnostic tests of leptospirosis using different approaches. MAbs were conjugated to peroxidase and evaluated in a native protein enzyme-linked immunosorbent assay (ELISA) with intact and heat-treated leptospiral cells, conjugated to fluorescein isothiocyanate (FITC) for indirect immunofluorescence with intact and methanol fixed cells and were used for LipL32 immunoprecipitation from leptospiral cells. rLipL32 MAbs conjugated to peroxidase or used as primary antibody bound to intact and heat-treated cells in ELISA, proving that they could be used in enzyme immunoassays for detection of the native protein. In immunofluorescence assay, MAbs labeled bacterial cells either intact or methanol fixed. Two MAbs were able to immunoprecipitate the native protein from live and motile leptospiral cells and, adsorbed onto magnetic beads, captured intact bacteria from artificially contaminated human sera for detection by polymerase chain reaction (PCR) amplification. Results of this study suggest that the MAbs produced can be useful for the development of diagnostic tests based on detection of LipL32 leptospiral antigen in biological fluids.

Serological Evidence of Lyssavirus Infection among Bats in Nagaland, a North-Eastern State in India.

PubMed

Mani, R S; Dovih, D P; Ashwini, M A; Chattopadhyay, B; Harsha, P K; Garg, K M; Sudarshan, S; Puttaswamaiah, R; Ramakrishnan, U; Madhusudana, S N

2017-06-01

Bats are known to be reservoirs of several medically important viruses including lyssaviruses. However, no systematic surveillance for bat rabies has been carried out in India, a canine rabies endemic country with a high burden of human rabies. Surveillance for rabies virus (RABV) infection in bats was therefore carried out in Nagaland, a north-eastern state in India at sites with intense human-bat interfaces during traditional bat harvests. Brain tissues and sera from bats were tested for evidence of infection due to RABV. Brain tissues were subjected to the fluorescent antibody test for detection of viral antigen and real-time reverse transcriptase PCR for presence of viral RNA. Bat sera were tested for the presence of rabies neutralizing antibodies by the rapid fluorescent focus inhibition test. None of the bat brains tested (n = 164) were positive for viral antigen or viral RNA. However, rabies neutralizing antibodies were detected in 4/78 (5·1%) bat sera tested, suggesting prior exposure to RABV or related lyssaviruses. The serological evidence of lyssaviral infection in Indian bats may have important implications in disease transmission and rabies control measures, and warrant extensive bat surveillance to better define the prevalence of lyssaviral infection in bats.

Peritonsillar abscess and cellulitis and their relation to a positive antigen detection test for streptococcal infection.

PubMed

Risberg, Stefan; Engfeldt, Peter; Hugosson, Svante

2010-10-01

The microbiological cause of peritonsillar abscess and the role of group A β-haemolytic Streptococcus (GAS) are unclear. We performed a retrospective study at the ear, nose and throat clinic (ENT) of Orebro University Hospital, Sweden, and included 376 events of peritonsillitis between 2002 and 2004. We determined if the patients had visited a primary healthcare centre (PHCC) within 30 days prior to inclusion. The results of the rapid antigen detection test for GAS (Strep A) taken at the PHCC were compared with the occurrence of peritonsillar abscess (PTA) and peritonsillar cellulitis (PTC). A Strep A test was performed in 61% (229/376) of the events studied. Strep A was positive in 22% of PTA events and in 35% of PTC events (p = 0.036). Of 48,000 Strep A tests taken in primary healthcare, mainly for sore throat, 22% were positive. We examined the relationship between age, the incidence of PTA, and positive Strep A tests. We also determined if there was a monthly correlation between number of positive Strep A tests and number of PTA events. We found no significant correlations. In conclusion, our findings indicate that GAS does not play a major role in the development of PTA/PTC.

Hepatitis B virus surface antigen and anti-hepatitis C virus rapid tests underestimate hepatitis prevalence among HIV-infected patients.

PubMed

Hønge, Bl; Jespersen, S; Medina, C; Té, Ds; da Silva, Zj; Ostergaard, L; Laursen, Al; Wejse, C; Krarup, H; Erikstrup, C

2014-10-01

In the case of coinfection with HIV and hepatitis B virus (HBV) and/or hepatitis C virus (HCV), hepatic disease progression is often accelerated, with higher rates of liver cirrhosis and liver-related mortality. We aimed to evaluate the performance of the rapid tests used routinely to detect HBV surface antigen (HBsAg) and anti-HCV among HIV-infected patients in Guinea-Bissau. Blood samples from HIV-infected patients in Guinea-Bissau were stored after testing for HBsAg and anti-HCV with rapid tests. Samples were subsequently re-tested for HBsAg and anti-HCV in Denmark. Two rapid tests were used in Guinea-Bissau: HBsAg Strip Ref 2034 (VEDA.LAB, Alençon, France; sensitivity 62.3%; specificity 99.2%) and HEPA-SCAN (Bhat Bio-Tech, Bangalore, India; sensitivity 57.1%; specificity 99.7%). In the two tests the ability to obtain the correct outcome depended on the antigen and antibody concentrations, respectively. Sex, age, CD4 cell count and antiretroviral therapy status did not differ between false negative and true positive samples in either of the tests. The study is limited by a low number of anti-HCV positive samples. New diagnostic rapid tests should always be evaluated in the setting in which they will be used before implementation. © 2014 British HIV Association.

ELISA Methods for the Detection of Ebolavirus Infection.

PubMed

Cross, Robert W; Ksiazek, Thomas G

2017-01-01

Ebola viruses are high-priority pathogens first discovered in rural Africa associated with sporadic outbreaks of severe hemorrhagic disease in humans and nonhuman primates. Little is known about the disease ecology or the prevalence of past exposure of human populations to any of the five species of the genus Ebolavirus. The use of immunologic means of detection for either virus antigens or the host's immune response to antigen associated with prior infections offers a powerful approach at understanding the epidemiology and epizootiology of these agents. Here we describe methods for preparing antigen detection sandwich enzyme-linked immunosorbent assays (ELISAs) as well as IgG and IgM ELISAs for the detection of ebolavirus antigens or antibodies in biological samples.