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...

Antigenic differences in the surfaces of hyphae and rhizoids in allomyces.

PubMed

Fultz, S A; Sussman, A S

1966-05-06

Immunofluorescent techniques have demonstrated a difference in surface components of hyphae and rhizoids of Allomyces macrogynus. An antigenic component detected on the rhizoidal surface may be present, but masked, in the hyphal-wall matrix material. The system also allows visualization of the hyphal wall during aging, when changes from a smooth to a fissured surface are noted, and differences in adsorptive properties occur.

Rh blood phenotyping (D, E, e, C, c) microarrays using multichannel surface plasmon resonance imaging.

PubMed

Pipatpanukul, Chinnawut; Takeya, Sasaki; Baba, Akira; Amarit, Ratthasart; Somboonkaew, Armote; Sutapun, Boonsong; Kitpoka, Pimpun; Kunakorn, Mongkol; Srikhirin, Toemsak

2018-04-15

The application of Surface Plasmon Resonance Imaging (SPRi) for the detection of transmembrane antigen of the Rhesus (Rh) blood group system is demonstrated. Clinically significant Rh blood group system antigens, including D, C, E, c, and e, can be simultaneously identified via solid phase immobilization assay, which offers significant time savings and assay simplification. Red blood cells (RBCs) flowed through the micro-channel, where a suitable condition for Rh blood group detection was an RBC dilution of 1:10 with a stop-flow condition. Stop flow showed an improvement in specific binding compared to continuous flow. Rh antigens required a longer incubation time to react with the immobilized antibody than A and B antigens due to the difference in antigen type and their location on the RBC. The interaction between the immobilized antibodies and their specific antigenic counterpart on the RBC showed a significant difference in RBC removal behavior using shear flow, measured from the decay of the SPR signal. The strength of the interaction between the immobilized antibody and RBC antigen was determined from the minimum wall shear stress required to start the decay process in the SPR signal. For a given range of immobilized antibody surface densities, the Rh antigen possesses a stronger interaction than A, B, and AB antigens. Identification of 82 samples of ABO and Rh blood groups using SPRi showed good agreement with the standard micro-column agglutination technique. A wider coverage of antigenic recognition for RBC when using the solid phase immobilization assay was demonstrated for the RBC with the antigenic site located on the transmembrane protein of the clinically significant Rh antigen. Given the level of accuracy and precision, the technique showed potential for the detection of the Rh minor blood group system. Copyright © 2017 Elsevier B.V. All rights reserved.

[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.

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.

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

PubMed Central

Owen, Peter; Salton, Milton R. J.

1977-01-01

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions. Images PMID:144722

Membrane asymmetry and expression of cell surface antigens of Micrococcus lysodeikticus established by crossed immunoelectrophoresis.

PubMed

Owen, P; Salton, M R

1977-12-01

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.

B-cell acquisition of antigen: Sensing the surface.

PubMed

Knight, Andrew M

2015-06-01

B-cell antigen receptor (BCR) recognition and acquisition of antigen by B cells is the essential first step in the generation of effective antibody responses. As B-cell-mediated antigen presentation is also believed to play a significant role in the activation of CD4(+) Th-cell responses, considerable effort has focused on clarifying the nature of antigen/BCR interactions. Following earlier descriptions of interactions of soluble antigens with the BCR, it is now clear that B cells also recognize, physically extract and present antigens that are tethered to, or integral components of, the surfaces or extracellular matrix of other cells. In this issue of the European Journal of Immunology, Zeng et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] examine how the physical property or "stiffness" of the surface displaying antigens to B cells influences the B-cell response. This commentary reports that antigen tethered on "less stiff" surfaces induces increased B-cell activation and antibody responses. I then infer how "sensing the surface" by B cells may represent a new component of the immune system's ability to detect "damage," and how this understanding may influence approaches to clinical therapies where immune activity is either unwanted or desired. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed Central

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

2017-01-01

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

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

Optical biosensors using surface plasmon resonance

NASA Astrophysics Data System (ADS)

Homola, Jiri; Brynda, Eduard; Tobiska, Petr; Tichy, Ivo; Skvor, Jiri

1999-12-01

We present a surface plasmon resonance sensor base on prism excitation of surface plasmons and spectral interrogation. For specific detection of biomolecular analytes, multilayers of monoclonal antibodies are immobilized on the surface of the sensor. Detection of biomolecular analytes such as human (beta) -2)-microglobulin, choriogonadotropin, hepatitis B surface antigen, salmonella enteritidis is demonstrated.

SPR platform based on image acquisition for HER2 antigen detection

NASA Astrophysics Data System (ADS)

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

2017-01-01

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

The sebaceous gland antigen defined by the OM-1 monoclonal antibody is expressed at high density on the surface of ovarian carcinoma cells.

PubMed

de Kretser, T A; Thorne, H J; Jacobs, D J; Jose, D G

1985-09-01

A monoclonal antibody, designated OM-1, was raised against ovarian serous papillary cystadenocarcinoma (stage IV) cells. This antibody was found to react strongly with primary and metastatic ovarian serous cystadenocarcinomas and endometrioid carcinomas but the antigen detected was either absent or at very low levels in ovarian mucinous adenocarcinomas, clear cell carcinomas, benign serous and mucinous cystadenomas and Brenner tumours. The OM-1 antibody gave no detectable reaction with 93 other human tumours, including examples of breast and colon adenocarcinomas. In normal tissues the OM-1 antibody reacted with normal sebaceous gland cells, lung type II pneumocytes and placental syncytial trophoblasts. In the normal ovary OM-1 reactivity was confined to extremely weak staining of the surface epithelium. No reaction with any other ovarian cell type could be detected. No evidence of reaction with other normal cell populations present in 24 adult and seven foetal tissues was found. The antigen detected is compared with other ovarian tumour-associated antigens. The OM-1 antibody is likely to prove of value in the detection and diagnosis of ovarian carcinoma.

Quantitative Detection of Prostatic-Specific Antigens by Using Scanning Electron Microscopy for the Analysis of Protein Chips.

PubMed

Lee, Jisu; Jung, Moon Youn; Park, Hyung Ju

2017-04-01

We reported that quantitative detection of prostatic-specific antigen (PSA), which is the biomarker of prostate cancer, could be carried out by calculating the number density and the area ratio of gold nanoparticle probes on the surface of silicon oxide chips. When chips selectively activated with PSA were immersed in the gold nanoparticles conjugated with prostatic specific antigens-poly clonal antibodies (PSA-pAb), it was possible to observe changes in the number density and the area ratio of gold nanoparticles on the surface of the chips according to the concentration of PSA with scanning electron microscopy (SEM) images. As PSA concentration increased, the number density and the area ratio of gold nanoparticle probes on the surfaces of the chips increased accordingly. Conversely, with lower concentration, the number density and the area ratio of gold nanoparticle probes on the surfaces decreased at a certain ratio. We observed the correlations between PSA concentration and number density, area ratio of gold nanoparticle probes through the analysis of SEM images. In addition, it was confirmed that the sizes of the gold nanoparticles affected the detection limit of the number density and the area ratio of gold nanoparticle probes on the surface.

Detection of antisperm antibodies: their localization to human sperm antigens that are transferred to the surface of zona-free hamster oocytes during the sperm penetration assay.

PubMed

Wiley, L M; Obasaju, M F; Overstreet, J W; Cross, N L; Hanson, F W; Chang, R J

1987-08-01

The authors have developed an extension of the sperm penetration assay for detecting serum immunoglobulins to sperm antigens that are transferred to the plasma membrane of a sperm-penetrated hamster oocyte. After the hamster oocytes have been scored for sperm penetration by observing for the presence of swollen sperm heads, they are incubated in serum followed by either a 20-minute treatment with rhodamine-conjugated protein A (which binds to most subclasses of IgA, IgG, and IgM) or a 2-hour incubation in guinea pig serum (complement). Positive fluorescence indicates that the serum contains antibodies to sperm antigens that were transferred to the surface of an oocyte during gamete fusion. Complement-mediated lysis indicates that the immunoglobulin that is bound can also fix complement. The advantages of this assay for detection of serum antisperm antibodies are that it is an extension of a widely used assay, is rapid and requires readily available reagents and equipment, can detect most subclasses of IgA, IgG, and IgM, detects antibodies to those sperm antigens that may be transferred to the oocyte during fertilization, and indicates whether the detected antisperm antibodies can mediate complement-dependent lysis of the fertilized oocyte.

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.

A biochip-based combined immunoassay for detection of serological status of Borrelia burgdorferi in Lyme borreliosis.

PubMed

Huang, Na-Li; Ye, Lei; Lv, Hui; Du, Yi-Xin; Schneider, Marion; Fan, Li-Bin; Du, Wei-Dong

2017-09-01

Dithiobis (succinimidyl undecanoate) modified gold surface biochip were used as a combined immunoassay platform for concurrently detecting immune responses to Borrelia burgdorferi (B. burgdorferi) sensu lato antigens, flagellin, outer surface protein C, variable major protein-like sequence proteins, and 3 VlsE protein IR 6 peptides. The peptides represented intrinsic Borrelia genospecies: B. burgdorferi sensu stricto, B. garinii, and B. afzelii, respectively. Fourier transform infrared spectroscopy was utilized to validate the surface chemical characteristics on the modified gold surface. The limits in detection of IgG antibody on the biochips were as little as 0.39μg/ml for anti-VlsE and 0.78μg/ml for anti-flagellin and anti-OspC, respectively. Samples from 56 neuroborreliosis (NB) patients and 114 healthy individuals were analyzed by the combined biochip. We found that the seroprevalences of IgM or IgG antibody against the 6 antigens were contributed to increased overall sensitivity by the multiplex immunobiochip assay. Serum combined positive rates of the 6 antigens in the patients were 92.86% for IgM antibody and 91.07% for IgG antibody. Part of the patients bore antibody responses against the 3 VlsE IR 6 variant peptides, indicating that Lyme borreliosis would attribute to consequence of multiple infections by one or more Borrelia burgdorferi strains. Concurrent assessment for both IgM and IgG antibodies against the protein antigens and B. burgdorferi IR 6 peptides in the sera of NB patients was beneficial from the biochip format, enabling detection of expanded serologic infection status and therapy strategy-making more efficiently. The combined biochip-based immunoassay, as a potential substitution of ELISA, provided a promising approach to extend the detection spectrum of infectious antibodies against a panel of Borrelia antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

PubMed Central

Dazzo, F B; Hubbell, D H

1975-01-01

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin. Images PMID:55100

Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

NASA Astrophysics Data System (ADS)

Meng, Zhongji; Song, Ruihua; Chen, Yue; Zhu, Yang; Tian, Yanhui; Li, Ding; Cui, Daxiang

2013-03-01

A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.

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

NASA Astrophysics Data System (ADS)

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

2018-05-01

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

Mutations Associated With Occult Hepatitis B in HIV-Positive South Africans

PubMed Central

Powell, Eleanor A.; Gededzha, Maemu P.; Rentz, Michael; Rakgole, Nare J.; Selabe, Selokela G.; Seleise, Tebogo A.; Mphahlele, M. Jeffrey; Blackard, Jason T.

2015-01-01

Occult hepatitis B is characterized by the absence of hepatitis B surface antigen (HBsAg) but the presence of HBV DNA. Because diagnosis of hepatitis B virus (HBV) typically includes HBsAg detection, occult HBV remains largely undiagnosed. Occult HBV is associated with increased risk of hepatocellular carcinoma, reactivation to chronic HBV during immune suppression, and transmission during blood transfusion and liver transplant. The mechanisms leading to occult HBV infection are unclear, although viral mutations are likely a significant factor. In this study, sera from 394 HIV-positive South Africans were tested for HBV DNA and HBsAg. For patients with detectable HBV DNA, the overlapping surface and polymerase open reading frames (ORFs) were sequenced. Occult-associated mutations—those mutations found exclusively in individuals with occult HBV infection but not in individuals with chronic HBV infection from the same cohort or GenBank references—were identified. Ninety patients (22.8%) had detectable HBV DNA. Of these, 37 had detectable HBsAg, while 53 lacked detectable surface antigen. The surface and polymerase ORFs were cloned successfully for 19 patients with chronic HBV and 30 patients with occult HBV. In total, 235 occult-associated mutations were identified. Ten occult-associated mutations were identified in more than one patient. Additionally, 15 amino acid positions had two distinct occult-associated mutations at the same residue. Occult-associated mutations were common and present in all regions of the surface and polymerase ORFs. Further study is underway to determine the effects of these mutations on viral replication and surface antigen expression in vitro. PMID:25164924

Mutations associated with occult hepatitis B in HIV-positive South Africans.

PubMed

Powell, Eleanor A; Gededzha, Maemu P; Rentz, Michael; Rakgole, Nare J; Selabe, Selokela G; Seleise, Tebogo A; Mphahlele, M Jeffrey; Blackard, Jason T

2015-03-01

Occult hepatitis B is characterized by the absence of hepatitis B surface antigen (HBsAg) but the presence of HBV DNA. Because diagnosis of hepatitis B virus (HBV) typically includes HBsAg detection, occult HBV remains largely undiagnosed. Occult HBV is associated with increased risk of hepatocellular carcinoma, reactivation to chronic HBV during immune suppression, and transmission during blood transfusion and liver transplant. The mechanisms leading to occult HBV infection are unclear, although viral mutations are likely a significant factor. In this study, sera from 394 HIV-positive South Africans were tested for HBV DNA and HBsAg. For patients with detectable HBV DNA, the overlapping surface and polymerase open reading frames (ORFs) were sequenced. Occult-associated mutations-those mutations found exclusively in individuals with occult HBV infection but not in individuals with chronic HBV infection from the same cohort or GenBank references-were identified. Ninety patients (22.8%) had detectable HBV DNA. Of these, 37 had detectable HBsAg, while 53 lacked detectable surface antigen. The surface and polymerase ORFs were cloned successfully for 19 patients with chronic HBV and 30 patients with occult HBV. In total, 235 occult-associated mutations were identified. Ten occult-associated mutations were identified in more than one patient. Additionally, 15 amino acid positions had two distinct occult-associated mutations at the same residue. Occult-associated mutations were common and present in all regions of the surface and polymerase ORFs. Further study is underway to determine the effects of these mutations on viral replication and surface antigen expression in vitro. © 2014 Wiley Periodicals, Inc.

Analysis of density and epitopes of D antigen on the surface of erythrocytes from DEL phenotypic individuals carrying the RHD1227A allele.

PubMed

Gu, Juan; Sun, An-Yuan; Wang, Xue-Dong; Shao, Chao-Peng; Li, Zheng; Huang, Li-Hua; Pan, Zhao-Lin; Wang, Qing-Ping; Sun, Guang-Ming

2014-04-01

The characteristics of the D antigen are important as they influence the immunogenicity of D variant cells. Several studies on antigenic sites have been reported in normal D positive, weak D and partial D cases, including a comprehensive analysis of DEL types in Caucasians. The aim of this study was to assess D antigen density and epitopes on the erythrocyte surface of Asian type DEL phenotypic individuals carrying the RHD1227A allele in the Chinese population. A total of 154 DEL phenotypic individuals carrying the RHD1227A allele were identified through adsorption and elution tests and polymerase chain reaction analysis with sequence-specific primers in the Chinese population. D antigen density on the erythrocyte surface of these individuals was detected using a flow cytometric method. An erythrocyte sample with known D antigen density was used as a standard. Blood samples from D-negative and D-positive individuals were used as controls. In addition, D antigen epitopes on the erythrocyte surface of DEL individuals carrying the RHD1227A allele were investigated with 18 monoclonal anti-D antibodies specific for different D antigen epitopes. The means of the median fluorescence intensity of D antigen on the erythrocyte membrane surface of D-negative, D-positive and DEL individuals were 2.14±0.25, 193.61±11.43 and 2.45±0.82, respectively. The DEL samples were estimated to have approximately 22 D antigens per cell. The samples from all 154 DEL individuals reacted positively with 18 monoclonal anti-D antibodies specific for different D antigen epitopes. In this study, D antigen density on the erythrocyte surface of DEL individuals carrying the RHD1227A allele was extremely low, there being only very few antigenic molecules per cell, but the D antigen epitopes were grossly complete.

An immuno-biosensor system based on quartz crystal microbalance for avian influenza virus detection

NASA Astrophysics Data System (ADS)

Liu, Shengping; Chen, Guoming; Zhou, Qi; Wei, Yunlong

2007-12-01

For the quick detection of Avian Influenza Virus (AIV), a biosensor based on Quartz Crystal Microbalance (QCM) was fabricated according to the specific bonding principle between antibody and antigen. Staphylococcal Protein A (SPA) was extracted from Staphylococcus and purified. Then SPA was coated on the surface of QCM for immobilizing AIV monoclonal antibodies. The use of AIV monoclonal antibody could enhance the specificity of the immuno-biosensor. A multi-channel piezoelectricity detection system for the immuno-biosensor was developed. The system can work for the quick detection of AIV antigen in the case of the entirely aqueous status owe to one special oscillating circuit designed in this work. The optimum conditions of SPA coating and AIV monoclonal antibody immobilization were investigated utilizing the multi-channel detection system. The preliminary application of the immuno-biosensor system for detection of AIV was evaluated. Results indicate that the immuno-biosensor system can detect the AIV antigens with a linear range of 3-200ng/ml. The system can accomplish the detection of AIV antigens around 40 minutes.

An improved Abbott ARCHITECT assay for the detection of hepatitis B virus surface antigen (HBsAg).

PubMed

Lou, Sheng C; Pearce, Sandra K; Lukaszewska, Teresa X; Taylor, Russell E; Williams, Gregg T; Leary, Thomas P

2011-05-01

The sensitive and accurate detection of hepatitis B virus surface antigen (HBsAg) is critical to the identification of infection and the prevention of transfusion transmitted disease. Improvement in HBsAg assay sensitivity is essential to reduce the window to detect an acute HBV infection. Additionally, the sensitive detection of HBsAg mutants that continue to evolve due to vaccine escape, immune selection and an error prone reverse transcriptase is a necessity. A fully automated HBsAg prototype assay on the Abbott ARCHITECT instrument was developed to improve sensitivity and mutant detection. This magnetic microparticle-based assay utilizes anti-HBsAg monoclonal antibodies to capture antigen present in serum or plasma. Captured antigen is then detected using anti-HBsAg antibody conjugated with the chemiluminescent compound, acridinium. The sensitivity of the ARCHITECT HBsAg prototype assay was improved as compared to the current ARCHITECT, PRISM, and competitor HBsAg assays. The enhancement in assay sensitivity was demonstrated by the use of commercially available HBV seroconversion panels. The prototype assay detected more panel members (185 of 383) vs. the current ARCHITECT (171), PRISM (181), or competitor HBsAg assays (73/140 vs. 62/140, respectively). The ARCHITECT prototype assay also efficiently detected all mutants evaluated. Finally, the sensitivity improvement did not compromise the specificity of the assay (99.94%). An improved Abbott ARCHITECT HBsAg prototype assay with enhanced detection of HBsAg and HBsAg mutants, as well as equivalent specificity was developed for the detection, diagnosis, and management of HBV infection. Copyright © 2011 Elsevier B.V. All rights reserved.

Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

PubMed

Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

2012-01-01

This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

[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.

Surface Plasmon Resonance Label-Free Monitoring of Antibody Antigen Interactions in Real Time

ERIC Educational Resources Information Center

Kausaite, Asta; van Dijk, Martijn; Castrop, Jan; Ramanaviciene, Almira; Baltrus, John P.; Acaite, Juzefa; Ramanavicius, Arunas

2007-01-01

Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label-free detection of biologically active compounds. This method allows one to monitor binding events in real time without…

Trypanosoma congolense: proliferative responses and interleukin production in lymph node cells of infected cattle.

PubMed

Lutje, V; Mertens, B; Boulangé, A; Williams, D J; Authié, E

1995-09-01

T-cell-mediated immune responses to defined antigens of Trypanosoma congolense were measured in cattle undergoing primary infection. The antigens used were the variable surface glycoprotein and two invariant antigens, a 33-kDa cysteine protease (congopain) and a recombinant form of a 69-kDa heat-shock protein. Proliferative responses were highest during the second week postinfection and were detected in cells obtained from the lymph node draining the site of infection but not in peripheral blood mononuclear cells. Production of IL-2 and IFN-gamma was measured in supernatants from antigen-stimulated lymph node cell cultures. Expression of IL-2, IL-4, and IFN-gamma mRNA was detected in antigen-stimulated lymph node cells by reverse transcription-polymerase chain amplification.

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

PubMed

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

2017-02-15

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

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.

A Molecular Simulation Study of Antibody-Antigen Interactions on Surfaces for the Rational Design of Next-Generation Antibody Microarrays

NASA Astrophysics Data System (ADS)

Bush, Derek B.

Antibody microarrays constitute a next-generation sensing platform that has the potential to revolutionize the way that molecular detection is conducted in many scientific fields. Unfortunately, current technologies have not found mainstream use because of reliability problems that undermine trust in their results. Although several factors are involved, it is believed that undesirable protein interactions with the array surface are a fundamental source of problems where little detail about the molecular-level biophysics are known. A better understanding of antibody stability and antibody-antigen binding on the array surface is needed to improve microarray technology. Despite the availability of many laboratory methods for studying protein stability and binding, these methods either do not work when the protein is attached to a surface or they do not provide the atomistic structural information that is needed to better understand protein behavior on the surface. As a result, molecular simulation has emerged as the primary method for studying proteins on surfaces because it can provide metrics and views of atomistic structures and molecular motion. Using an advanced, coarse-grain, protein-surface model this study investigated how antibodies react to and function on different types of surfaces. Three topics were addressed: (1) the stability of individual antibodies on surfaces, (2) antibody binding to small antigens while on a surface, and (3) antibody binding to large antigens while on a surface. The results indicate that immobilizing antibodies or antibody fragments in an upright orientation on a hydrophilic surface can provide the molecules with thermal stability similar to their native aqueous stability, enhance antigen binding strength, and minimize the entropic cost of binding. Furthermore, the results indicate that it is more difficult for large antigens to approach the surface than small antigens, that multiple binding sites can aid antigen binding, and that antigen flexiblity simultaneously helps and hinders the binding process as it approaches the surface. The results provide hope that next-generation microarrays and other devices decorated with proteins can be improved through rational design.

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.

Kinetics of antibody-induced modulation of respiratory syncytial virus antigens in a human epithelial cell line

PubMed Central

Sarmiento, Rosa E; Tirado, Rocio G; Valverde, Laura E; Gómez-Garcia, Beatriz

2007-01-01

Background The binding of viral-specific antibodies to cell-surface antigens usually results in down modulation of the antigen through redistribution of antigens into patches that subsequently may be internalized by endocytosis or may form caps that can be expelled to the extracellular space. Here, by use of confocal-laser-scanning microscopy we investigated the kinetics of the modulation of respiratory syncytial virus (RSV) antigen by RSV-specific IgG. RSV-infected human epithelial cells (HEp-2) were incubated with anti-RSV polyclonal IgG and, at various incubation times, the RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs) and intracellular viral proteins were detected by indirect immunoflourescence. Results Interaction of anti-RSV polyclonal IgG with RSV HEp-2 infected cells induced relocalization and aggregation of viral glycoproteins in the plasma membrane formed patches that subsequently produced caps or were internalized through clathrin-mediated endocytosis participation. Moreover, the concentration of cell surface RSV Ag-Abs and intracellular viral proteins showed a time dependent cyclic variation and that anti-RSV IgG protected HEp-2 cells from viral-induced death. Conclusion The results from this study indicate that interaction between RSV cell surface proteins and specific viral antibodies alter the expression of viral antigens expressed on the cells surface and intracellular viral proteins; furthermore, interfere with viral induced destruction of the cell. PMID:17608950

Limitations of a localized surface plasmon resonance sensor on Salmonella detection

USDA-ARS?s Scientific Manuscript database

We have designed a localized surface plasmon resonance (LSPR) biosensor to perform the whole cell detection of Salmonella using gold nanoparticls fabricated by oblique angle deposition technique. The LSPR sensor showed a plasmon peak shift due to the Salmonella antigen and anti-Salmonella antibody r...

Detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology.

PubMed

Zijlmans, H J; Bonnet, J; Burton, J; Kardos, K; Vail, T; Niedbala, R S; Tanke, H J

1999-02-01

A novel luminescent reporter for the sensitive detection of antigens in tissue sections or on cell membranes is described. It consists of submicron-size phosphor crystals (0.2-0.4 microm), which are surface labeled with avidin or antibodies and capable of binding specifically to antigens on intact cells or in tissue sections. These phosphor reporters exhibit two-photon, anti-Stokes luminescence by up-converting infrared to visible light and are named Up-converting Phosphor Technology (UPT). They typically consist of yttriumoxysulfides doped with two different lanthanides exhibiting photostable, strong emission in the visible (blue, green, and red) upon excitation in the infrared. This report describes the conjugation of phosphor particles to NeutrAvidin with the subsequent use of this conjugate in a model system consisting of prostate-specific antigen in tissue sections and the CD4 membrane antigen on human lymphocytes. An epi-illumination fluorescence microscope was adapted to provide near-IR excitation using a xenon lamp for visualization of the visible emission. Advantages of UPT are (i) permanent, strong, anti-Stokes emission of discrete wavelengths; (ii) unmatched contrast in biological specimens due to the absence of autofluorescence upon excitation with IR light; (iii) simultaneous detection of multiple target analytes; and (iv) low-cost microscope modifications. The new methodology has not only high potential value in diagnostic pathology as described here, but may offer advantages for the detection of proteins or nucleic acids when applied to molecular biology, genomic research, virology, and microbiology. Copyright 1999 Academic Press.

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

PubMed

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

2007-06-01

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

Detection of egg yolk antibodies reflecting Salmonella enteritidis infections using a surface plasmon resonance biosensor.

PubMed

Thomas, Ekelijn; Bouma, Annemarie; van Eerden, Ellen; Landman, Wil J M; van Knapen, Frans; Stegeman, Arjan; Bergwerff, Aldert A

2006-08-31

A surface plasmon resonance (SPR) biosensor assay was developed on the basis of a lipopolysaccharide antigen of Salmonella enterica serovar enteritidis (S. enterica serovar enteritidis) to detect egg yolk antibodies against S. enterica serovar enteritidis. This biosensor assay was compared to two commercial ELISA kits based on LPS antigen and flagellar antigen. A number of 163 egg yolk and combined egg white and yolk samples from chickens experimentally infected with S. enterica serovar enteritidis and 90 egg yolk and combined egg white and yolk samples from uninfected chickens were analyzed. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 82% and a diagnostic specificity of 100%. The within-day coefficient of variation of a positive internal-control egg yolk was 1%. The SPR biosensor assay was able to detect antibodies in a significantly higher percentage of known positive samples than the commercial ELISA's. The anticipated use of the SPR biosensor assay is to determine the S. enterica serovar enteritidis serostatus of non-vaccinated layer hens.

Patterns of protective associations differ for antibodies to P. falciparum-infected erythrocytes and merozoites in immunity against malaria in children.

PubMed

Chan, Jo-Anne; Stanisic, Danielle I; Duffy, Michael F; Robinson, Leanne J; Lin, Enmoore; Kazura, James W; King, Christopher L; Siba, Peter M; Fowkes, Freya Ji; Mueller, Ivo; Beeson, James G

2017-12-01

Acquired antibodies play an important role in immunity to P. falciparum malaria and are typically directed towards surface antigens expressed by merozoites and infected erythrocytes (IEs). The importance of specific IE surface antigens as immune targets remains unclear. We evaluated antibodies and protective associations in two cohorts of children in Papua New Guinea. We used genetically-modified P. falciparum to evaluate the importance of PfEMP1 and a P. falciparum isolate with a virulent phenotype. Our findings suggested that PfEMP1 was the dominant target of antibodies to the IE surface, including functional antibodies that promoted opsonic phagocytosis by monocytes. Antibodies were associated with increasing age and concurrent parasitemia, and were higher among children exposed to a higher force-of-infection as determined using molecular detection. Antibodies to IE surface antigens were consistently associated with reduced risk of malaria in both younger and older children. However, protective associations for antibodies to merozoite surface antigens were only observed in older children. This suggests that antibodies to IE surface antigens, particularly PfEMP1, play an earlier role in acquired immunity to malaria, whereas greater exposure is required for protective antibodies to merozoite antigens. These findings have implications for vaccine design and serosurveillance of malaria transmission and immunity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

NASA Astrophysics Data System (ADS)

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

2017-01-01

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

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.

Recognition of Typhus Group Rickettsia-Infected Targets by Human Lymphokine-Activated Killer Cells

DTIC Science & Technology

1988-09-01

rick- Similar problems in detection of antigens of Theileria parva ettsia-specific cell surface antigens by performing polyacryl- (7) or influenza virus...infected with the protozoan parasite Theileria parva: workers in our laboratory are now in the process of cloning parasite strain specificity and class I

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.

Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance.

PubMed

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili; Fathi, Farzaneh; Ezzati Nazhad Dolatabadi, Jafar

2017-06-01

Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique. Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control. Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells. Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method.

Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance

PubMed Central

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili; Fathi, Farzaneh; Ezzati Nazhad Dolatabadi, Jafar

2017-01-01

Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique. Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control. Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells. Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method. PMID:28761820

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.

Design of a sensor for the blood AB0 group antibodies detection

NASA Astrophysics Data System (ADS)

Kolesov, D. V.; Kiselev, G. A.; Moiseev, M. A.; Kudrinskiy, A. A.; Yaminskiy, I. V.

2012-02-01

Control the content of the blood group antibodies in the plasma of the recipient is an important task in modern transplantation. In this paper we proposed to use micromechanical cantilever sensors for detection of the low concentrations of AB0 blood group antibodies in serum. The technique of chemical modification of cantilever surface to create the receptor layer was developed. The apparatus, which provides data acquisition from multiple microconsoles simultaneously was created. We carried out experiments by the detection in a solution the β antibodies with a concentration of 300 times less than the native content of antibodies in the blood. Change in surface stress due to formation of antigen-antibody complexes on the cantilever surface was 0.075 N/m. The resulting lateral strain, apparently, induced by repulsion between the complexes during the sorption of antibodies in layer of antigens, immobilized on the surface. The possibility of regeneration of sensory layer for repeated measurements was shown.

[Imaging of surface cell antigens on the tumor sections of lymph nodes using fluorescence quantum dots].

PubMed

Rafalovskaia-Orlovskaia, E P; Gorgidze, L A; Gladkikh, A A; Tauger, S M; Vorob'ev, I A

2012-01-01

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.

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

PubMed

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

2017-04-01

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

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.

Detection of immune deposits in glomeruli: the masking effect on antigenicity of formalin in the presence of proteins.

PubMed

Hed, J; Eneström, S

1981-01-01

Formalin is known to mask the antigenicity of immune deposits in glomeruli but not of surface immunoglobulins of isolated lymphocytes. We have shown in mice with experimental passive anti-GBM glomerulonephritis that formalin masks the antigenicity of GBM-bound immunoglobulins only if the tissue is fixed before sectioning. The presence of a high concentration of normal bovine serum during fixation of cryostat sections masks the antigenicity of immune deposits, whereas formalin alone has no obvious effect. The same results were obtained with human immunoglobulins (IgG, IgM and IgA) bound to tissue sections. Protease treatment with pepsin and trypsin restored the ability of the immunoglobulins to be stained. The masking effect seems to be due to extensive cross-linking of environmental proteins which prevents fluorescent conjugates reaching their antigens. Methods for detecting immunoglobulins in tissues must, therefore, take into consideration the influence of fixatives not only on epitopes but also on the environment in which the antigenic determinants are localised.

SEROLOGICAL RESPONSES TO CRYPTOSPORIDIUM ANTIGENS AMONG USERS OF SURFACE VERSUS UNDERGROUND DRINKING WATER SOURCES

EPA Science Inventory

Cryptosporidium oocysts have been detected in source and treated drinking waters in the United States and elsewhere. Enhanced enteric disease surveillance, initiated following detection of oocysts, has not often detected elevated rates of infection or of symptoms compatible with...

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.

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

PubMed

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

2005-01-01

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

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.

Detection of low-level environmental chemical allergy by a long-term sensitization method.

PubMed

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

2008-07-30

Multiple chemical sensitivity (MCS) is characterized by various signs, including neurological disorders and allergy. Exposure may occur through a major event, such as a chemical spill, or from long-term contact with chemicals at low levels. We are interested in the allergenicity of MCS and the detection of low-level chemical-related hypersensitivity. We used long-term sensitization followed by low-dose challenge to evaluate sensitization by well-known Th2 type sensitizers (trimellitic anhydride (TMA) and toluene diisocyanate (TDI)) and a Th1 type sensitizer (2,4-dinitrochlorobenzene (DNCB)). After topically sensitizing BALB/c mice (9 times in 3 weeks) and challenging them with TMA, TDI or DNCB, we assayed their auricular lymph nodes (LNs) for number of lymphocytes, surface antigen expression of B cells, and local cytokine production, and measured antigen-specific serum IgE levels. TMA and TDI induced marked increases in levels of antigen-specific serum IgE and of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) produced by ex vivo restimulated lymph node cells. DNCB induced a marked increase in Th1 cytokine (IL-2, IFN-gamma, and TNF-alpha) levels, but antigen-specific serum IgE levels were not elevated. All chemicals induced significant increases in number of lymphocytes and surface antigen expression of B cells. Our mouse model enabled the identification and characterization of chemical-related allergic reactions at low levels. This long-term sensitization method would be useful for detecting environmental chemical-related hypersensitivity.

Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

PubMed

Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

2004-10-01

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

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

PubMed Central

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

1987-01-01

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

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.

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

PubMed

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

2014-07-09

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

Surface plasmon-enhanced fluorescence on Au nanohole array for prostate-specific antigen detection

PubMed Central

Zhang, Qingwen; Wu, Lin; Wong, Ten It; Zhang, Jinling; Liu, Xiaohu; Zhou, Xiaodong; Bai, Ping; Liedberg, Bo; Wang, Yi

2017-01-01

Localized surface plasmon (LSP) has been widely applied for the enhancement of fluorescence emission for biosensing owing to its potential for strong field enhancement. However, due to its small penetration depth, LSP offers limited fluorescence enhancement over a whole sensor chip and, therefore, insufficient sensitivity for the detection of biomolecules, especially large molecules. We demonstrate the simultaneous excitation of LSP and propagating surface plasmon (PSP) on an Au nanohole array under Kretschmann configuration for the detection of prostate-specific antigen with a sandwich immunoassay. The proposed method combines the advantages of high field enhancement by LSP and large surface area probed by PSP field. The simulated results indicated that a maximum enhancement of electric field intensity up to 1,600 times can be achieved under the simultaneous excitation of LSP and PSP modes. The sandwich assay of PSA carried out on gold nanohole array substrate showed a limit of detection of 140 fM supporting coexcitation of LSP and PSP modes. The limit of detection was approximately sevenfold lower than that when only LSP was resonantly excited on the same substrate. The results of this study demonstrate high fluorescence enhancement through the coexcitation of LSP and PSP modes and pave a way for its implementation as a highly sensitive bioassay. PMID:28392689

Label-free detection of surface markers on stem cells by oblique-incidence reflectivity difference microscopy

PubMed Central

Lo, Kai-Yin; Sun, Yung-Shin; Landry, James P.; Zhu, Xiangdong; Deng, Wenbin

2012-01-01

Conventional fluorescent microscopy is routinely used to detect cell surface markers through fluorophore-conjugated antibodies. However, fluorophore-conjugation of antibodies alters binding properties such as strength and specificity of the antibody in ways often uncharacterized. The binding between antibody and antigen might not be in the native situation after such conjugation. Here, we present an oblique-incidence reflectivity difference (OI-RD) microscope as an effective method for label-free, real-time detection of cell surface markers and apply such a technique to analysis of Stage-Specific Embryonic Antigen 1 (SSEA1) on stem cells. Mouse stem cells express SSEA1 on their surfaces and the level of SSEA1 decreases when the cells start to differentiate. In this study, we immobilized mouse stem cells and non-stem cells (control) on a glass surface as a microarray and reacted the cell microarray with unlabeled SSEA1 antibodies. By monitoring the reaction with an OI-RD microscope in real time, we confirmed that the SSEA1 antibodies only bind to the surface of the stem cells while not to the surface of non-stem cells. From the binding curves, we determined the equilibrium dissociation constant (Kd) of the antibody with the SSEA1 markers on the stem cell surface. The results concluded that OI-RD microscope can be used to detect binding affinities between cell surface markers and unlabeled antibodies bound to the cells. The information could be another indicator to determine the cell stages. PMID:21781038

Gold nanoparticles mediated colorimetric assay for HIV-Tat protein detection

NASA Astrophysics Data System (ADS)

Hashwan, Saeed S. Ba; Ruslinda, A. Rahim; Fatin, M. F.; Gopinath, Subash C. B.; Thivina, V.; Tony, V. C. S.; Arshad, M. K. Md.; Hashim, U.

2016-07-01

Gold-nanoparticle (AuNP) based colorimetric assays have been formulated for different biomolecular interactions. With this assay the probe such as antibody immobilized on the Au surface and in the presence of appropriate binding partner (antigen), will interact with each other on the Au surface. By following this strategy, herein we formulated a detection system with two anti-HIV-Tat antibodies, Mono (McAb) - and polyclonal (PcAb) by immobilizing them independently with different AuNPs. Under this condition, these two antibodies are under dispersed condition, and in the presence of HIV-Tat antigen, these molecules will be connected and forms the aggregation of AuNPs. This strategy yield rapid results, can be monitored by the spectral changes in UV-Vis spectrophotometry. Experiments were performed with two different methods using two anti-HIV-Tats monoclonal and one Polyclonal antibody against the antigen HIV-Tat. Between these methods conjugation of HIV-Tat and McAb on the AuNP followed by addition of PcAb yielded better results.

Nanoporous gold as a solid support for protein immobilization and development of an electrochemical immunoassay for prostate specific antigen and carcinoembryonic antigen

PubMed Central

Pandey, Binod; Demchenko, Alexei V.; Stine, Keith J.

2013-01-01

Nanoporous gold (NPG) was utilized as a support for immobilizing alkaline phosphatase (ALP) conjugated to monoclonal antibodies against either prostate specific antigen (PSA) or carcinoembryonic antigen (CEA). The antibody-ALP conjugates were coupled to self-assembled monolayers of lipoic acid and used in direct kinetic assays. Using the enzyme substrate p-aminophenylphosphate, the product p-aminophenol was detected by its oxidation near 0.1 V (vs. Ag|AgCl) using square wave voltammetry. The difference in peak current arising from oxidation of p-aminophenol before and after incubation with biomarker increased with biomarker concentration. The response to these two biomarkers was linear up to 10 ng mL-1 for CEA and up to 30 ng mL-1 for PSA. The effect of interference on the PSA assay was studied using bovine serum albumin (BSA) as a model albumin protein. The effect of interference from a serum matrix was examined for the PSA assay using newborn calf serum. A competitive version of the immunoassay using antigen immobilized onto the NPG surface was highly sensitive at lower antigen concentration. Estimates of the surface coverage of the antibody-ALP conjugates on the NPG surface are presented. PMID:23935216

A placebo controlled observer blind immunocytochemical and histologic study of epithelium adjacent to anogenital warts in patients treated with systemic interferon alpha in combination with cryotherapy or cryotherapy alone.

PubMed Central

Handley, J M; Maw, R D; Horner, T; Lawther, H; Walsh, M; Dinsmore, W W

1992-01-01

OBJECTIVE--To examine biopsy specimens of tissue immediately adjacent to anogenital (AG) warts which had been treated with either cryotherapy plus subcutaneous interferon (IFN) alpha 2a or cryotherapy alone, for histological features of (a) human papilloma virus (HPV) infection (b) localised cellular immune responses, to further characterise any cellular immune infiltrates with tissue immunocytochemistry, and to relate any histological, immunocytochemical findings to the treatment response of nearby AG warts. DESIGN--A randomised placebo controlled observer blind study. SETTING--Genitourinary Medicine clinic, Department of Immunopathology, Royal Victoria Hospital, Belfast, N. Ireland. SUBJECTS--Thirty patients with AG warts; 16 treated with IFN alpha 2a plus cryotherapy, and 14 treated with cryotherapy alone. OUTCOME MEASURES--(1) Light microscopic features associated with HPV infection and local cellular immune responses. (2) Indirect immunofluorescence detection of the following cell surface markers: HLA DR, alpha one antitrypsin, CD1, CD3, CD4, CD8, CD22. (3) Clinical response of AG warts to treatment. RESULTS--In pre-treatment biopsies only non specific indicators of HPV infection (acanthosis, 29/30 biopsies, and hyperkeratosis, 7/30 biopsies) were seen on light microscopy. Mononuclear cells were seen both throughout the upper dermis and centred around dermal blood vessels in 19/30 (63.3%) biopsies, and infiltrating into the epidermis in 12/30 (40%) biopsies. On indirect immunofluorescence CD3, CD8, CD4 antigen was detected on the surface of cells throughout the upper dermis in 24/29 (82.7%), 15/29 (51.7%), and 3/29 (10.3%), of biopsy specimens respectively. CD3 antigen, CD8 antigen and CD4 antigen was detected on the surface of cells infiltrating into the epidermis in 18/29 (62%), 7/29 (24.1%), and 6/29 (20.7%) of biopsy specimens respectively. CD1 antigen was seen on the surface of dendritic cells throughout the epidermis in all specimens; CD1 positive cells infiltrated into the upper dermis in 5/29 (17.2%). HLA DR was detected on the surface of dendritic cells throughout the epidermis in 22/29 (75.9%) of specimens, and on the surface of cells scattered both diffusely throughout the upper dermis and centred around dermal blood vessels in all specimens. Alpha one antitrypsin (A1AT) antigen was seen on the surface of cells in the upper dermis in 6/29 (20.7%) of biopsy specimens; no cells expressing CD22 surface antigen were seen. The nature of this local cellular immune response was not altered by treatment of nearby warts with either cryotherapy alone or cryotherapy plus systemic IFN alpha 2a, or related to the therapeutic outcome of these warts. CONCLUSIONS--(1) No convincing histological evidence of HPV infection was seen in epithelium surrounding AG warts. (2) A predominantly T cell-mediated immune response (the target of which is uncertain) was seen in this perilesional epithelium. (3) In the dosage regimens used in this study, treatment of AG warts with either systemic IFN alpha 2a plus cryotherapy or cryotherapy alone did not appear to augment localised cellular immune responses (against any presumed subclinical HPV infection) in epithelium surrounding AG warts. Images PMID:1316307

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

PubMed

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

2012-08-21

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

Detection of Salmonella enteritidis Using a Miniature Optical Surface Plasmon Resonance Biosensor

NASA Astrophysics Data System (ADS)

Son, J. R.; Kim, G.; Kothapalli, A.; Morgan, M. T.; Ess, D.

2007-04-01

The frequent outbreaks of foodborne illness demand rapid detection of foodborne pathogens. Unfortunately, conventional methods for pathogen detection and identification are labor-intensive and take days to complete. Biosensors have shown great potential for the rapid detection of foodborne pathogens. Surface plasmon resonance (SPR) sensors have been widely adapted as an analysis tool for the study of various biological binding reactions. SPR biosensors could detect antibody-antigen bindings on the sensor surface by measuring either a resonance angle or refractive index value. In this study, the feasibility of a miniature SPR sensor (Spreeta, TI, USA) for detection of Salmonella enteritidis has been evaluated. Anti-Salmonella antibodies were immobilized on the gold sensor surface by using neutravidin. Salmonella could be detected by the Spreeta biosensor at concentrations down to 105 cfu/ml.

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.

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

PubMed

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

2018-06-01

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

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

NASA Astrophysics Data System (ADS)

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

2014-12-01

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

Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae

PubMed Central

Hathroubi, S.; Hancock, M. A.; Langford, P. R.; Tremblay, Y. D. N.; Labrie, J.

2015-01-01

Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. PMID:26483403

Rapid detection of hepatitis B virus surface antigen by an agglutination assay mediated by a bispecific diabody against both human erythrocytes and hepatitis B virus surface antigen.

PubMed

Chen, Yu-Ping; Qiao, Yuan-Yuan; Zhao, Xiao-Hang; Chen, Hong-Song; Wang, Yan; Wang, Zhuozhi

2007-06-01

Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens.

Tracking serum antibody response to viral antigens with arrayed imaging reflectometry

NASA Astrophysics Data System (ADS)

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

2009-02-01

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

Development of nanoparticle applications in cell imaging, bioassay and reactive oxygen species detection based on surface-enhanced raman spectroscopy

NASA Astrophysics Data System (ADS)

Huang, Yiming

Surface-enhanced Raman scattering (SERS) has been developed over forty years with a wide variety of applications. Signals enhanced from single molecule absorbed on the surface of metallic nanoparticles can be up to 14-order-of-magnitude. This is due to the resonance between the optical field and surface plasmon of the metal substrate. Nanoshells have been chosen as substrates since they do not need to pre-aggregate due to their tunable optical property. We developed Raman imaging system by incorporating functionalized nanoshells, cells and SERS. Nanoshells have been coated with different self-assembled monolayers containing poly(ethylene glycol) (PEG) molecules. Probes have been designed by coating nanoshells with Raman active PEG molecules and delivered into macrophage cells. The imaging technique requires less preparation and provides the information of nanoshells in semi-quantitative way in vitro. We developed half-sandwich bioassay by detecting low volume of antigens on nitrocellulose membrane, detected by SERS. Antibodies were grafted to the surface of nanoshells and were conjugated to the antigens on the nitrocellulose membrane for detection. Raman active PEGs were conjugated onto the metal substrate for recognition and quantification. The benefits of this assay are that it is faster, easier to execute and requires less volume of antigen to conjugate onto the substrate. We also developed reactive oxygen species (ROS) sensors by incubating PEGs and either 4-nitrobenzenethiol (4-NBT) or 4-mercaptophenol (4-MP) on the surface of nanoshells. By analyzing the changes of SERS spectrum, the production of hydroxyl radicals produced in the Fenton reaction can be tracked in low concentrations. The sensors were designed to track ROS production within cells when they are under oxidative stress. The methods developed in this thesis are versatile, and can be broadly applied to the study of different subtracts, such as gold colloid.

Linearized hepatitis B surface antigen and hepatitis B core-related antigen in the natural history of chronic hepatitis B.

PubMed

Seto, W-K; Wong, D K-H; Fung, J; Huang, F-Y; Liu, K S-H; Lai, C-L; Yuen, M-F

2014-11-01

Changes in two novel HBV serological markers, linearized hepatitis B surface antigen (HQ-HBsAg) and hepatitis B core-related antigen (HBcrAg), in the natural history of chronic hepatitis B (CHB) have not been well characterized. Serum HQ-HBsAg and HBcrAg levels of 404 Asian treatment-naïve CHB patients were analysed in a cross-sectional manner. Patients were categorized into five groups: immune tolerant (IT group, n=52), immune clearance (IC group, n=105), hepatitis B e antigen (HBeAg)-negative hepatitis (ENH group, n=97), HBeAg-negative quiescent group (ENQ group, n=95) and CHB with hepatitis B surface antigen (HBsAg) seroclearance (SC group, n=55). HQ-HBsAg and HBcrAg were measured and correlated with HBV DNA, HBsAg, HBV genotype and clinical parameters. HQ-HBsAg showed good correlation with HBsAg, especially in the ENQ group (r=0.874, p<0.001). Correlation of HQ-HBsAg with HBV DNA was less prominent and weakest in the ENH group (r=0.268, p 0.008). HBcrAg correlated best with HBV DNA in the ENQ group (r=0.537, p<0.001). In the ENQ group, 42.1% of patients had undetectable HBcrAg; this subgroup of patients, when compared with those with detectable HBcrAg, had significantly lower median HBV DNA (3.17/4.48 log IU/mL, p<0.001) and HBsAg (5.05/5.96 log mIU/mL, p<0.001) levels. Forty per cent of the SC group patients had detectable HQ-HBsAg and/or HBcrAg up to 42 months after HBsAg seroclearance. When comparing anti-HBs positivity and median time after HBsAg seroclearance in the SC group with and without detectable HQ-HBsAg/HBcrAg, there was no significant difference (22.7% and 36.4%, respectively, p 0.284, and 76.5 and 93.2 months, respectively, p 0.245). HQ-HBsAg and HBcrAg showed unique patterns of distribution throughout the five disease phases of CHB, including high detectability rates after HBsAg seroclearance, opening up different possibilities for their applicability. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Nanoparticle-based flow virometry for the analysis of individual virions

PubMed Central

Arakelyan, Anush; Fitzgerald, Wendy; Margolis, Leonid; Grivel, Jean-Charles

2013-01-01

While flow cytometry has been used to analyze the antigenic composition of individual cells, the antigenic makeup of viral particles is still characterized predominantly in bulk. Here, we describe a technology, “flow virometry,” that can be used for antigen detection on individual virions. The technology is based on binding magnetic nanoparticles to virions, staining the virions with monoclonal antibodies, separating the formed complexes with magnetic columns, and characterizing them with flow cytometers. We used this technology to study the distribution of two antigens (HLA-DR and LFA-1) that HIV-1 acquires from infected cells among individual HIV-1 virions. Flow virometry revealed that the antigenic makeup of virions from a single preparation is heterogeneous. This heterogeneity could not be detected with bulk analysis of viruses. Moreover, in two preparations of the same HIV-1 produced by different cells, the distribution of antigens among virions was different. In contrast, HIV-1 of two different HIV-1 genotypes replicating in the same cells became somewhat antigenically similar. This nanotechnology allows the study of virions in bodily fluids without virus propagation and in principle is not restricted to the analysis of HIV, but can be applied to the analysis of the individual surface antigenic makeup of any virus. PMID:23925291

Genetic diversity of K-antigen gene clusters of Escherichia coli and their molecular typing using a suspension array.

PubMed

Yang, Shuang; Xi, Daoyi; Jing, Fuyi; Kong, Deju; Wu, Junli; Feng, Lu; Cao, Boyang; Wang, Lei

2018-04-01

Capsular polysaccharides (CPSs), or K-antigens, are the major surface antigens of Escherichia coli. More than 80 serologically unique K-antigens are classified into 4 groups (Groups 1-4) of capsules. Groups 1 and 4 contain the Wzy-dependent polymerization pathway and the gene clusters are in the order galF to gnd; Groups 2 and 3 contain the ABC-transporter-dependent pathway and the gene clusters consist of 3 regions, regions 1, 2 and 3. Little is known about the variations among the gene clusters. In this study, 9 serotypes of K-antigen gene clusters (K2ab, K11, K20, K24, K38, K84, K92, K96, and K102) were sequenced and correlated with their CPS chemical structures. On the basis of sequence data, a K-antigen-specific suspension array that detects 10 distinct CPSs, including the above 9 CPSs plus K30, was developed. This is the first report to catalog the genetic features of E. coli K-antigen variations and to develop a suspension array for their molecular typing. The method has a number of advantages over traditional bacteriophage and serum agglutination methods and lays the foundation for straightforward identification and detection of additional K-antigens in the future.

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

PubMed

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

2018-04-17

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

Optoacoustic detection of viral antigens using targeted gold nanorods

NASA Astrophysics Data System (ADS)

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

2009-02-01

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

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

NASA Astrophysics Data System (ADS)

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

2015-10-01

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

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

NASA Astrophysics Data System (ADS)

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

2013-12-01

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

Localized Surface Plasmon Resonance (LSPR)-Coupled Fiber-Optic Nanoprobe for the Detection of Protein Biomarkers.

PubMed

Wei, Jianjun; Zeng, Zheng; Lin, Yongbin

2017-01-01

Here is presented a miniaturized, fiber-optic (FO) nanoprobe biosensor based on the localized surface plasmon resonance (LSPR) at the reusable dielectric-metallic hybrid interface with a robust, gold nano-disk array at the fiber end facet. The nanodisk array is directly fabricated using electron beam lithography (EBL) and metal lift-off process. The free prostate-specific antigen (f-PSA) has been detected with a mouse anti-human prostate-specific antigen (PSA) monoclonal antibody (mAb) as a specific receptor linked with a self-assembled monolayer (SAM) at the LSPR-FO facet surfaces. Experimental investigation and data analysis found near field refractive index (RI) sensitivity at ~226 nm/RIU with the LSPR-FO nanoprobe, and demonstrated the lowest limit of detection (LOD) at 100 fg/mL (~3 fM) of f-PSA in PBS solutions. The SAM shows insignificant nonspecific binding to the target biomarkers in the solution. The control experimentation using 5 mg/mL bovine serum albumin in PBS and nonspecific surface test shows the excellent specificity and selectivity in the detection of f-PSA in PBS. These results indicate important progress toward a miniaturized, multifunctional fiber-optic technology that integrates informational communication and sensing function for developing a high-performance, label-free, point-of-care (POC) device.

Design and Preparation of Nanoparticle Dimers for SERS Detection

DTIC Science & Technology

2012-09-10

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

Array biosensor: recent developments

NASA Astrophysics Data System (ADS)

Golden, Joel P.; Rowe-Taitt, Chris A.; Feldstein, Mark J.; Ligler, Frances S.

1999-05-01

A fluorescence-based immunosensor has been developed for simultaneous analyses of multiple samples for 1 to 6 different antigens. A patterned array of recognition antibodies immobilized on the surface of a planar waveguide is used to 'capture' analyte present in samples. Bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. A new design for a fluidics distribution system is shown, as well as results from assays for physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D- dimer, a marker of sepsis and thrombotic disorders.

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1.

PubMed

Howe, Daniel K; Gaji, Rajshekhar Y; Marsh, Antoinette E; Patil, Bhagyashree A; Saville, William J; Lindsay, David S; Dubey, J P; Granstrom, David E

2008-05-01

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages of their life cycle in opossums.

Surface plasmon resonance label-free monitoring of antibody antigen interactions in real time

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

Kausaite, A.; van Dijk, M.; Castrop, J.

2007-01-01

Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label-free detection of biologically active compounds. This method allows one to monitor binding events in real time without labeling. The system can therefore be used to determine both affinity and rate constants for interactions between various types of molecules. Here, we describe the application of a surface plasmon resonance biosensor for label-free investigation of the interaction between an immobilized antigen bovine serum albumin (BSA) and antibodymore » rabbit anti-cow albumin IgG1 (anti-BSA). The formation of a self-assembled monolayer (SAM) over a gold surface is introduced into this laboratory training protocol as an effective immobilization method, which is very promising in biosensing systems based on detection of affinity interactions. In the next step, covalent attachment via artificially formed amide bonds is applied for the immobilization of proteins on the formed SAM surface. These experiments provide suitable experience for postgraduate students to help them understand immobilization of biologically active materials via SAMs, fundamentals of surface plasmon resonance biosensor applications, and determination of non-covalent biomolecular interactions. The experiment is designed for master and/or Ph.D. students. In some particular cases, this protocol might be adoptable for bachelor students that already have completed an extended biochemistry program that included a background in immunology.« less

Adsorption of virus-like particles on ion exchange surface: Conformational changes at different pH detected by dual polarization interferometry.

PubMed

Yang, Yanli; Mengran Yu; Zhang, Songping; Ma, Guanghui; Su, Zhiguo

2015-08-21

Disassembling of virus-like particles (VLPs) like hepatitis B virus surface antigen (HB-VLPs) during chromatographic process has been identified as a major cause of loss of antigen activity. In this study, dual polarization interferometry (DPI) measurement, together with chromatography experiments, were performed to study the adsorption and conformational change of HB-VLPs on ion exchange surface at three different pHs. Changes in pH values of buffer solution showed only minimal effect on the HB-VLPs assembly and antigen activity, while significantly different degree of HB-VLPs disassembling was observed after ion exchange chromatography (IEC) at different pHs, indicating the conformational change of HB-VLPs caused mainly by its interactions with the adsorbent surface. By creating an ion exchange surface on chip surface, the conformational changes of HB-VLPs during adsorption to the surface were monitored in real time by DPI for the first time. As pH increased from 7.0 to 9.0, strong electrostatic interactions between oppositely charged HB-VLPs and the ion exchange surface make the HB-VLPs spread thinly or even adsorbed in disassembled formation on the surface as revealed by significant decrease in thickness of the adsorbed layer measured by DPI. Such findings were consistent with the results of IEC experiments operated at different pHs, that more disassembled HB-VLPs were detected in the eluted proteins at pH 9.0. At low pH like pH 5.0, however, possible bi-layer adsorption was involved as evidenced by an adsorbed layer thickness higher than average diameter of the HB-VLPs. The "lateral" protein-protein interactions might be unfavorable and would make additional contribution to the disassembling of HB-VLPs besides the primary mechanism related to the protein-surface interactions; therefore, the lowest antigen activity was observed after IEC at pH 5.0. Such real-time information on conformational change of VLPs is helpful for better understanding the real mechanism for the disassembling of VLPs on the solid-liquid interface. Copyright © 2015 Elsevier B.V. All rights reserved.

Biofluidic Transport and Molecular Recognition in Polymer Microdevices

DTIC Science & Technology

2005-04-29

flexible membrane separating the particles and reservoir. B. Using photopolymerizable wires, an electrolysis pump was fabricated on a microdevice. It...Antigen detection was accomplished by grafting the approximate antibody or sensing compound via acrylation and polymerization to the surface. Figure 14...were detected with assay times of approximately 10 minutes. Figure 15 shows detection data for a compound (glucagon) that is impossible to detect by

A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode.

PubMed

Kong, Fen-Ying; Xu, Mao-Tian; Xu, Jing-Juan; Chen, Hong-Yuan

2011-10-15

In this paper, gold nanoparticle-thionine-reduced graphene oxide (GNP-THi-GR) nanocomposites were prepared to design a label-free immunosensor for the sensitive detection of carcinoembryonic antigen (CEA). The nanocomposites with good biocompatibility, excellent redox electrochemical activity and large surface area were coated onto the glassy carbon electrode (GCE) surface and then CEA antibody (anti-CEA) was immobilized on the electrode to construct the immunosensor. The morphologies and electrochemistry of the formed nanocomposites were investigated by using scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrometry, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) studies demonstrated that the formation of antibody-antigen complexes decreased the peak current of THi in the GNP-THi-GR nanocomposites. The decreased currents were proportional to the CEA concentration in the range of 10-500 pg/mL with a detection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determination of CEA at very low levels. Copyright © 2011 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2016-04-01

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-01-01

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

Hepatitis B serological markers and plasma DNA concentrations

PubMed Central

Price, Huw; Dunn, David; Zachary, Tamale; Vudriko, Tobias; Chirara, Michael; Kityo, Cissy; Munderi, Paula; Spyer, Moira; Hakim, James; Gilks, Charles; Kaleebu, Pontiano; Pillay, Deenan; Gilson, Richard

2017-01-01

Objectives: To examine hepatitis B (HBV) serological markers and plasma DNA concentrations in a large group of untreated HBV/HIV-coinfected individuals in two sub-Saharan settings. Design: Baseline analysis of a randomized controlled trial. Methods: DART was a large trial of treatment monitoring practices in HIV-infected adults with advanced disease starting antiretroviral therapy at centres in Kampala or Entebbe, Uganda (n = 2317) and Harare, Zimbabwe (n = 999). HBV serological markers [antibody to HBV core antigen, HBV surface antigen (HBsAg), antibody to HBV surface antigen, HBV ‘e’ antigen (HBeAg), and antibody to hepatitis B ‘e’ antigen] and plasma HBV DNA viral load were measured retrospectively on stored baseline samples. Logistic regression was used to examine associations with baseline demographic and clinical factors. Results: The rate of HBsAg positivity was significantly higher in Zimbabwe than Uganda (12.2 vs. 7.7%, adjusted odds ratio = 1.54, P < 0.001) despite a similar prevalence of antibody to HBV core antigen (56.3 vs. 52.4%) in the two settings. Overall, HBsAg positivity was associated with male sex (adjusted odds ratio = 1.54, P < 0.001) but not with age, WHO disease stage, or CD4+ cell count. HBeAg was detected among 37% of HBsAg-positive patients, with higher rates among those with advanced WHO stage (P = 0.02). Also in HBsAg-positive patients, HBV DNA was undetectable in 21%, detectable but below the level of quantification in 14%, and quantifiable in 65%. A total of 96% of HBeAg-positive and 70% of HBeAg-negative patients had detectable HBV DNA; 92 and 28% of patients, respectively, had HBV DNA viral load more than 2000 IU/ml. Conclusion: High rates of HBV coinfection were observed, highlighting the importance of ensuring that coinfected patients receive an antiretroviral regimen, whether first-line or not, that is active against both viruses. PMID:28328795

Methods and compositions for diagnosing and preventing a group B streptococcal infection

DOEpatents

Brady, Linda Jeannine [Gainesville, FL; Seifert, Kyle N [Harrisonburg, VA; Adderson, Elisabeth E [Memphis, TN; Bohnsack, John F [Salt Lake City, UT

2009-09-15

The present invention provides a group B streptococcal (GBS) surface antigen, designated epsilon antigen, that is co-expressed with the delta antigen on a subset of serotype III GBS. Epsilon is expressed on more pathogenic Restriction Digest Pattern (RDP) III-3 GBS, but not on RDP types 1, 2, or 4. Accordingly, the present invention provides compositions and methods for detecting a group B streptococcus serotype III, RDP III-3 strain. Vaccines and methods of identifying agents which inhibit adhesion of a group B streptococcal cell to a host cell are also provided.

A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues.

PubMed Central

Casey, T. T.; Cousar, J. B.; Collins, R. D.

1988-01-01

Routine fixation and paraffin embedding destroys many hematopoietic and lymphoid differentiation antigens detected by flow cytometry or frozen section immunohistochemistry. On the other hand, morphologic evaluation is difficult in flow cytometric or frozen section studies. A simplified three-step plastic embedding system using acetone-fixed tissues embedded in glycol-methacrylate (GMA) resin has been found to provide both excellent morphologic and antigenic preservation. With our system, a wide variety of antigens are detected in plastic sections without trypsinization or prolonged embedding procedures; pan-B (CD19, CD22), pan-T (CD7, CD5, CD3, CD2), T-subset (CD4, CD8, CD1, CD25) markers as well as surface immunoglobulin and markers for myeloid and mononuclear-phagocyte cells are preserved. In summary, modifications of plastic embedding techniques used in this study simplify the procedure, apparently achieve excellent antigenic preservation, and facilitate evaluation of morphologic details in relation to immunocytochemical markers. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:3282442

Concurrent detection of secreted products from human lymphocytes by microengraving: cytokines and antigen-reactive antibodies

PubMed Central

Bradshaw, Elizabeth M.; Kent, Sally C.; Tripuraneni, Vinay; Orban, Tihamer; Ploegh, Hidde L.; Hafler, David A.; Love, J. Christopher

2008-01-01

Cell surface determinants, cytokines and antibodies secreted by hematopoietic cells are used to classify their lineage and function. Currently available techniques are unable to elucidate multiple secreted proteins while also assigning phenotypic surface-displayed markers to the individual living cells. Here, a soft lithographic method, microengraving, was adapted for the multiplexed interrogation of populations of individual human peripheral blood mononuclear cells for secreted cytokines (IFN-γ and IL-6), antigen-specific antibodies, and lineage-specific surface-expressed markers. Application of the method to a clinical sample from a recent onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that ~0.58% of circulating CD19+ B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2–3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products. PMID:18675591

A laser scanning confocal imaging-surface plasmon resonance system application in real time detection of antibody-antigen interaction

NASA Astrophysics Data System (ADS)

Zhang, H. Y.; Yang, L. Q.; Liu, W. M.

2011-12-01

The laser scanning confocal microscope (LSCM) offers several advantages over conventional optical microscopy, but most LSCM work is qualitative analysis and it is very hard to achieve quantitative detection directly with the changing of the fluorescent intensity. A new real time sensor system for the antibody-antigen interaction detection was built integrating with a LSCM and a wavelength-dependent surface plasmon resonance (SPR) sensor. The system was applied to detect the bonding process of human IgG and fluorescent-labeled affinity purified antibody in real time. The fluorescence images changing is well with that of SPR wavelengths in real time, and the trend of the resonance wavelength shift with the concentrations of antibody is similar to that of the fluorescent intensity changing. The results show that SPR makes up the short of quantificational analysis with LSCM with the high spatial resolution. The sensor system shows the merits of the of the LSCM and SPR synergetic application, which are of great importance for practical application in biosensor and life science for interesting local interaction.

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

PubMed Central

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

2014-01-01

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

Porous silicon photoluminescence biosensor for rapid and sensitive detection of toxins

NASA Astrophysics Data System (ADS)

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

2017-08-01

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

Immobilization of proteins on glow discharge treated polymers

NASA Astrophysics Data System (ADS)

Kiaei, D.; Safranj, A.; Chen, J. P.; Johnston, A. B.; Zavala, F.; Deelder, A.; Castelino, J. B.; Markovic, V.; Hoffman, A. S.

Certain glow discharge-treated surfaces have been shown to enhance retention of adsorbed proteins. On the basis of this phenomenon, we have investigated the possibility of immobilizing (a) albumin for developing thromboresistant and non-fouling surfaces, (b) antibodies for immuno-diagnostic assays and (c) enzymes for various biosensors and industrial bioprocesses. Albumin retention was highest on surfaces treated with tetrafluoroethylene (TFE) compared to untreated surfaces or other glow discharge treatments studied. Preadsorption of albumin on TFE-treated surfaces resulted in low fibrinogen adsorption and platelet adhesion. IgG retention was also highest on TFE-treated surfaces. The lower detection limits of both malaria antigen and circulating anodic antigen of the schistosomiasis worm were enhanced following glow discharge treatment of the assay plates with TFE. Both TFE and tetrachloroethylene (TCE) glow discharge treated surfaces showed high retention of adsorbed horseradish peroxidase (HRP). However, the retained specific activity of HRP after adsorption on TCE-treated surfaces was remarkably higher than on TFE-treated surfaces.

Gold nanoparticles paper as a SERS bio-diagnostic platform.

PubMed

Ngo, Ying Hui; Then, Whui Lyn; Shen, Wei; Garnier, Gil

2013-11-01

Bioactive papers are usually challenged by four major limitations: sensitivity, selectivity, simplicity and strength (4S). Gold nanoparticles (AuNPs) treated paper has previously been demonstrated as a Surface Enhanced Raman Scattering (SERS) active substrate, capable of addressing the 4S issues. In this study, AuNPs on paper substrate were functionalized by a series of biomolecules to develop a generic SERS platform for antibody-antigen detection. The functionalization steps were performed by taking advantage of the high affinity association between Streptomyces avidinii-derived protein, streptavidin, and biotin. Streptavidin was firstly bound onto the AuNPs treated paper using biotinylated-thiol. Subsequently, desired biotinylated-antibody was bound onto the streptavidin. SERS spectra of each functionalization step were obtained to ensure specific adsorption of the bio-molecules. The binding interaction of the antibody with its specific antigen was detected using SERS. Shifts of Raman band associated with α-helix and β-sheet structures indicated structural modification of the antibody upon interaction with its antigen. Predominant tryptophan and tyrosine residue bands were also detected, confirming the presence of antigen. Reproducible spectral features were quantified as AuNP papers were subjected to different concentrations of antigen; the spectra intensity increased as a function of the antigen concentration. The retention of AuNPs on paper remained constant after all the consecutive washing and functionalization steps. The feasibility of AuNPs paper as a low-cost and generic SERS platform for bio-diagnostic applications was demonstrated. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Stabilizing short-lived Schiff base derivatives of 5-aminouracils that activate mucosal-associated invariant T cells

NASA Astrophysics Data System (ADS)

Mak, Jeffrey Y. W.; Xu, Weijun; Reid, Robert C.; Corbett, Alexandra J.; Meehan, Bronwyn S.; Wang, Huimeng; Chen, Zhenjun; Rossjohn, Jamie; McCluskey, James; Liu, Ligong; Fairlie, David P.

2017-03-01

Mucosal-associated invariant T (MAIT) cells are activated by unstable antigens formed by reactions of 5-amino-6-D-ribitylaminouracil (a vitamin B2 biosynthetic intermediate) with glycolysis metabolites such as methylglyoxal. Here we show superior preparations of antigens in dimethylsulfoxide, avoiding their rapid decomposition in water (t1/2 1.5 h, 37 °C). Antigen solution structures, MAIT cell activation potencies (EC50 3-500 pM), and chemical stabilities are described. Computer analyses of antigen structures reveal stereochemical and energetic influences on MAIT cell activation, enabling design of a water stable synthetic antigen (EC50 2 nM). Like native antigens, this antigen preparation induces MR1 refolding and upregulates surface expression of human MR1, forms MR1 tetramers that detect MAIT cells in human PBMCs, and stimulates cytokine expression (IFNγ, TNF) by human MAIT cells. These antigens also induce MAIT cell accumulation in mouse lungs after administration with a co-stimulant. These chemical and immunological findings provide new insights into antigen properties and MAIT cell activation.

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.

Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

PubMed

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-07-01

The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

Recombinant antibodies and their use in biosensors.

PubMed

Zeng, Xiangqun; Shen, Zhihong; Mernaugh, Ray

2012-04-01

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

Simultaneous multicolor detection system of the single-molecular microbial antigen by total internal reflection fluorescence microscopy with fluorescent nanocrystal quantum dots

NASA Astrophysics Data System (ADS)

Hoshino, Akiyoshi; Fujioka, Kouki; Yamamoto, Mayu; Manabe, Noriyoshi; Yasuhara, Masato; Suzuki, Kazuo; Yamamoto, Kenji

2005-11-01

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystals QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

Affinity ranking of antibodies using flow cytometry: application in antibody phage display-based target discovery.

PubMed

Geuijen, Cecilia A W; Clijsters-van der Horst, Marieke; Cox, Freek; Rood, Pauline M L; Throsby, Mark; Jongeneelen, Mandy A C; Backus, Harold H J; van Deventer, Els; Kruisbeek, Ada M; Goudsmit, Jaap; de Kruif, John

2005-07-01

Application of antibody phage display to the identification of cell surface antigens with restricted expression patterns is often complicated by the inability to demonstrate specific binding to a certain cell type. The specificity of an antibody can only be properly assessed when the antibody is of sufficient high affinity to detect low-density antigens on cell surfaces. Therefore, a robust and simple assay for the prediction of relative antibody affinities was developed and compared to data obtained using surface plasmon resonance (SPR) technology. A panel of eight anti-CD46 antibody fragments with different affinities was selected from phage display libraries and reformatted into complete human IgG1 molecules. SPR was used to determine K(D) values for these antibodies. The association and dissociation of the antibodies for binding to CD46 expressed on cell surfaces were analysed using FACS-based assays. We show that ranking of the antibodies based on FACS data correlates well with ranking based on K(D) values as measured by SPR and can therefore be used to discriminate between high- and low-affinity antibodies. Finally, we show that a low-affinity antibody may only detect high expression levels of a surface marker while failing to detect lower expression levels of this molecule, which may lead to a false interpretation of antibody specificity.

Amplification of the antigen-antibody interaction from quartz crystal microbalance immunosensors via back-filling immobilization of nanogold on biorecognition surface.

PubMed

Tang, Dian-Quan; Zhang, Da-Jun; Tang, Dian-Yong; Ai, Hua

2006-10-20

A new quartz crystal microbalance immunoassay method based on a novel transparent immunoaffinity reactor was developed for clinical immunoassay. To construct such an affinity reactor, resonators with a frequency of 10 MHz were fabricated by affinity binding of functionalized gold nanoparticles (nanogold) to quartz crystal with immobilized specific ligand for the label-free analysis of the affinity reaction between a ligand and its receptor. [Recombinant human tumor markers, carcinoembryonic antigen (CEA) was chosen as a model ligand.] The binding of target molecules onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was proportional to the CEA concentration in the range of 3.0-50 ng/ml with a detection limit of 1.5 ng/ml at a signal/noise ration of 3. A glycine-HCl solution (pH 2.3) was used to release antigen-antibody complexes from the biorecognition surface. Good reusability was exhibited. Moreover, spiking various levels of CEA into normal human sera was diagnosed using the proposed immunoassay. Analytical results show the precision of the developed immunoassay is acceptable, implying a promising alternative approach for detecting CEA in clinical immunoassay. Compared with the conventional enzyme-linked immunosorbent assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.

Production and Characterization of a Monoclonal Antibody Raised Against Surface Antigens from Mycelium of Gaeumannomyces graminis var. tritici: Evidence for an Extracellular Polyphenol Oxidase.

PubMed

Thornton, C R; Dewey, F M; Gilligan, C A

1997-01-01

ABSTRACT A murine monoclonal antibody (MAb) of immunoglobulin class M (IgM) was raised against surface antigens from Gaeumannomyces graminis var. tritici and, by enzyme-linked immunosorbent assay, recognized isolates of G. graminis var. tritici, G. graminis var. avenae and G. graminis var. graminis. Characterization of the antigen by heat and protease treatments showed that the epitope recognized by the MAb was a protein. Antigen production was detected only in live mycelia. Immunofluorescence studies showed that the antigen was associated with both the broad melanized macrohyphae and hyaline mycelia of G. graminis var. tritici. Secretion of antigen into an aqueous minimal medium was promoted only by exposure of live mycelia to certain phenolic substrates, including monophenols ortho-, para-, and meta-cresol; 3,4,5-trihydroxybenzoic acid (gallic acid); and phenolic amino acid L-3-(3,4-dihydroxyphenyl) alanine (L-DOPA). Antigen secretion was not promoted by 3-(4-hydroxyphenyl) alanine (L-tyrosine). The MAb reacted strongly with purified enzyme laccase (polyphenol oxidase, EC 1.10.3.2) but did not recognize purified tyrosinase (monophenol oxidase, EC 1.14.18.1). Moreover, chemicals that bind to copper and inhibit copper-containing enzymes such as laccase completely inhibited antigen secretion in response to L-DOPA. The MAb was tested for specificity against a wide range of fungi, common yeast species, and gram positive and negative bacteria. It did not recognize antigens from a broad range of unrelated fungi, including Gliocladium roseum, Fusarium sp., Phoma exigua, Phialophora fastigiata, Penicillium crustosum, Pythium ultimum, Rhizopus stolonifer, Rhizoctonia carotae, R. oryzae, R. tuliparum, and Trichoderma viride, nor did it recognize surface antigens from yeasts or bacteria. The MAb cross-reacted with antigens from Botrytis spp., Chaetomium globosum, R. cerealis, and R. solani. However, secretion of antigen by R. solani and R. cerealis was not promoted by L-DOPA, and secretion by C. globosum in response to the phenolic amino acid was significantly less compared to G. graminis var. tritici.

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

PubMed

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

2000-12-15

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

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

Detection of hepatitis B surface antigen in pregnant women attending a public hospital for delivery: implication for vaccination strategy in Bangladesh.

PubMed

Rumi, M A; Begum, K; Hassan, M S; Hasan, S M; Azam, M G; Hasan, K N; Shirin, M; Khan, A K

1998-08-01

Routine antenatal hepatitis B surface antigen (HBsAg) screening and immunization of risk babies is very effective in preventing perinatal transmission of hepatitis B virus (HBV). We studied 1,800 parturients attending a public hospital to assess the rationale for such vaccination in Bangladesh. In one in every 29 deliveries (63 of 1,800 or 3.5%), the mother was found to be HBsAg positive. All were asymptomatic and many (41 of 63 or 65%) without risk factors would remain undetected if HBsAg screening were performed on selected groups. Most of the HBsAg-positive mothers (54 of 63 or 85.7%) were found to be chronic carriers and 30.2% (19 of 63) were also hepatitis B e antigen (HBeAg) positive, indicating high infectivity. Although 23 cord blood were positive for HBsAg or HBeAg, none were positive for IgM antibody to hepatitis B core antigen (IgM anti-HBc), suggesting transplacental transmission of the antigens rather than intrauterine infection. These findings are discussed in relation to the cost-effectiveness of routine prenatal screening and immunization of risk babies compared with universal infant immunization.

A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G

USDA-ARS?s Scientific Manuscript database

A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite...

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

PubMed

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

2011-11-07

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

[Production of marker-free plants expressing the gene of the hepatitis B virus surface antigen].

PubMed

Rukavtsova, E B; Gaiazova, A R; Chebotareva, E N; Bur'ianova, Ia I

2009-08-01

The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants. A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco and tomato transgenic lines synthesizing this antigen at a level of 0.01-0.05% of the total soluble protein were obtained. The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers.

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

PubMed

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

2014-10-07

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

A novel approach for osteocalcin detection by competitive ELISA using porous silicon as a substrate.

PubMed

Rahimi, Fereshteh; Mohammadnejad Arough, Javad; Yaghoobi, Mona; Davoodi, Hadi; Sepehri, Fatemeh; Amirabadizadeh, Masood

2017-11-01

In this study, porous silicon (PSi) was utilized instead of prevalent polystyrene platforms, and its capability in biomolecule screening was examined. Here, two types of porous structure, macroporous silicon (Macro-PSi) and mesoporous silicon (Meso-PSi), were produced on silicon wafers by electrochemical etching using different electrolytes. Moreover, both kinds of fresh and oxidized PSi samples were investigated. Next, osteocalcin as a biomarker of the bone formation process was used as a model biomarker, and the colorimetric detection was performed by competitive enzyme-linked immunosorbent assay (ELISA). Both Macro-PSi and Meso-PSi substrates in the oxidized state, specifically the Meso-porous structure, were reported to have higher surface area to volume ratio, more capacitance of surface-antigen interaction, and more ability to capture antigen in comparison with the prevalent platforms. Moreover, the optical density signal of osteocalcin detected by the ELISA technique was notably higher than the common platforms. Based on the findings of this study, PSi can potentially be used in the ELISA to achieve better results and consequently more sensitivity. A further asset of incorporating such a nanometer structure in the ELISA technique is that the system response to analyte concentration could be maintained by consuming lower monoclonal antibody (or antigen) and consequently reduces the cost of the experiment. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

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

PubMed

Sharma, Anshul; Kaushal, Ankur; Kulshrestha, Saurabh

2017-07-01

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

Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells.

PubMed

Kwong, Gabriel A; Radu, Caius G; Hwang, Kiwook; Shu, Chengyi J; Ma, Chao; Koya, Richard C; Comin-Anduix, Begonya; Hadrup, Sine Reker; Bailey, Ryan C; Witte, Owen N; Schumacher, Ton N; Ribas, Antoni; Heath, James R

2009-07-22

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.

Antibody response against HERV-W env surface peptides differentiates multiple sclerosis and neuromyelitis optica spectrum disorder.

PubMed

Arru, Giannina; Sechi, Elia; Mariotto, Sara; Farinazzo, Alessia; Mancinelli, Chiara; Alberti, Daniela; Ferrari, Sergio; Gajofatto, Alberto; Capra, Ruggero; Monaco, Salvatore; Deiana, Giovanni A; Caggiu, Elisa; Mameli, Giuseppe; Sechi, Leonardo A; Sechi, Gian Pietro

2017-01-01

A specific humoral immune response against HERV-W envelope surface (env-su) glycoprotein antigens has been reported in serum of patients with multiple sclerosis (MS). However, it has not been evaluated to date in patients with neuromyelitis optica spectrum disorder (NMOSD). The objective of this paper is to investigate whether antibody (Ab) response against HERV-W env-su antigenic peptides differs between NMOSD and MS. Serum samples were collected from 36 patients with NMOSD, 36 patients with MS and 36 healthy control individuals (HCs). An indirect ELISA was set up to detect specific Abs against HERV-W env-su peptides. Our data showed that two antigenic peptides, particularly HERV-Wenv 93-108 and HERV-Wenv 248-262, were statistically significantly present only in serum of MS compared to NMOSD and HCs. Thus, the specific humoral immune response against HERV-W env-su glycoprotein antigens found in MS is widely missing in NMOSD. Increased circulating serum levels of these HERV-W Abs may be suitable as additional biomarkers to better differentiate MS from NMOSD.

Engineered Recombinant Single-Chain Fragment Variable Antibody for Immunosensors

PubMed Central

Shen, Zhihong; Mernaugh, Raymond L.; Yan, Heping; Yu, Lei; Zhang, Ying; Zeng, Xiangqun

2008-01-01

A recombinant single-chain fragment variable (scFv) antibody (designated A10B) was engineered to contain two histidines within the linker peptide used to join the scFv heavy and light chains. A piezoimmunosensor using the scFv was successfully developed. A10B scFv bound to the gold piezoimmunosensor surface were correctly oriented, retained antigen-binding activity, and coupled at high surface concentration. These results, and results obtained from an earlier study using an scFv containing a linker cysteine, suggest that the location on the linker sequence in which the amino acids were incorporated was well tolerated by the scFv and did not interfere with scFv antigen-binding activity. The scFv-modified QCM sensor was thoroughly characterized and used to specifically detect antigen in crude serum sample and had a sensitivity of 2.3 ± 0.15 nM (n = 4) with a linear range over 2.3 × 10−9–3.3 × 10−8 M. The piezoimmunosensor was also used to study the kinetics and thermodynamics of antigen/scFv antibody binding. PMID:16255580

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

Multiplex detection of IgG and IgM to Rift Valley fever virus nucleoprotein, nonstructural proteins, and glycoprotein in ovine and bovine

USDA-ARS?s Scientific Manuscript database

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were...

Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice.

PubMed

Joan, Stella Siaw Xiu; Pui-Fong, Jee; Song, Adelene Ai-Lian; Chang, Li-Yen; Yusoff, Khatijah; AbuBakar, Sazaly; Rahim, Raha Abdul

2016-05-01

An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

Label-free protein assay based on a nanomechanical cantilever array

NASA Astrophysics Data System (ADS)

Arntz, Y.; Seelig, J. D.; Lang, H. P.; Zhang, J.; Hunziker, P.; Ramseyer, J. P.; Meyer, E.; Hegner, M.; Gerber, Ch

2003-01-01

We demonstrate continuous label-free detection of two cardiac biomarker proteins (creatin kinase and myoglobin) using an array of microfabricated cantilevers functionalized with covalently anchored anti-creatin kinase and anti-myoglobin antibodies. This method allows biomarker proteins to be detected via measurement of surface stress generated by antigen-antibody molecular recognition. Reference cantilevers are used to eliminate thermal drifts, undesired chemical reactions and turbulences from injections of liquids by calculating differential deflection signals with respect to sensor cantilevers. The sensitivity achieved for myoglobin detection is below 20 µg ml-1. Both myoglobin and creatin kinase could be detected independently using cantilevers functionalized with the corresponding antibodies, in unspecific protein background. This approach permits the use of up to seven different antigen-antibody reactions simultaneously, including an additional thermomechanical and chemical in situ reference. Applications lie in the field of early and rapid diagnosis of acute myocardial infarction.

Chemiluminescence assay for the detection of biological warfare agents

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

Langry, K; Horn, J

A chemiluminescent homogeneous immunoassay and a hand-size multiassay reader are described that could be used for detecting biological materials. The special feature of the assay is that it employs two different antibodies that each bind to a unique epitope on the same antigen. Each group of epitope-specific antibodies has linked to it an enzyme of a proximal-enzyme pair. One enzyme of the pair utilizes a substrate in high concentration to produce a second substrate required by the second enzyme. This new substrate enables the second enzyme to function. The reaction of the second enzyme is configured to produce light. Thismore » chemiluminescence is detected with a charge-coupled device (CCD) camera. The proximal pair enzymes must be in close proximity to one another to allow the second enzyme to react with the product of the first enzyme. This only occurs when the enzyme-linked antibodies are attached to the antigen, whether antigen is a single protein with multiple epitopes or the surface of a cell with a variety of different antigens. As a result of their juxtaposition, the enzymes produce light only in the presence of the biological material. A brief description is given as to how this assay could be utilized in a personal bio-agent detector system.« less

Functionalized vertical GaN micro pillar arrays with high signal-to-background ratio for detection and analysis of proteins secreted from breast tumor cells.

PubMed

Choi, Mun-Ki; Kim, Gil-Sung; Jeong, Jin-Tak; Lim, Jung-Taek; Lee, Won-Yong; Umar, Ahmad; Lee, Sang-Kwon

2017-11-02

The detection of cancer biomarkers has recently attracted significant attention as a means of determining the correct course of treatment with targeted therapeutics. However, because the concentration of these biomarkers in blood is usually relatively low, highly sensitive biosensors for fluorescence imaging and precise detection are needed. In this study, we have successfully developed vertical GaN micropillar (MP) based biosensors for fluorescence sensing and quantitative measurement of CA15-3 antigens. The highly ordered vertical GaN MP arrays result in the successful immobilization of CA15-3 antigens on each feature of the arrays, thereby allowing the detection of an individual fluorescence signal from the top surface of the arrays owing to the high regularity of fluorophore-tagged MP spots and relatively low background signal. Therefore, our fluorescence-labeled and CA15-3 functionalized vertical GaN-MP-based biosensor is suitable for the selective quantitative analysis of secreted CA15-3 antigens from MCF-7 cell lines, and helps in the early diagnosis and prognosis of serious diseases as well as the monitoring of the therapeutic response of breast cancer patients.

Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

PubMed Central

Owens, Gemma L.; Sheard, Victoria E.; Kalaitsidou, Milena; Blount, Daniel; Lad, Yatish; Cheadle, Eleanor J.; Edmondson, Richard J.; Kooner, Gurdeep; Gilham, David E.

2018-01-01

Chimeric antigen receptor (CAR) T cells represent a novel targeted approach to overcome both quantitative and qualitative shortfalls of the host immune system relating to the detection and subsequent destruction of tumors. The identification of antigens expressed specifically on the surface of tumor cells is a critical first step in the ability to utilize CAR T cells for the treatment of cancer. The 5T4 is a tumor-associated antigen which is expressed on the cell surface of most solid tumors including ovarian cancer. Matched blood and tumor samples were collected from 12 patients with ovarian cancer; all tumors were positive for 5T4 expression by immunohistochemistry. Patient T cells were effectively transduced with 2 different anti-5T4 CAR constructs which differed in their affinity for the target antigen. Co-culture of CAR T cells with matched autologous tumor disaggregates resulted in antigen-specific secretion of IFN-gamma. Furthermore, assessment of the efficacy of anti-5T4 CAR T cells in a mouse model resulted in therapeutic benefit against established ovarian tumors. These results demonstrate proof of principle that 5T4 is an attractive target for immune intervention in ovarian cancer and that patient T cells engineered to express a 5T4-specific CAR can recognize and respond physiologically to autologous tumor cells. PMID:29239915

[Cellular immunophenotypes in 97 adults with acute leukemia].

PubMed

Piedras, J; López-Karpovitch, X; Cárdenas, M R

1997-01-01

To analyze hematopoietic cell surface antigen reactivity in acute leukemia (AL) by flow cytometry and identify acute mixed-lineage leukemias (AMLL) employing the most widely accepted criteria. Ninety seven patients with de novo AL were studied. Cell surface antigens were investigated with monoclonal antibodies directed to: B lymphoid (CD10, CD19, CD20, CD21, CD22); T lymphoid (CD2, CD3, CD5, CD7); and myeloid (CD13, CD14, CD15, CD33, CD41) cell lineages. Maturation cell-associated antigens (CD34, HLA-DR and TdT) were also studied. Twelve patients unclassified by cytomorphology could be classified by immunophenotype. Using cytomorphologic, cytochemical and immunophenotypic data, 54 cases corresponded to acute lymphoblastic leukemia (ALL) and 43 were acute myeloblastic leukemia (AML). In All there were 63% B lineage, 15% T, 7% T/B, 6% undifferentiated and 9% mixed-lineage (coexpression of two or more myeloid-associated antigens). In AML, myeloid immunophenotype was observed in 86% undifferentiated in 2%, and mixed-lineage in 12% (coexpression of two or more lymphoid-associated antigens). In addition, 26% of ALL cases and 12% of AML cases expressed a single myeloid and lymphoid antigen respectively. The most common aberrant antigens in ALL and AML were CD13 and CD7 respectively. The highest frequency of CD34 antigen expression (90%) was detected in patients with AMLL. Flow cytometric immunophenotypic analysis allowed to: a) establish diagnosis in cytomorphologically unclassified cases; b) identify AMLL with a frequency similar to that reported in other series; and c) confirm the heterogeneity of AL.

The Dynamics of a SEIR-SIRC Antigenic Drift Influenza Model.

PubMed

Adi-Kusumo, Fajar

2017-06-01

We consider the dynamics of an influenza model with antigenic drift mechanism. Antigenic drift is an antigen mutation on the skin surface of the influenza virus that do not produce a new virus strain. The mutation produces the same virus but with slightly different antigens that cannot be recognized by the immune receptors formed by the previous infection. There are some type of influenza that involve the interaction between two populations such as human and animal. In this paper, we construct an influenza model with antigenic drift mechanism on the human population that has interaction with the animal population. The animal population is assumed to follow the SEIR epidemic model. Our model is motivated by the fact that some of the influenza cases in human come from the animal such as the swine and the avian. The transmission parameter that shows number of contact between the susceptible human and the infectious animals are important to study. The parameter plays an important role to detect the cycle of infection of the disease. The other important parameters are the seasonality degree, which shows the pathogen appearance and disappearance via annual migration, and the infection rate on the human population. We employ the bifurcation theory to analyze the behavior of the system and to detect the cycle of infection types when the parameters values are varied.

[Autoantibodies in Paraneoplastic Neurological Syndrome].

PubMed

Kawachi, Izumi

2018-04-01

Paraneoplastic neurological syndromes (PNS) are caused by immune responses against neuronal antigens expressed by the tumor. Based on the immunological pathomechanisms and responsiveness of treatments, onconeuronal antibodies are divided into two categories: 1) antibodies against neural intracellular antigens and 2) antibodies against neuronal surface or synaptic antigens. The recent discovery of onconeuronal antibodies have radically changed concepts of CNS autoimmunity, including PNS. The recognition of PNS provides a foundation for the early detection of underlying tumors and initiations of prompt treatments, which can result in substantial improvement. We here review the characteristic onconeuronal antibodies, including anti-Hu, anti-Ma2, and anti-N-methyl-D-aspartate receptor, and discuss the algorithm for the diagnosis of PNS.

Human Antibodies to a Mr 155,000 Plasmodium falciparum Antigen Efficiently Inhibit Merozoite Invasion

NASA Astrophysics Data System (ADS)

Wahlin, Birgitta; Wahlgren, Mats; Perlmann, Hedvig; Berzins, Klavs; Bjorkman, Anders; Patarroyo, Manuel E.; Perlmann, Peter

1984-12-01

IgG from a donor clinically immune to Plasmodium falciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, <1 μ g/ml) than the original IgG preparation. The major antibody in this eluate was directed against a parasite-derived antigen of Mr 155,000 (Pf 155) deposited by the parasite in the erythrocyte membrane in the course of invasion. A detailed study of IgG fractions from 11 donors with acute P. falciparum malaria or clinical immunity revealed the existence of an excellent correlation between their capacities to stain the surface of infected erythrocytes, their titers in reinvasion inhibition, and the presence of antibodies to Pf 155 as detected by immunoblotting. No such correlations were seen when the IgG fractions were analyzed for immunofluorescence of intracellular parasites or for the presence of antibodies to other parasite antigens as detected by immunoprecipitation of [35S]methionine-labeled and NaDodSO4/PAGE-separated parasite extracts. The results suggest that Pf 155 has an important role in the process of erythrocyte infection and that host antibodies to this antigen may efficiently interfere with this process.

High-contrast grating resonators for label-free detection of disease biomarkers

PubMed Central

Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

2016-01-01

A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI. PMID:27265624

High-contrast grating resonators for label-free detection of disease biomarkers

NASA Astrophysics Data System (ADS)

Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

2016-06-01

A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI.

Magnetic bead-sensing-platform-based chemiluminescence resonance energy transfer and its immunoassay application.

PubMed

Qin, Guoxin; Zhao, Shulin; Huang, Yong; Jiang, Jing; Ye, Fanggui

2012-03-20

A competitive immunoassay based on chemiluminescence resonance energy transfer (CRET) on the magnetic beads (MBs) is developed for the detection of human immunoglobulin G (IgG). In this protocol, carboxyl-modified MBs were conjugated with horseradish peroxidase (HRP)-labeled goat antihuman IgG (HRP-anti-IgG) and incubated with a limited amount of fluorescein isothiocyanate (FITC)-labeled human IgG to immobilize the antibody-antigen immune complex on the surface of the MBs, which was further incubated with the target analyte (human IgG) for competitive immunoreaction and separated magnetically to remove the supernatant. The chemiluminescence (CL) buffer (containing luminol and H(2)O(2)) was then added, and the CRET from donor luminol to acceptor FITC in the immunocomplex on the surface of MBs occured immediately. The present protocol was evaluated for the competitive immunoassay of human IgG, and a linear relationship between CL intensity ratio (R = I(425)/I(525)) and human IgG concentration in the range of 0.2-4.0 nM was obtained with a correlation coefficient of 0.9965. The regression equation was expressed as R = 1.9871C + 2.4616, and a detection limit of 2.9 × 10(-11) M was obtained. The present method was successfully applied for the detection of IgG in human serum. The results indicate that the present protocol is quite promising for the application of CRET in immunoassays. It could also be developed for detection of other antigen-antibody immune complexes by using the corresponding antigens and respective antibodies.

42 CFR 486.302 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-10-01

...) Gangrenous bowel or perforated bowel and/or intra-abdominal sepsis. (ii) Viral: (A) HIV infection by serologic or molecular detection. (B) Rabies. (C) Reactive Hepatitis B Surface Antigen. (D) Retroviral infections including HTLV I/II. (E) Viral Encephalitis or Meningitis. (F) Active Herpes simplex, varicella...

Cellular immune responses in patients with hepatitis B surface antigen seroclearance induced by antiviral therapy

PubMed Central

2011-01-01

Background The mechanisms by which chronic hepatitis B is completely resolved through antiviral therapy are unknown, and the contribution of acquired T cell immunity to hepatitis B surface antigen (HBsAg) seroclearance has not been investigated. Therefore, we measured the T-cell responses to core and envelope antigens in patients with HBsAg seroclearance. Methods Fourteen subjects with HBsAg seroclearance following antiviral treatment for chronic hepatitis B, 7 HBeAg-positive immunotolerant HBV carriers and 9 HBeAg-negative inactive HBsAg carriers were recruited. HBV-specific T-cell responses to recombinant HBV core (rHBcAg) and envelope (rHBsAg) proteins and pools of core and envelope peptides were measured using an ELISPOT assay detecting interferon-gamma and intracellular cytokine staining (ICS) assays detecting interferon-gamma or interleukin 2. Results Interferon-gamma ELISPOT assays showed a low frequency of weak responses to the rHBsAg and S peptide pool in the HBsAg seroclearance group, and the response frequency to the rHBcAg and the C peptide pool was higher than to the rHBsAg (P < 0.001) and S peptide pool (P = 0.001) respectively. A higher response frequency to C than S peptide pools was confirmed in the interferon-gamma ICS assays for both CD4+ (P = 0.033) and CD8+ (P = 0.040) T cells in the HBsAg seroclearance group. The responses to C and S antigens in the inactive carriers were similar. Conclusions There was a low frequency of CD4+ and CD8+ T cell immune responses to envelope antigens in Chinese subjects with HBsAg seroclearance following antiviral therapy. It is unlikely that these immune responses are responsible for HBsAg seroclearance in these subjects. PMID:21320337

Expression and immunological characterisation of Eimeria tenella glycosylphosphatidylinositol-anchored surface antigen-5

NASA Astrophysics Data System (ADS)

Ho, Sue-Kim; Nathan, Sheila; Wan, Kiew-Lian

2016-11-01

Eimeria tenella is the most pathogenic of the Eimeria species that infect chickens and causes huge economic losses to the poultry industry. The glycosylphosphatidylinositol-anchored surface antigen-5 (SAG5) found on the surface of the parasite has been shown to activate the chicken's immune system. In this study, recombinant SAG5 was expressed, purified and used to investigate the immune-inducing characteristics of the molecule. Chickens were immunized with purified recombinant SAG5 and sera were subjected to Enzyme-linked Immunosorbant Assay (ELISA). Results indicated that specific antibodies against rSAG5 were produced, with IgG detected at a higher level compared to IgA and IgM. Information on the immunological responses elicited by SAG5 provides essential knowledge that will contribute towards the effort to develop more effective strategies against coccidiosis.

Using reduced graphene oxide-Ca:CdSe nanocomposite to enhance photoelectrochemical activity of gold nanoparticles functionalized tungsten oxide for highly sensitive prostate specific antigen detection.

PubMed

Wang, Xueping; Xu, Rui; Sun, Xu; Wang, Yaoguang; Ren, Xiang; Du, Bin; Wu, Dan; Wei, Qin

2017-10-15

An ultrasensitive sandwich-type photoelectrochemical (PEC) immunosensor was constructed for the detection of prostate specific antigen (PSA). In this work, Au-nanoparticle-loaded tungsten oxide (WO 3 -Au) hybrid composites was applied as PEC sensing platform, while Ca ions doped CdSe equipped on the conducting framework of reduced graphene oxide (rGO-Ca:CdSe) nanocomposites were employed as the signal amplification probe. As for WO 3 -Au, massive Au nanoparticles were formed on the surface of WO 3 without any additional reducing agent, providing a novel nanocarriers for anchoring plenty of the primary antibodies due to the large specific surface area and good biocompatibility by chemical bonding between Au nanoparticles and -NH 2 of antibodies. Besides, the incorporation of the rGO and the doping of Ca ions could improve the conductivity and hinder the recombination of electron-hole pairs of CdSe nanoparticles effectively, thereby enhancing the photocurrent conversion efficiency. Based on the sandwich immunoreaction, the primary antibody was immobilized onto WO 3 -Au substrate, after the formed rGO-Ca:CdSe labels were captured onto the electrode surface via the specific antibody-antigen interaction, the photocurrent intensity could be further enhanced due to the sensitization effect. Under the optimal conditions, the proposed PEC immunosensor shows a linear relationship between photocurrent variation and the logarithm of PSA concentration in the wide range of 5pgmL -1 to 50ngmL -1 with a low detection limit of 2.6pgmL -1 (S/N=3). Moreover, it also presented good stability and acceptable specificity, indicating the potential applications in clinical diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

Antibodies to Plasmodium falciparum antigens predict a higher risk of malaria but protection from symptoms once parasitemic.

PubMed

Greenhouse, Bryan; Ho, Benjamin; Hubbard, Alan; Njama-Meya, Denise; Narum, David L; Lanar, David E; Dutta, Sheetij; Rosenthal, Philip J; Dorsey, Grant; John, Chandy C

2011-07-01

Associations between antibody responses to Plasmodium falciparum antigens and protection against symptomatic malaria have been difficult to ascertain, in part because antibodies are potential markers of both exposure to P. falciparum and protection against disease. We measured IgG responses to P. falciparum circumsporozoite protein, liver-stage antigen 1, apical-membrane antigen 1 (AMA-1), and merozoite surface proteins (MSP) 1 and 3, in children in Kampala, Uganda, and measured incidence of malaria before and after antibody measurement. Stronger responses to all 5 antigens were associated with an increased risk of clinical malaria (P < .01) because of confounding with prior exposure to P. falciparum. However, with use of another assessment, risk of clinical malaria once parasitemic, stronger responses to AMA-1, MSP-1, and MSP-3 were associated with protection (odds ratios, 0.34, 0.36, and 0.31, respectively, per 10-fold increase; P < .01). Analyses assessing antibodies in combination suggested that any protective effect of antibodies was overestimated by associations between individual responses and protection. Using the risk of symptomatic malaria once parasitemic as an outcome may improve detection of associations between immune responses and protection from disease. Immunoepidemiology studies designed to detect mechanisms of immune protection should integrate prior exposure into the analysis and evaluate multiple immune responses.

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

Nano-particle enhanced impedimetric biosensor for detedtion of foodborne pathogens

NASA Astrophysics Data System (ADS)

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

2007-03-01

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

Surface design of antibody-immobilized thermoresponsive cell culture dishes for recovering intact cells by low-temperature treatment.

PubMed

Kobayashi, Jun; Hayashi, Masaki; Ohno, Takahiro; Nishi, Masanori; Arisaka, Yoshinori; Matsubara, Yoshinori; Kakidachi, Hiroshi; Akiyama, Yoshikatsu; Yamato, Masayuki; Horii, Akihiro; Okano, Teruo

2014-11-01

Antibody-immobilized thermoresponsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [poly(IPAAm-co-CIPAAm)]-grafted cell culture surfaces were designed to enhance both the initial adhesion of weakly adhering cells and the ability of cells to detach in response to low temperature through the regulation of affinity binding between immobilized antibodies and antigens on the cellular surface. Ty-82 cells and neonatal normal human dermal fibroblasts (NHDFs), which express CD90 on the cell surface, adhered to anti-CD90 antibody-immobilized thermoresponsive surfaces at 37°C, a condition at which the grafted thermoresponsive polymer chains shrank. Adherent Ty-82 cells were detached from the surfaces by lowering the temperature to 20°C and applying external forces, such as pipetting, whereas cultured NHDF sheets spontaneously detached themselves from the surface in response to reduced temperature alone. When the temperature was decreased to 20°C, the swelling of grafted thermoresponsive polymer chains weakened the affinity binding between immobilized antibody and antigen on the cells due to the increasing steric hindrance of the polymer chains around the antigen-recognition site of the immobilized antibodies. No contamination was detected on cells harvested from covalently immobilized antibodies on the culture surfaces by low-temperature treatment, whereas a carryover of the antibody and avidin from the avidin-biotin binding surface was observed. Furthermore, the initial adhesion of adipose tissue-derived cells, which adhere weakly to PIPAAm-grafted surfaces, was enhanced on the antibody-immobilized thermoresponsive surfaces. © 2013 Wiley Periodicals, Inc.

Application of hepatitis B core particles produced by human primary hepatocellular carcinoma (PLC/342) propagated in nude mice to the determination of anti-HBc by passive hemagglutination.

PubMed

Miyamoto, K; Itoh, Y; Tsuda, F; Matsui, T; Tanaka, T; Miyamoto, H; Naitoh, S; Imai, M; Usuda, S; Nakamura, T

1986-05-22

Human primary hepatocellular carcinoma (PLC/342), carried by nude mice, produces hepatitis B core particles as well as hepatitis B surface antigen particles. Core particles purified form PLC/342 tumors displayed epitopes of hepatitis B core antigen (HBcAg) but not epitopes of hepatitis B e antigen (HBeAg) on their surface, unlike core particles prepared from Dane particles, derived from plasma of asymptomatic carriers, that expressed epitopes of both HBcAg and HBeAg. Core particles obtained from PLC/342 tumors were applied to the determination of antibody to HBcAg (anti-HBc) by passive hemagglutination. The assay detected anti-HBc not only in individuals with persistent infection with hepatitis B virus and in those who had recovered from transient infection, but also in patients with acute type B hepatitis, indicating that it can detect anti-HBc of either IgG or IgM class. A liberal availability of core particles from tumors carried by nude mice, taken together with an easy applicability of the method, would make the passive hemagglutination for anti-HBc a valuable tool in clinical and epidemiological studies, especially in places where sophisticated methods are not feasible.

Trypanosoma congolense: tissue distribution of long-term T- and B-cell responses in cattle.

PubMed

Lutje, V; Taylor, K A; Boulangé, A; Authié, E

1995-11-01

Memory T- and B-cell responses to trypanosome antigens were measured in peripheral blood mononuclear cells, spleen and lymph node cells obtained from four trypanotolerant N'Dama cattle which had been exposed to six experimental infections with Trypanosoma congolense. These cattle were treated with trypanocidal drugs following each infection and had remained aparasitemic for 3 years prior to this study. The antigens used were whole trypanosome lysate, variable surface glycoprotein, a 33-kDa cysteine protease (congopain) and a 70-kDa heat-shock protein. As parameters of T-cell-mediated immunity, we measured T-cell proliferation and IFN-gamma production. Lymph node cells, spleen cells and peripheral blood mononuclear cells all proliferated to a mitogenic stimulus (concanavalin A) but only lymph node cells responded to trypanosome antigens. Similarly, IFN-gamma was produced by both lymph node and spleen cells stimulated with concanavalin A but only by lymph node cells stimulated with variable surface glycoprotein and whole trypanosome lysate. T. congolense-specific antibodies were detected in sera and in supernatants of cultured lymph node and spleen cells after in vitro stimulation with lipopolysaccharide and recombinant bovine interleukin-2. In conclusion, we have demonstrated that memory T- and B-cell responses are detectable in various lymphoid organs in cattle 3 years following infection and treatment with T. congolense.

Antibody-antigenic peptide interactions monitored by SPR and QCM-D. A model for SPR detection of IA-2 autoantibodies in human serum.

PubMed

Ayela, Cedric; Roquet, Francoise; Valera, Lionel; Granier, Claude; Nicu, Liviu; Pugnière, Martine

2007-06-15

This work reports on a complementary use of surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D) technologies to study interactions between a peptide antigen and polyclonal antibodies, in an experimental format suitable for diagnostic assays of autoimmune diseases. In the chosen model, a synthetic peptide from the juxtamembrane region of IA-2 (a type 1 diabetes associated antigen) was immobilized by an optimized chemical protocol applicable to both BIACORE and QCM-D sensors. A thorough study of the peptide immobilization was performed to optimize the signal-to-noise ratio using mixed self-assembled monolayers (SAM) on a gold surface. Introduction of polyethylene glycol (EG(6)) chains into mixed SAM layers and addition of an anionic surfactant to the human serum reduced non-specific binding without modifying the viscoelasticity properties of the layer. Under our conditions, the antibody SPR detection limit was determined to be 0.2 nM in diluted human serum. This value is in agreement with the reported rank distribution of IA-2 antibodies in diabetic patient sera. Label-free and real-time technologies such as SPR and/or QCM-D could be precious tools in future diagnostic assays.

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

Using Phage Lytic Enzymes to Destroy Pathogenic and BW Bacteria

DTIC Science & Technology

2005-07-14

against antibiotic resistant Enterococcus faecalis and Enterococcus faecium . J Bacteriol. 186:4808-12. Cheng, Q., D. Nelson, S. Zhu, and V.A...Lysins from Enterococcus faecalis RU-654 3. Fischetti, Vincent A. Schuch, Raymond Lytic Enzymes and spore surface antigens for detection and

Baculovirus display of functional antibody Fab fragments.

PubMed

Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

Fabrication of fiber-optic localized surface plasmon resonance sensor and its application to detect antibody-antigen reaction of interferon-gamma

NASA Astrophysics Data System (ADS)

Jeong, Hyeon-Ho; Erdene, Norov; Lee, Seung-Ki; Jeong, Dae-Hong; Park, Jae-Hyoung

2011-12-01

A fiber-optic localized surface plasmon (FO LSPR) sensor was fabricated by gold nanoparticles (Au NPs) immobilized on the end-face of an optical fiber. When Au NPs were formed on the end-face of an optical fiber by chemical reaction, Au NPs aggregation occurred and the Au NPs were immobilized in various forms such as monomers, dimers, trimers, etc. The component ratio of the Au NPs on the end-face of the fabricated FO LSPR sensor was slightly changed whenever the sensors were fabricated in the same condition. Including this phenomenon, the FO LSPR sensor was fabricated with high sensitivity by controlling the density of Au NPs. Also, the fabricated sensors were measured for the resonance intensity for the different optical systems and analyzed for the effect on sensitivity. Finally, for application as a biosensor, the sensor was used for detecting the antibody-antigen reaction of interferon-gamma.

Expression of the Major Surface Antigen of Plasmodium knowlesi Sporozoites in Yeast

NASA Astrophysics Data System (ADS)

Sharma, Shobhona; Godson, G. Nigel

1985-05-01

The circumsporozoite protein, a surface antigen of the sporozoite stage of the monkey malarial parasite Plasmodium knowlesi, was expressed in the yeast Saccharomyces cerevisiae by using an expression vector containing the 5' regulatory region of the yeast alcohol dehydrogenase I gene. It was necessary to eliminate the entire 5' upstream region of the parasite DNA to obtain the expression of this protein. Only the circumsporozoite precursor protein was produced by the yeast transformants, as detected by immunoblotting. About 55 and 20 percent of the circumsporozoite protein produced in yeast was associated with the 25,000g and 150,000g particulate fractions, respectively. The protein could be solubilized in Triton X-100 and was stable in solubilized extracts.

Probing the effects of surface hydrophobicity and tether orientation on antibody-antigen binding

NASA Astrophysics Data System (ADS)

Bush, Derek B.; Knotts, Thomas A.

2017-04-01

Antibody microarrays have the potential to revolutionize molecular detection for many applications, but their current use is limited by poor reliability, and efforts to change this have not yielded fruitful results. One difficulty which limits the rational engineering of next-generation devices is that little is known, at the molecular level, about the antibody-antigen binding process near solid surfaces. Atomic-level structural information is scant because typical experimental techniques (X-ray crystallography and NMR) cannot be used to image proteins bound to surfaces. To overcome this limitation, this study uses molecular simulation and an advanced, experimentally validated, coarse-grain, protein-surface model to compare fab-lysozyme binding in bulk solution and when the fab is tethered to hydrophobic and hydrophilic surfaces. The results show that the tether site in the fab, as well as the surface hydrophobicity, significantly impacts the binding process and suggests that the optimal design involves tethering fabs upright on a hydrophilic surface. The results offer an unprecedented, molecular-level picture of the binding process and give hope that the rational design of protein-microarrays is possible.

Neuronal surface antigen antibodies in limbic encephalitis

PubMed Central

Graus, F; Saiz, A; Lai, M; Bruna, J; López, F; Sabater, L; Blanco, Y; Rey, M J.; Ribalta, T; Dalmau, J

2008-01-01

Objective: To report the frequency and type of antibodies against neuronal surface antigens (NSA-ab) in limbic encephalitis (LE). Methods: Analysis of clinical features, neuropathologic findings, and detection of NSA-ab using immunochemistry on rat tissue and neuronal cultures in a series of 45 patients with paraneoplastic (23) or idiopathic (22) LE. Results: NSA-ab were identified in 29 patients (64%; 12 paraneoplastic, 17 idiopathic). Thirteen patients had voltage-gated potassium channels (VGKC)-ab, 11 novel NSA (nNSA)-ab, and 5 NMDA receptor (NMDAR)-ab. nNSA-ab did not identify a common antigen and were more frequent in paraneoplastic than idiopathic LE (39% vs 9%; p = 0.03). When compared with VGKC-ab or NMDAR-ab, the nNSA associated more frequently with intraneuronal antibodies (11% vs 73%; p = 0.001). Of 12 patients (9 nNSA-ab, 2 VGKC-ab, 1 NMDAR-ab) with paraneoplastic LE and NSA-ab, concomitant intraneuronal antibodies occurred in 9 (75%). None of these 12 patients improved with immunotherapy. The autopsy of three of them showed neuronal loss, microgliosis, and cytotoxic T cell infiltrates in the hippocampus and amygdala. These findings were compatible with a T-cell mediated neuronal damage. In contrast, 13 of 17 (76%) patients with idiopathic LE and NSA-ab (8 VGKC-ab, 4 NMDAR-ab, 1 nNSA-ab) and 1 of 5 (20%) without antibodies had clinical improvement (p = 0.04). Conclusions: In paraneoplastic limbic encephalitis (LE), novel antibodies against neuronal surface antigens (nNSA-ab) occur frequently, coexist with antibodies against intracellular antigens, and these cases are refractory to immunotherapy. In idiopathic LE, the likelihood of improvement is significantly higher in patients with NSA-ab than in those without antibodies. GLOSSARY GAD = glutamic acid decarboxylase; LE = limbic encephalitis; NMDAR = N-methyl-D-aspartate receptor; NSA = neuronal surface antigens; nNSA = novel NSA; SCLC = small-cell lung cancer; VGKC = voltage-gated potassium channels; WBC = white blood cells. PMID:18794496

Electrophoretically deposited reduced graphene oxide platform for food toxin detection

NASA Astrophysics Data System (ADS)

Srivastava, Saurabh; Kumar, Vinod; Ali, Md Azahar; Solanki, Pratima R.; Srivastava, Anchal; Sumana, Gajjala; Saxena, Preeti Suman; Joshi, Amish G.; Malhotra, B. D.

2013-03-01

Reduced graphene oxide (RGO) due to its excellent electrochemical properties and large surface area, has recently aroused much interest for electrochemical biosensing application. Here, the chemically active RGO has been synthesized and deposited onto an indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. This novel platform has been utilized for covalent attachment of the monoclonal antibodies of aflatoxin B1 (anti-AFB1) for food toxin (AFB1) detection. The electron microscopy, X-ray diffraction, and UV-visible studies reveal successful synthesis of reduced graphene oxide while the XPS and FTIR studies suggest its carboxylic functionalized nature. The electrochemical sensing results of the anti-AFB1/RGO/ITO based immunoelectrode obtained as a function of aflatoxin concentration show high sensitivity (68 μA ng-1 mL cm-2) and improved detection limit (0.12 ng mL-1). The association constant (ka) for antigen-antibody interaction obtained as 5 × 10-4 ng mL-1 indicates high affinity of antibodies toward the antigen (AFB1).Reduced graphene oxide (RGO) due to its excellent electrochemical properties and large surface area, has recently aroused much interest for electrochemical biosensing application. Here, the chemically active RGO has been synthesized and deposited onto an indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. This novel platform has been utilized for covalent attachment of the monoclonal antibodies of aflatoxin B1 (anti-AFB1) for food toxin (AFB1) detection. The electron microscopy, X-ray diffraction, and UV-visible studies reveal successful synthesis of reduced graphene oxide while the XPS and FTIR studies suggest its carboxylic functionalized nature. The electrochemical sensing results of the anti-AFB1/RGO/ITO based immunoelectrode obtained as a function of aflatoxin concentration show high sensitivity (68 μA ng-1 mL cm-2) and improved detection limit (0.12 ng mL-1). The association constant (ka) for antigen-antibody interaction obtained as 5 × 10-4 ng mL-1 indicates high affinity of antibodies toward the antigen (AFB1). Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr32242d

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.

Sero-prevalence of hepatitis B virus infection in Balochistan Province of Pakistan.

PubMed

Sheikh, Nadeem S; Sheikh, Azeem S; Sheikh, Aqleem A; Yahya, Samira; Lateef, Majid

2011-01-01

The objective was to evaluate the sero-prevalence of hepatitis B surface antigen (HBsAg) and IgM antibodies to hepatitis core antigen in Balochistan Province of Pakistan. A cross-sectional, population-based study. Place and time of the study: The study was conducted in Balochistan from 1 st January 2004 to 31 st December, 2008. The screening areas included Barkhan, Eashani, Khuzdar, Kodi Zikriani, Kohlu, Rakhni and Turbat. A total of 15,260 subjects were enrolled; 11,900 (78%) agreed to undergo screening. Fresh serum samples were tested for the presence of hepatitis B surface antigen and IgM antibodies to hepatitis B core antigen. HBsAg was detected in 1166 (9.8%) while anti-HBc IgM was found in 117 (10.0%). HBsAg positivity was seen in 875 (12.7%) males and 291 (5.8%) females. The prevalence of hepatitis B in Balochistan varies from 3.3% in Khuzdar to 17.0% in Kodi Zikriani. It is utmost important to educate the public, to take proper measures to control the spread of infection and vaccination in order to interrupt transmission of this threatening public health problem in Balochistan province of Pakistan.

Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis.

PubMed

Knapp, W; Strobl, H; Majdic, O

1994-12-15

New technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro-)myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferentiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to be used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB-1), CD79b (B29), CD87 (uPA-R), and CD117 (c-kit).

Longitudinal evaluation of humoral immune response and merozoite surface antigen diversity in calves naturally infected with Babesia bovis, in São Paulo, Brazil.

PubMed

Matos, Carlos António; Gonçalves, Luiz Ricardo; Alvarez, Dasiel Obregón; Freschi, Carla Roberta; Silva, Jenevaldo Barbosa da; Val-Moraes, Silvana Pompeia; Mendes, Natalia Serra; André, Marcos Rogério; Machado, Rosangela Zacarias

2017-01-01

Babesiosis is an economically important infectious disease affecting cattle worldwide. In order to longitudinally evaluate the humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis among naturally infected calves in Taiaçu, Brazil, serum and DNA samples from 15 calves were obtained quarterly, from their birth to 12 months of age. Anti-B. bovis IgG antibodies were detected by means of the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) was used to investigate the genetic diversity of B. bovis, based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 2, 4 and 5 sequences of the genes msa-1, msa-2b and msa-2c were obtained. The present study demonstrated that the msa-1 and msa-2b genes sequences amplified from blood DNA of calves positive to B. bovis from Taiaçu were genetically distinct, and that msa-2c was conserved. All animals were serologically positive to ELISA and IFAT, which used full repertoire of parasite antigens in despite of the genetic diversity of MSAs.

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

PubMed Central

Halloran, Phil; Schirrmacher, Volker; Festenstein, Hilliard

1974-01-01

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

Research Update: Nanoscale surface potential analysis of MoS2 field-effect transistors for biomolecular detection using Kelvin probe force microscopy

NASA Astrophysics Data System (ADS)

Kim, Min Hyung; Park, Heekyeong; Lee, Hyungbeen; Nam, Kihwan; Jeong, Seokhwan; Omkaram, Inturu; Yoon, Dae Sung; Lee, Sei Young; Kim, Sunkook; Lee, Sang Woo

2016-10-01

We used high-resolution Kelvin probe force microscopy (KPFM) to investigate the immobilization of a prostate specific antigen (PSA) antibody by measuring the surface potential (SP) on a MoS2 surface over an extensive concentration range (1 pg/ml-100 μg/ml). After PSA antibody immobilization, we demonstrated that the SP on the MoS2 surface characterized by KPFM strongly correlated to the electrical signal of a MoS2 bioFET. This demonstration can not only be used to optimize the immobilization conditions for captured molecules, but can also be applied as a diagnostic tool to complement the electrical detection of a MoS2 FET biosensor.

Oral delivery of plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to high doses.

PubMed

Lindh, Ingrid; Bråve, Andreas; Hallengärd, David; Hadad, Ronza; Kalbina, Irina; Strid, Åke; Andersson, Sören

2014-04-25

During early infection with human immunodeficiency virus type 1 (HIV-1), there is a rapid depletion of CD4(+) T-cells in the gut-associated lymphoid tissue (GALT) in the gastrointestinal tract. Therefore, immediate protection at these surfaces is of high priority for the development of an HIV-1 vaccine. Thus, transgenic plants expressing HIV-1 antigens, which are exposed to immune competent cells in the GALT during oral administration, can be interesting as potential vaccine candidates. In the present study, we used two HIV-1 p24 antigen-expressing transgenic plant systems, Arabidopsis thaliana and Daucus carota, in oral immunization experiments. Both transgenic plant systems showed a priming effect in mice and induced humoral immune responses, which could be detected as anti-p24-specific IgG in sera after an intramuscular p24 protein boost. Dose-dependent antigen analyses using transgenic A. thaliana indicated that low p24 antigen doses were superior to high p24 antigen doses. Copyright © 2014. Published by Elsevier Ltd.

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

SPR based immunosensor for detection of Legionella pneumophila in water samples

NASA Astrophysics Data System (ADS)

Enrico, De Lorenzis; Manera, Maria G.; Montagna, Giovanni; Cimaglia, Fabio; Chiesa, Maurizio; Poltronieri, Palmiro; Santino, Angelo; Rella, Roberto

2013-05-01

Detection of legionellae by water sampling is an important factor in epidemiological investigations of Legionnaires' disease and its prevention. To avoid labor-intensive problems with conventional methods, an alternative, highly sensitive and simple method is proposed for detecting L. pneumophila in aqueous samples. A compact Surface Plasmon Resonance (SPR) instrumentation prototype, provided with proper microfluidics tools, is built. The developed immunosensor is capable of dynamically following the binding between antigens and the corresponding antibody molecules immobilized on the SPR sensor surface. A proper immobilization strategy is used in this work that makes use of an important efficient step aimed at the orientation of antibodies onto the sensor surface. The feasibility of the integration of SPR-based biosensing setups with microfluidic technologies, resulting in a low-cost and portable biosensor is demonstrated.

Shrink-induced graphene sensor for alpha-fetoprotein detection with low-cost self-assembly and label-free assay

NASA Astrophysics Data System (ADS)

Sando, Shota; Zhang, Bo; Cui, Tianhong

2017-12-01

Combination of shrink induced nano-composites technique and layer-by-layer (LbL) self-assembled graphene challenges controlling surface morphology. Adjusting shrink temperature achieves tunability on graphene surface morphology on shape memory polymers, and it promises to be an alternative in fields of high-surface-area conductors and molecular detection. In this study, self-assembled graphene on a shrink polymer substrate exhibits nanowrinkles after heating. Induced nanowrinkles on graphene with different shrink temperature shows distinct surface roughness and wettability. As a result, it becomes more hydrophilic with higher shrink temperatures. The tunable wettability promises to be utilized in, for example, microfluidic devices. The graphene on shrink polymer also exhibits capability of being used in sensing applications for pH and alpha-fetoprotein (AFP) detection with advantages of label free and low cost, due to self-assembly technique, easy functionalization, and antigen-antibody reaction on graphene surface. The detection limit of AFP detection is down to 1 pg/mL, and therefore the sensor also has a significant potential for biosensing as it relies on low-cost self-assembly and label-free assay.

Development and potential applications of microarrays based on fluorescent nanocrystal-encoded beads for multiplexed cancer diagnostics

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Grinevich, Regina; Efimov, Anton E.; Nabiev, Igor; Sukhanova, Alyona

2014-05-01

Advanced multiplexed assays have recently become an indispensable tool for clinical diagnostics. These techniques provide simultaneous quantitative determination of multiple biomolecules in a single sample quickly and accurately. The development of multiplex suspension arrays is currently of particular interest for clinical applications. Optical encoding of microparticles is the most available and easy-to-use technique. This technology uses fluorophores incorporated into microbeads to obtain individual optical codes. Fluorophore-encoded beads can be rapidly analyzed using classical flow cytometry or microfluidic techniques. We have developed a new generation of highly sensitive and specific diagnostic systems for detection of cancer antigens in human serum samples based on microbeads encoded with fluorescent quantum dots (QDs). The designed suspension microarray system was validated for quantitative detection of (1) free and total prostate specific antigen (PSA) in the serum of patients with prostate cancer and (2) carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA 15-3) in the serum of patients with breast cancer. The serum samples from healthy donors were used as a control. The antigen detection is based on the formation of an immune complex of a specific capture antibody (Ab), a target antigen (Ag), and a detector Ab on the surface of the encoded particles. The capture Ab is bound to the polymer shell of microbeads via an adapter molecule, for example, protein A. Protein A binds a monoclonal Ab in a highly oriented manner due to specific interaction with the Fc-region of the Ab molecule. Each antigen can be recognized and detected due to a specific microbead population carrying the unique fluorescent code. 100 and 231 serum samples from patients with different stages of prostate cancer and breast cancer, respectively, and those from healthy donors were examined using the designed suspension system. The data were validated by comparing with the results of the "gold standard" enzyme-linked immunosorbent assay (ELISA). They have shown that our approach is a good alternative to the diagnostics of cancer markers using conventional assays, especially in early diagnostic applications.

Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella

PubMed Central

2014-01-01

Background Autotransporter proteins represent a treasure trove for molecular engineers who modify Gram-negative bacteria for the export or secretion of foreign proteins across two membrane barriers. A particularly promising direction is the development of autotransporters as antigen display or secretion systems. Immunologists have been using ovalbumin as a reporter antigen for years and have developed sophisticated tools to detect specific T cells that respond to ovalbumin. Although ovalbumin-expressing bacteria are being used to trace T cell responses to colonizing or invading pathogens, current constructs for ovalbumin presentation have not been optimized. Results The activation of T helper cells in response to ovalbumin was improved by displaying the OVA-CD4 reporter epitope as a multimer on the surface of Salmonella and fused to the autotransporter MisL. Expression was optimized by including tandem in vivo promoters and two post-segregational killing systems for plasmid stabilization. Conclusions The use of an autotransporter protein to present relevant epitope repeats on the surface of bacteria, combined with additional techniques favoring stable and efficient in vivo transcription, optimizes antigen presentation to T cells. The technique of multimeric epitope surface display should also benefit the development of new Salmonella or other enterobacterial vaccines. PMID:24898796

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

Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

PubMed

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-09-08

Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

Cellular Pathway(S) of Antigen Processing and Presentation in Fish APC: Endosomal Involvement and Cell-Free Antigen Presentation

PubMed Central

Vallejo, Abbe N.; Miller, Norman W.; Harvey, Nancy E.; Cuchens, Marvin A.; Warr, Gregory W.

1992-01-01

Studies were conducted to address further the role(s) of antigen processing and presentation in the induction of immune responses in a phylogenetically lower vertebrate, specifically a teleost, the channel catfish. In particular, studies were aimed at determining the subcellular compartments involved in antigen degradation by channel catfish antigen-presenting cells (APC) as well as ascertaining the reexpression of immunogenic peptides on the surfaces of APC. The results showed that exogenous protein antigens were actively endocytosed by APC as detected by flow cytometry. Use of radiolabeled antigen and subcellular fractionation protocols also showed that antigen localized in endosomes/lysosomes. Furthermore, there was an apparent redistribution of antigen between these organelles and the plasma membrane during the course of antigen pulsing. Functional assays for the induction of in vitro antigen-specific proliferation of immune catfish peripheral blood leukocytes (PBL) showed that membrane preparations from antigen-pulsed autologous APC were highly stimulatory. The magnitude of responses elicited with such membrane preparations was very similar to that of PBL cultures stimulated with native antigen-pulsed and fixed intact APC or prefixed intact APC incubated with a peptide fragment of the nominal antigen. Current data further corroborate our previous findings that steps akin to antigen processing and presentation are clearly important in the induction of immune responses in lower vertebrates like fish, in a manner similar to that seen in mammalian systems. Consequently, it would appear that many immune functions among the diverse taxa of vertebrates are remarkably conserved. PMID:1343103

2012 annual literature review of donor-specific HLA antibodies after organ transplantation.

PubMed

Kaneku, Hugo

2012-01-01

From the articles reviewed in the present chapter, we observed: 1. The frequency of de novo donor-specific human leukocyte antigen (HLA) antibodies (DSA) detection in different organs is very similar: ranging between 15% and 23% in kidney, 23% in pancreas, and 18% in intestinal transplant patients. Apparently, all organs can elicit humoral responses after transplantation at comparable rates. 2. Although rates of de novo DSA formation after kidney transplantation are very similar across different centers--between 15% and 23%--, the mean time to the first detection of de novo DSA is markedly variable between centers (from 8 months to 4 years). Some differences found in the studies that may account for this could be the age of patients (studies including pediatric patients tend to show longer time to DSA detection compared to studies only including adults patients), patients' race, and maintenance immunosuppression regimens. 3. In most organs, alloantibodies against class II HLA--and especially against HLA-DQ antigens--are the most common DSA detected. This finding supports previous studies, but the explanation remains unclear. Poor HLA-DQ matching, paucity of class II HLA antigen expression on cell surface, and technical factors related to the detection of these antibodies (mean fluorescence intensity cutoff, multiple beads with the same antigen, denatured protein on single antigen beads) are some of the potential explanations that need further investigation. 4. Recent focus on histological changes during rejection in the presence of DSA that are independent of C4d deposition may change how antibody-mediated rejection is diagnosed in the near future. 5. More studies are looking into the importance of DSA in non-kidney transplants and now evidence shows that DSA may not only affect survival and rejection rates, but may also be associated with organ-specific lesions like fibrosis and biliary complications in livers or capillaritis in lungs.

Rapid Detection of Hepatitis B Virus Surface Antigen by an Agglutination Assay Mediated by a Bispecific Diabody against Both Human Erythrocytes and Hepatitis B Virus Surface Antigen▿

PubMed Central

Chen, Yu-Ping; Qiao, Yuan-Yuan; Zhao, Xiao-Hang; Chen, Hong-Song; Wang, Yan; Wang, Zhuozhi

2007-01-01

Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens. PMID:17442848

Manual-slide-engaged paper chip for parallel SERS-immunoassay measurement of clenbuterol from swine hair.

PubMed

Zheng, Tingting; Gao, Zhigang; Luo, Yong; Liu, Xianming; Zhao, Weijie; Lin, Bingcheng

2016-02-01

Clenbuterol (CL), as a feed additive, has been banned in many countries due to its potential threat to human health. In detection of CL, a fast, low-cost technique with high accuracy and specificity would be ideal for its administrative on-field inspections. Among the attempts to pursue a reliable detection tool of CL, a technique that combines surface enhanced Raman spectroscopy (SERS) and immunoassay, is close to meet the requirements as above. However, multiple steps of interactions between CL analyte, antibody, and antigen are involved in this method, and under conventional setup, the operation of SERS/immunoassay were unwieldy. In this paper, to facilitate a more manageable sample manipulation for SERS-immunoassay measurement, a 3D paper chip was suggested. A switch-on-chip multilayered (abbreviated as SoCM-) microfluidic paper-based analysis device (μPad) was fabricated to provide operators with manual switches on the interactions between different microfluids. Besides, on a detection slip we made on the main body of our SoCM-μPad, antigen was anchored in pattern. With this architecture, multistep interactions between the CL analyte in swine hair extract and the SERS probe-modified antibody and antigen, were managed for on-chip SERS-immunoassay detection. This would be very attractive for fast, cheap, accurate, and on-site specific detection of CL from real samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

PubMed Central

Hubert, Rene S.; Vivanco, Igor; Chen, Emily; Rastegar, Shiva; Leong, Kahan; Mitchell, Steve C.; Madraswala, Rashida; Zhou, Yanhong; Kuo, James; Raitano, Arthur B.; Jakobovits, Aya; Saffran, Douglas C.; Afar, Daniel E. H.

1999-01-01

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging. PMID:10588738

Neuronal Surface Autoantibodies in Neuropsychiatric Disorders: Are There Implications for Depression?

PubMed Central

Zong, Shenghua; Hoffmann, Carolin; Mané-Damas, Marina; Molenaar, Peter; Losen, Mario; Martinez-Martinez, Pilar

2017-01-01

Autoimmune diseases are affecting around 7.6–9.4% of the general population. A number of central nervous system disorders, including encephalitis and severe psychiatric disorders, have been demonstrated to associate with specific neuronal surface autoantibodies (NSAbs). It has become clear that specific autoantibodies targeting neuronal surface antigens and ion channels could cause severe mental disturbances. A number of studies have focused or are currently investigating the presence of autoantibodies in specific mental conditions such as schizophrenia and bipolar disorders. However, less is known about other conditions such as depression. Depression is a psychiatric disorder with complex etiology and pathogenesis. The diagnosis criteria of depression are largely based on symptoms but not on the origin of the disease. The question which arises is whether in a subgroup of patients with depression, the symptoms might be caused by autoantibodies targeting membrane-associated antigens. Here, we describe how autoantibodies targeting membrane proteins and ion channels cause pathological effects. We discuss the physiology of these antigens and their role in relation to depression. Finally, we summarize a number of studies detecting NSAbs with a special focus on cohorts that include depression diagnosis and/or show depressive symptoms. PMID:28725222

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

A 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.

Characterization of porcine CD205

USDA-ARS?s Scientific Manuscript database

Dendritic cells (DC) express a cell-surface receptor, CD205, that plays a role in antigen capture and delivery to the endocytic pathway. Besides DCs, high CD205 expression is also detected on thymic epithelial cells, but B cells, macrophages, and T cells have limited or no expression. CD205 has be...

Invited Review Article: Review of centrifugal microfluidic and bio-optical disks

PubMed Central

Nolte, David D.

2009-01-01

Spinning biodisks have advantages that make them attractive for specialized biochip applications. The two main classes of spinning biodisks are microfluidic disks and bio-optical compact disks (BioCD). Microfluidic biodisks take advantage of noninertial pumping for lab-on-a-chip devices using noninertial valves and switches under centrifugal and Coriolis forces to distribute fluids about the disks. BioCDs use spinning-disk interferometry, under the condition of common-path phase quadrature, to perform interferometric label-free detection of molecular recognition and binding. The optical detection of bound molecules on a disk is facilitated by rapid spinning that enables high-speed repetitive sampling to eliminate 1∕f noise through common-mode rejection of intensity fluctuations and extensive signal averaging. Multiple quadrature classes have been developed, such as microdiffraction, in-line, phase contrast, and holographic adaptive optics. Thin molecular films are detected through the surface dipole density with a surface height sensitivity for the detection of protein spots that is approximately 1 pm. This sensitivity easily resolves a submonolayer of solid-support immobilized antibodies and their antigen targets. Fluorescence and light scattering provide additional optical detection techniques on spinning disks. Immunoassays have been applied to haptoglobin using protein A∕G immobilization of antibodies and to prostate specific antigen. Small protein spots enable scalability to many spots per disk for high-throughput and highly multiplexed immonoassays. PMID:19895047

Determination of neuronal antibodies in suspected and definite Creutzfeldt-Jakob disease.

PubMed

Grau-Rivera, Oriol; Sánchez-Valle, Raquel; Saiz, Albert; Molinuevo, José Luis; Bernabé, Reyes; Munteis, Elvira; Pujadas, Francesc; Salvador, Antoni; Saura, Júlia; Ugarte, Antonio; Titulaer, Maarten; Dalmau, Josep; Graus, Francesc

2014-01-01

Creutzfeldt-Jakob disease (CJD) and autoimmune encephalitis with antibodies against neuronal surface antigens (NSA-abs) may present with similar clinical features. Establishing the correct diagnosis has practical implications in the management of care for these patients. To determine the frequency of NSA-abs in the cerebrospinal fluid of patients with suspected CJD and in patients with pathologically confirmed (ie, definite) CJD. A mixed prospective (suspected) and retrospective (definite) CJD cohort study was conducted in a reference center for detection of NSA-abs. The population included 346 patients with suspected CJD and 49 patients with definite CJD. Analysis of NSA-abs in cerebrospinal fluid with brain immunohistochemistry optimized for cell-surface antigens was performed. Positive cases in the suspected CJD group were further studied for antigen specificity using cell-based assays. All definite CJD cases were comprehensively tested for NSA-abs, with cell-based assays used for leucine-rich glioma-inactivated 1 (LGI1), contactin-associated protein-like 2 (CASPR2), N-methyl-d-aspartate (NMDA), and glycine (GlY) receptors. Neuronal surface antigens were detected in 6 of 346 patients (1.7%) with rapid neurologic deterioration suggestive of CJD. None of these 6 patients fulfilled the diagnostic criteria for probable or possible CJD. The target antigens included CASPR2, LGI1, NMDAR, aquaporin 4, Tr (DNER [δ/notch-like epidermal growth factor-related receptor]), and an unknown protein. Four of the patients developed rapidly progressive dementia, and the other 2 patients had cerebellar ataxia or seizures that were initially considered to be myoclonus without cognitive decline. The patient with Tr-abs had a positive 14-3-3 test result. Small cell lung carcinoma was diagnosed in the patient with antibodies against an unknown antigen. All patients improved or stabilized after appropriate treatment. None of the 49 patients with definite CJD had NSA-abs. A low, but clinically relevant, number of patients with suspected CJD had potentially treatable disorders associated with NSA-abs. In contrast, none of 49 patients with definite CJD had NSA-abs, including NMDAR-abs, GlyR-abs, LGI1-abs, or CASPR2-abs. These findings suggest that cerebrospinal fluid NSA-abs analysis should be included in the diagnostic workup of patients with rapidly progressive central nervous system syndromes, particularly when they do not fulfill the diagnostic criteria of probable or possible CJD.

Conservation of myeloid surface antigens on primate granulocytes.

PubMed

Letvin, N L; Todd, R F; Palley, L S; Schlossman, S F; Griffin, J D

1983-02-01

Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

Development of a highly sensitive bioluminescent enzyme immunoassay for hepatitis B virus surface antigen capable of detecting divergent mutants.

PubMed

Minekawa, Takayuki; Takehara, Shizuka; Takahashi, Masaharu; Okamoto, Hiroaki

2013-08-01

Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals.

Evaluation of a newly designed sandwich enzyme linked immunosorbent assay for the detection of hydatid antigen in serum, urine and cyst fluid for diagnosis of cystic echinococcosis.

PubMed

Chaya, Dr; Parija, Subhash Chandra

2013-07-01

Cystic echinococcosis (CE) is a zoonotic disease of humans with variable clinical manifestations. Imaging and immunological methods are currently the mainstay of diagnosis of this disease. Although the immunological tests for detection of anti-echinococcal antibodies have several disadvantages, they are widely being used. Antigen is far more superior than antibody detection test as they can provide a specific parasitic diagnosis. A sandwich enzyme linked immunosorbent assay (ELISA) was designed using antibodies to 24 kDa urinary hydatid antigen for the detection of hydatid antigens in urine, serum and cyst fluid specimens. The performance of this novel test was compared with that of other hydatid antibody detection ELISA and enzyme immune transfer blot (EITB) using radiological and surgical confirmation as the gold standard. The antigen detection ELISA showed 100% sensitivity and specificity when tested with cyst fluid. On testing urine and serum, the antigen detection ELISA was found to be more specific than antibody detection ELISA. EITB was found to be the most sensitive and specific test. ELISA using polyclonal antibodies against 24 kDa urinary hydatid protein was moderately sensitive to detect hydatid antigen in serum and urine. Hence polyclonal antibodies to 24 kDa urinary hydatid antigen can be used as an alternative source of antibody to detect hydatid antigen in serum, urine and cyst fluid. In the present study, EITB was found to be highly specific test for detection of hydatid antibodiesin serum. 24 kDa protein was found to be specific and of diagnostic value in CE.

Non-invasive optical detection of HBV based on serum surface-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Zheng, Zuci; Wang, Qiwen; Weng, Cuncheng; Lin, Xueliang; Lin, Yao; Feng, Shangyuan

2016-10-01

An optical method of surface-enhanced Raman spectroscopy (SERS) was developed for non-invasive detection of hepatitis B surface virus (HBV). Hepatitis B virus surface antigen (HBsAg) is an established serological marker that is routinely used for the diagnosis of acute or chronic hepatitis B virus(HBV) infection. Utilizing SERS to analyze blood serum for detecting HBV has not been reported in previous literature. SERS measurements were performed on two groups of serum samples: one group for 50 HBV patients and the other group for 50 healthy volunteers. Blood serum samples are collected from healthy control subjects and patients diagnosed with HBV. Furthermore, principal components analysis (PCA) combined with linear discriminant analysis (LDA) were employed to differentiate HBV patients from healthy volunteer and achieved sensitivity of 80.0% and specificity of 74.0%. This exploratory work demonstrates that SERS serum analysis combined with PCA-LDA has tremendous potential for the non-invasive detection of HBV.

A novel liquid-phase piezoelectric immunosensor for detecting Schistosoma japonicum circulating antigen.

PubMed

Wen, Zhili; Wang, Shiping; Wu, Zhaoyang; Shen, Guoli

2011-09-01

A new liquid-phase piezoelectric immunosensor (LP-PEIS), which can detect Schistosoma japonicum (Sj) circulating antigens (SjCAg) quantificationally, was developed. The IgG antibodies were purified from the sera of rabbits which had been infected or immunized by Sj and were immobilized on the surface of piezoelectric quartz crystal in LP-PEIS by staphylococcal protein A (SPA). It was used to detect SjCAg in sera of rabbits which had been infected by Sj in order to acquire some optimum conditions for detecting SjCAg. Finally, the LP-PEIS with optimum conditions was used to detect SjCAg in sera of patients who had been infected by Sj, and was compared with sandwich ELISA. A lot of optimum conditions of LP-PEIS for detecting SjCAg had been acquired. In the detection of patients' sera with acute Schistosomiasis, LP-PEIS has higher positive rate (100%) and lower false positive rate (3.0%) than sandwich ELISA (92.8%, 6.0%). However, there were no significant difference between LP-PEIS and sandwich ELISA. LP-PEIS can quantificationally detect SjCAg in patients' sera as well as sandwich ELISA. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Response of dairy calves to vaccinia viruses that express foreign genes.

PubMed Central

Gillespie, J H; Geissinger, C; Scott, F W; Higgins, W P; Holmes, D F; Perkus, M; Mercer, S; Paoletti, E

1986-01-01

Repeated intradermal inoculations of calves with wild-type vaccinia virus and recombinant vaccinia viruses expressing human hepatitis B virus surface antigen and herpes simplex virus, type 1, glycoprotein D produced characteristic pox lesions at each site of injection. In some instances, calves were inoculated as many as five times at intervals from 4 to 7 weeks. The lesions invariably were more severe after the second inoculation. Subsequent inoculations produced a less severe area of redness, swelling, necrosis, and scab formation. No other signs of illness, such as an elevation in temperature, were noted in the calves. Vaccinia virus was isolated in low titers from scabs taken at various times after inoculation. No lesions were formed at the sites injected with tissue culture fluid and cellular debris at the same time that virus inoculations were made. Calf contact controls remained normal through the 8-week exposure in isolation units with calves inoculated twice with vaccinia virus. No neutralizing antibody to vaccinia virus was detected in the contact controls. In contrast, the virus-inoculated calves developed neutralizing antibody to vaccinia virus and to herpes simplex virus glycoprotein D in serum. In all cattle, a second inoculation significantly enhanced the neutralizing antibody response within 1 week, suggesting that an anamnestic response had occurred. No antibody to hepatitis B virus surface antigen was elicited in calves after repeated inoculations with vaccinia recombinants that express hepatitis B virus surface antigen and are known to elicit in rabbits antibodies reactive with hepatitis B virus surface antigen. Images PMID:3700615

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

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

Pneumococcal surface protein A (PspA) is effective at eliciting T cell-mediated responses during invasive pneumococcal disease in adults

PubMed Central

Baril, L; Dietemann, J; Essevaz-Roulet, M; Béniguel, L; Coan, P; Briles, D E; Guy, B; Cozon, G

2006-01-01

Humoral immune response is essential for protection against invasive pneumococcal disease and this property is the basis of the polysaccharide-based anti-pneumococcal vaccines. Pneumococcal surface protein A (PspA), a cell-wall-associated surface protein, is a promising component for the next generation of pneumococcal vaccines. This PspA antigen has been shown to stimulate an antibody-based immunity. In the present study, we evaluated the capacity of PspA to stimulate CD4+ T cells which are needed for the correct development of a B cell based immune response in humans. Cellular immunity to PspA was evaluated by whole-blood culture with different pneumococcal antigens, followed by flow cytometric detection of activated CD4+CD25+ T cells. T cell-mediated immune responses to recombinant PspA proteins were assessed in acute-phase and convalescent blood from adults with invasive pneumococcal disease and in blood from healthy subjects. All cases had detectable antibodies against PspA on admission. We found that invasive pneumococcal disease induced transient T cell depletion but adaptive immune responses strengthened markedly during convalescence. The increased production of both interleukin (IL)-10 and interferon (IFN)-γ during convalescence suggests that these cytokines may be involved in modulating antibody-based immunity to pneumococcal disease. We demonstrated that PspA is efficient at eliciting T cell immune responses and antibodies to PspA. This study broadens the applicability of recombinant PspA as potent pneumococcal antigen for vaccination against S. pneumoniae. PMID:16879247

Identification of a Streptococcus agalactiae protein antigen associated with bovine mastitis isolates.

PubMed Central

Wanger, A R; Dunny, G M

1987-01-01

Immunoblotting was used to analyze the immune response of cows to Streptococcus agalactiae. Antibody from the milk of cows immunized (via the superficial inguinal lymph node) with formalinized S. agalactiae cells or from the milk of cows with S. agalactiae mastitis reacted strongly with a group of high-molecular-weight proteinaceous antigens. The two most predominant antigenic polypeptides in this group had apparent molecular weights of 97,000 and 104,000. Because the data indicated that these two antigens, as well as several minor antigens sometimes observed in the 70- to 100-kilodalton size range, seemed to be different forms of the same protein, we refer to the entire group as Sas97/104. A monoclonal antibody that was reactive with Sas97/104 was derived and was used to purify the antigen by affinity chromatography. Whole-cell and colony blot enzyme-linked immunoassays with either the monoclonal antibody or a polyclonal serum sample raised against the affinity-purified antigen indicated that this antigen (or cross-reactive proteins with higher molecular weights) is present on the S. agalactiae strains that were freshly isolated from mastitic cows. However, the antigen was not detected in S. agalactiae of human origin, bovine strains of S. agalactiae maintained for a prolonged period in the laboratory, or other streptococci. The data are consistent with the notion that Sas97/104 is a surface antigen and does not correspond to previously described type-specific antigens of group B streptococci. Images PMID:3552991

Phosphine-free synthesis of high-quality reverse type-I ZnSe/CdSe core with CdS/CdxZn1 - xS/ZnS multishell nanocrystals and their application for detection of human hepatitis B surface antigen

NASA Astrophysics Data System (ADS)

Shen, Huaibin; Yuan, Hang; Niu, Jin Zhong; Xu, Shasha; Zhou, Changhua; Ma, Lan; Li, Lin Song

2011-09-01

Highly photoluminescent (PL) reverse type-I ZnSe/CdSe nanocrystals (NCs) and ZnSe/CdSe/CdS/CdxZn1 - xS/ZnS core/multishell NCs were successfully synthesized by a phosphine-free method. By this low-cost, 'green' synthesis route, more than 10 g of high-quality ZnSe/CdSe/CdS/CdxZn1 - xS/ZnS NCs were synthesized in a large scale synthesis. After the overgrowth of a CdS/CdxZn1 - xS/ZnS multishell on ZnSe/CdSe cores, the PL quantum yields (QYs) increased from 28% to 75% along with the stability improvement. An amphiphilic oligomer was used as a surface coating agent to conduct a phase transfer experiment, core/multishell NCs were dissolved in water by such surface modification and the QYs were still kept above 70%. The as-prepared water dispersible ZnSe/CdSe/CdS/CdxZn1 - xS/ZnS core/multishell NCs not only have high fluorescence QYs but also are extremely stable in various physiological conditions. Furthermore, a biosensor system (lateral flow immunoassay system, LFIA) for the detection of human hepatitis B surface antigen (HBsAg) was developed by using this water-soluble core/multishell NCs as a fluorescent label and a nitrocellulose filter membrane for lateral flow. The result showed that such ZnSe/CdSe/CdS/CdxZn1 - xS/ZnS core/multishell NCs were excellent fluorescent labels to detect HBsAg. The sensitivity of HBsAg detection could reach as high as 0.05 ng ml - 1.

Polyvalent heat-killed antigen for the diagnosis of infection with Legionella pneumophila.

PubMed Central

Fallon, R J; Abraham, W H

1982-01-01

A polyvalent antigen composed of heat-killed agar-grown Legionella pneumophila serogroups 1-4 suspended in a suspension of yolk-sac from embryonated hens' eggs has been examined for use in the indirect fluorescent antibody test for Legionella infection. The serological response detected by monovalent antigen correlated well with that detected by polyvalent antigen. The use of polyvalent antigen forms a useful screening test for the detection of antibody to L pneumophila, but positive results must be confirmed by test using monovalent antigen. PMID:7042762

Evaluation of the Potential Risk of Hepatitis B Virus Transmission in Skin Allografting.

PubMed

Wang, D; Xie, W; Chen, T; Dong, C; Zhao, C; Tan, H; Tian, H; Xie, Q

2015-01-01

Skin transplantation is associated with potential risk of infectious disease transmission; however, the exclusion of donors owing to hepatitis B virus (HBV) infection will worsen the shortage of allograft skin supply. We report a paired study to evaluate the potential risk of HBV transmission in skin allografting. The presence of HBV DNA in the serum and skin from 37 burn patients with chronic HBV infection (CHB) was monitored by a HBV polymerase chain reaction (PCR) and the positive rates were compared by Fisher's exact probability test. There was a high consistency in the HBV serology profile between HBV DNA PCR (83.78%) and the clinical HBV test. Only 2 patients who were positive for hepatitis B surface antigen, hepatitis B e antigen, and hepatitis B core antibody had detectable HBV DNA in the skin tissue; however, no hepatitis B surface antigen was detected as examined by immunohistochemistry staining. There was a significant difference between the positive rates of HBV DNA in the serum and skin (χc(2) = 27.03; P < .001). The potential risk for HBV transmission by skin allografting is very low. Given that China has a large population of patients with HBV, the acceptance of skin from donors with CHB to the skin bank would increase the number of tissue donations to meet the urgent medical need for skin transplantation. Copyright © 2015 Elsevier Inc. All rights reserved.

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy.

PubMed

Poiroux, Guillaume; Barre, Annick; van Damme, Els J M; Benoist, Hervé; Rougé, Pierre

2017-06-09

Aberrant O -glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O -glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola , and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O -glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors.

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy

PubMed Central

Poiroux, Guillaume; Barre, Annick; van Damme, Els J. M.; Benoist, Hervé; Rougé, Pierre

2017-01-01

Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors. PMID:28598369

Evaluation of a newly designed sandwich enzyme linked immunosorbent assay for the detection of hydatid antigen in serum, urine and cyst fluid for diagnosis of cystic echinococcosis

PubMed Central

Chaya, DR; Parija, Subhash Chandra

2013-01-01

Introduction: Cystic echinococcosis (CE) is a zoonotic disease of humans with variable clinical manifestations. Imaging and immunological methods are currently the mainstay of diagnosis of this disease. Although the immunological tests for detection of anti-echinococcal antibodies have several disadvantages, they are widely being used. Antigen is far more superior than antibody detection test as they can provide a specific parasitic diagnosis. Materials and Methods: A sandwich enzyme linked immunosorbent assay (ELISA) was designed using antibodies to 24 kDa urinary hydatid antigen for the detection of hydatid antigens in urine, serum and cyst fluid specimens. The performance of this novel test was compared with that of other hydatid antibody detection ELISA and enzyme immune transfer blot (EITB) using radiological and surgical confirmation as the gold standard. Results: The antigen detection ELISA showed 100% sensitivity and specificity when tested with cyst fluid. On testing urine and serum, the antigen detection ELISA was found to be more specific than antibody detection ELISA. EITB was found to be the most sensitive and specific test. Conclusions: ELISA using polyclonal antibodies against 24 kDa urinary hydatid protein was moderately sensitive to detect hydatid antigen in serum and urine. Hence polyclonal antibodies to 24 kDa urinary hydatid antigen can be used as an alternative source of antibody to detect hydatid antigen in serum, urine and cyst fluid. In the present study, EITB was found to be highly specific test for detection of hydatid antibodiesin serum. 24 kDa protein was found to be specific and of diagnostic value in CE. PMID:24470996

Multiplexed BioCD for prostate specific antigen detection

NASA Astrophysics Data System (ADS)

Wang, Xuefeng; Zhao, Ming; Nolte, David D.

2008-02-01

Specific protein concentrations in human body fluid can serve as diagnostic markers for some diseases, and a quantitative and high-throughput technique for multiplexed protein detection would speed up diagnosis and facilitate medical research. For this purpose, our group developed the BioCD, a spinning-disc interferometric biosensor on which antibody is immobilized. The detection system adopts a common-path scheme making it ultra stable. The scaling mass sensitivity is below 10 pg/mm for protein surface density. A 25000-spot antibody BioCD was fabricated to measure the concentration of prostate specific antigen (PSA), a protein indicating prostate cancer if its level is high. Statistical analysis of our immunoassay results projects that the detection limit of PSA would reach 20 pg/ml in a 2 mg/ml background solution. For future prospects, a multiplexed BioCD can be produced for simultaneous diagnosis of diverse diseases. For instance, 100 markers above 200 pg/ml could be measured on a single disc given that the detection limit is inversely proportional to square root of the number of spots.

Mapping epitopes and antigenicity by site-directed masking

NASA Astrophysics Data System (ADS)

Paus, Didrik; Winter, Greg

2006-06-01

Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to -lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with -lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops. backbone flexibility | Freund's adjuvant | conformational epitope | antisera

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets*

PubMed Central

Rhoden, John J.; Dyas, Gregory L.

2016-01-01

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. PMID:27022022

Dynamic of Immune Response induced in Hepatitis B Surface Antigen-transgenic Mice Immunized with a Novel Therapeutic Formulation

PubMed Central

Almeida, Freya M Freyre; Blanco, Aracelys; Trujillo, Heidy; Hernández, Dunia; García, Daymir; Alba, José S; Abad, Matilde López; Merino, Nelson; Lobaina, Yadira

2016-01-01

ABSTRACT The development of therapeutic vaccines against chronic hepatitis B requires the capacity of the formulation to subvert a tolerated immune response as well as the evaluation of histopathological damage resulting from the treatment. In the present study, the dynamicity of induced immune response to hepatitis B surface antigen (HBsAg) was evaluated in transgenic mice that constitutively express the HBsAg gene (HBsAg-tg mice). After immunization with a vaccine candidate containing both surface (HBsAg) and core (HBcAg) antigens of hepatitis B virus (HBV), the effect of vaccination on clearance of circulating HBsAg and the potential histological alterations were examined. Transgenic (tg) and non-transgenic (Ntg) mice were immunized by intranasal (IN) and subcutaneous (SC) routes simultaneously. A control group received phosphate-buffered saline (PBS) by IN route and aluminum by SC route. Positive responses, at both humoral and cellular levels, were obtained after five immunizations in HBsAg-tg mice. Such responses were delayed and of lower intensity in tg mice, compared to vaccinated Ntg mice. Serum IgG response was characterized by a similar IgG subclass pattern. Even when HBsAg-specific CD8+ T cell responses were clearly detectable by gamma-interferon ELISPOT assay, histopathological alterations were not detected in any organ, including the liver and kidneys. Our study demonstrated, that it is possible to subvert the immune tolerance against HBsAg in tg mice, opening a window for new studies to optimize the schedule, dose, and formulation to improve the immune response to the therapeutic vaccine candidate. These results can be considered a safety proof to support clinical developments for the formulation under study. How to cite this article Freyre FM, Blanco A, Trujillo H, Hernández D, García D, Alba JS, Lopez M, Merino N, Lobaina Y, Aguilar JC. Dynamic of Immune Response induced in Hepatitis B Surface Antigen-transgenic Mice Immunized with a Novel Therapeutic Formulation. Euroasian J Hepato-Gastroenterol 2016;6(1):25-30. PMID:29201720

Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model.

PubMed

Zhong, Zhaoyang; Wu, Wei; Wang, Dong; Wang, Dan; Shan, Jinlu; Qing, Yi; Zhang, Zhimin

2010-06-15

A new, highly sensitive electrochemical immunosensor with a sandwich-type immunoassay format was designed to quantify carcinoembryonic antigen (CEA), as a model tumor marker, using nanogold-enwrapped graphene nanocomposites (NGGNs) as trace labels in clinical immunoassays. The device consisted of a glassy carbon electrode coated with Prussian Blue (PB) on whose surface gold nanoparticles were electrochemically deposited to the further modified with the specific analyte-capturing molecule, anti-CEA antibodies. The immunoassay was performed using horseradish peroxidase (HRP)-conjugated anti-CEA as secondary antibodies attached on the NGGN surface (HRP-anti-CEA-NGGN). The method using HRP-anti-CEA-NGGNs as detection antibodies shows high signal amplification, and exhibits a dynamic working range of 0.05-350 ng/mL with a low detection limit of 0.01 ng/mL CEA (at 3s). The assayed results of serum samples with the sensor received an acceptable agreement with the reference values. Importantly, the methodology provides a promising ultrasensitive assay strategy for clinical applications. Copyright 2010 Elsevier B.V. All rights reserved.

Neuronal surface antigen antibodies in limbic encephalitis: clinical-immunologic associations.

PubMed

Graus, F; Saiz, A; Lai, M; Bruna, J; López, F; Sabater, L; Blanco, Y; Rey, M J; Ribalta, T; Dalmau, J

2008-09-16

To report the frequency and type of antibodies against neuronal surface antigens (NSA-ab) in limbic encephalitis (LE). Analysis of clinical features, neuropathologic findings, and detection of NSA-ab using immunochemistry on rat tissue and neuronal cultures in a series of 45 patients with paraneoplastic (23) or idiopathic (22) LE. NSA-ab were identified in 29 patients (64%; 12 paraneoplastic, 17 idiopathic). Thirteen patients had voltage-gated potassium channels (VGKC)-ab, 11 novel NSA (nNSA)-ab, and 5 NMDA receptor (NMDAR)-ab. nNSA-ab did not identify a common antigen and were more frequent in paraneoplastic than idiopathic LE (39% vs 9%; p = 0.03). When compared with VGKC-ab or NMDAR-ab, the nNSA associated more frequently with intraneuronal antibodies (11% vs 73%; p = 0.001). Of 12 patients (9 nNSA-ab, 2 VGKC-ab, 1 NMDAR-ab) with paraneoplastic LE and NSA-ab, concomitant intraneuronal antibodies occurred in 9 (75%). None of these 12 patients improved with immunotherapy. The autopsy of three of them showed neuronal loss, microgliosis, and cytotoxic T cell infiltrates in the hippocampus and amygdala. These findings were compatible with a T-cell mediated neuronal damage. In contrast, 13 of 17 (76%) patients with idiopathic LE and NSA-ab (8 VGKC-ab, 4 NMDAR-ab, 1 nNSA-ab) and 1 of 5 (20%) without antibodies had clinical improvement (p = 0.04). In paraneoplastic limbic encephalitis (LE), novel antibodies against neuronal surface antigens (nNSA-ab) occur frequently, coexist with antibodies against intracellular antigens, and these cases are refractory to immunotherapy. In idiopathic LE, the likelihood of improvement is significantly higher in patients with NSA-ab than in those without antibodies.

Mucosal tolerance disruption favors disease progression in an extraorbital lacrimal gland excision model of murine dry eye.

PubMed

Guzmán, Mauricio; Keitelman, Irene; Sabbione, Florencia; Trevani, Analía S; Giordano, Mirta N; Galletti, Jeremías G

2016-10-01

Dry eye is a highly prevalent immune disorder characterized by a dysfunctional tear film and a Th1/Th17 T cell response at the ocular surface. The specificity of these pathogenic effector T cells remains to be determined, but auto-reactivity is considered likely. However, we have previously shown that ocular mucosal tolerance to an exogenous antigen is disrupted in a scopolamine-induced murine dry eye model and that it is actually responsible for disease progression. Here we report comparable findings in an entirely different murine model of dry eye that involves resection of the extraorbital lacrimal glands but no systemic muscarinic receptor blockade. Upon ocular instillation of ovalbumin, a delayed breakdown in mucosal tolerance to this antigen was observed in excised but not in sham-operated mice, which was mediated by interferon γ- and interleukin 17-producing antigen-specific T cells. Consistently, antigen-specific regulatory T cells were detectable in sham-operated but not in excised mice. As for other models of ocular surface disorders, epithelial activation of the NF-κB pathway by desiccating stress was determinant in the mucosal immune outcome. Underscoring the role of mucosal tolerance disruption in dry eye pathogenesis, its prevention by a topical NF-κB inhibitor led to reduced corneal damage in excised mice. Altogether these results show that surgically originated desiccating stress also initiates an abnormal Th1/Th17 T cell response to harmless exogenous antigens that reach the ocular surface. This event might actually contribute to corneal damage and challenges the conception of dry eye as a strictly autoimmune disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antibody purification-independent microarrays (PIM) by direct bacteria spotting on TiO2-treated slides.

PubMed

De Marni, Marzia L; Monegal, Ana; Venturini, Samuele; Vinati, Simone; Carbone, Roberta; de Marco, Ario

2012-02-01

The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO(2) or epoxy slides (purification-independent microarray - PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10μg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31 days after spotting. We finally demonstrated that the method is suitable for other antigen-antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs. Copyright © 2011. Published by Elsevier Inc.

Environmental quantification of Pasteuria penetrans endospores using in situ antigen extraction and immunodetection with a monoclonal antibody.

PubMed

Schmidt, L M; Preston, J F; Dickson, D W; Rice, J D; Hewlett, T E

2003-05-01

Abstract Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.) that has attracted significant attention as a promising biocontrol agent. The inability to culture P. penetrans has invoked the need for a quantitative detection capability to facilitate biocontrol studies. A chemical extraction method using urea, dithiothreitol and CHES buffer (UDC) is shown to release soluble endospore envelope antigen from endospores present in complex matrices, generating an extract that can be used to determine the levels of spores when compared to a standard in an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody, MAb 2A41D10. Extractions can be performed in less than 1 h. Linear regression analysis routinely produced line fits with r(2)>0.90. Antigen extraction efficiency was not influenced by soil type. Three ELISA formats were analyzed for quantitative detection of P. penetrans endospores. A tertiary ELISA immunodetection system provided the lowest level of detection at approximately 300 spores per gram of soil. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots of soil extracts containing P. penetrans endospore antigen produced signature peptides bearing a common epitope characteristic of endospores of Pasteuria spp. MAb 2A41D10 was specific for Pasteuria spp. and did not react with extracts of Pasteuria-free soil or with spore extracts of native Gram-positive endospore-forming bacteria. Immunofluorescent microscopy revealed that MAb 2A41D10 recognizes an epitope uniformly distributed on the endospore surface. The development of a rapid extraction method and analysis of solubilized antigen by immunodetection has the potential for broad application in food and environmental microbiology.

Induction of anti-HBs in HB vaccine nonresponders in vivo by hepatitis B surface antigen-pulsed blood dendritic cells.

PubMed

Fazle Akbar, Sk Md; Furukawa, Shinya; Yoshida, Osamu; Hiasa, Yoichi; Horiike, Norio; Onji, Morikazu

2007-07-01

Antigen-pulsed dendritic cells (DCs) are now used for treatment of patients with cancers, however, the efficacy of these DCs has never been evaluated for prophylactic purposes. The aim of this study was (1) to prepare hepatitis B surface antigen (HBsAg)-pulsed human blood DCs, (2) to assess immunogenicity of HBsAg-pulsed DCs in vitro and (3) to evaluate the efficacy of HBsAg-pulsed DCs in hepatitis B (HB) vaccine nonresponders. Human peripheral blood DCs were cultured with HBsAg to prepare HBsAg-pulsed DCs. The expression of immunogenic epitopes of HBsAg on HBsAg-pulsed DCs was assessed in vitro. Finally, HBsAg-pulsed DCs were administered, intradermally to six HB vaccine nonresponders and the levels of antibody to HBsAg (anti-HBs) in the sera were assessed. HB vaccine nonresponders did not exhibit features of immediate, early or delayed adverse reactions due to administration of HBsAg-pulsed DCs. Anti-HBs were detected in the sera of all HB vaccine nonresponders within 28 days after administration of HBsAg-pulsed DCs. This study opens a new field of application of antigen-pulsed DCs for prophylactic purposes when adequate levels of protective antibody cannot be induced by traditional vaccination approaches.

Generation and selection of anti-flagellin monoclonal antibodies useful for serotyping Salmonella enterica.

PubMed

Hiriart, Yanina; Serradell, Maria; Martínez, Araci; Sampaolesi, Sofia; Maciel, Dolores Gonzalez; Chabalgoity, Jose Alejandro; Yim, Lucía; Algorta, Gabriela; Rumbo, Martin

2013-01-01

In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.

Sero-prevalence of Hepatitis B Virus Infection in Balochistan Province of Pakistan

PubMed Central

Sheikh, Nadeem S.; Sheikh, Azeem S.; Sheikh, Aqleem A.; Yahya, Samira; Rafi-U-Shan; Lateef, Majid

2011-01-01

Background/Aim: The objective was to evaluate the sero-prevalence of hepatitis B surface antigen (HBsAg) and IgM antibodies to hepatitis core antigen in Balochistan Province of Pakistan. Design of the study: A cross-sectional, population-based study. Place and time of the study: The study was conducted in Balochistan from 1st January 2004 to 31st December, 2008. The screening areas included Barkhan, Eashani, Khuzdar, Kodi Zikriani, Kohlu, Rakhni and Turbat. Materials and Methods: A total of 15,260 subjects were enrolled; 11,900 (78%) agreed to undergo screening. Fresh serum samples were tested for the presence of hepatitis B surface antigen and IgM antibodies to hepatitis B core antigen. Results: HBsAg was detected in 1166 (9.8%) while anti-HBc IgM was found in 117 (10.0%). HBsAg positivity was seen in 875 (12.7%) males and 291 (5.8%) females. The prevalence of hepatitis B in Balochistan varies from 3.3% in Khuzdar to 17.0% in Kodi Zikriani. Conclusions: It is utmost important to educate the public, to take proper measures to control the spread of infection and vaccination in order to interrupt transmission of this threatening public health problem in Balochistan province of Pakistan. PMID:21546720

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

Identification of biological agents using surface enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Paxon, Tracy L.; Duthie, R. Scott; Renko, Casey; Burns, Andrew A.; Lesaicherre, Marie L.; Mondello, Frank J.

2011-05-01

GE Global Research Center, in collaboration with Morpho Detection, Inc. has developed an assay scheme for the identification of biological agents using Surface Enhanced Raman Scattering (SERS). Specifically, unique spectroscopic signatures are generated using SERS tags consisting of individual glass-encapsulated gold nanoparticles and surfacebound reporter molecules. These SERS tags are modified with a capture moiety specific to the antigen of interest, and serve as a spectroscopic label in a bead-based sandwich assay. Assays are being developed for a variety of pathogens and this paper will focus on aspects of assay development, optimization, stabilization and validation. Results on the development of an assay to detect Ricin toxin will be presented, and preliminary feasibility studies for the detection of additional pathogens will be discussed.

Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

PubMed

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

2010-07-26

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

PubMed Central

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L.; Conrad, Daniel H.; Xu, Ping

2010-01-01

Background Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. Methods and Findings We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. Conclusions The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases. PMID:20668678

Cross-calibrating interferon-γ detection by using eletrochemical impedance spectroscopy and paraboloidal mirror enabled surface plasmon resonance interferometer

NASA Astrophysics Data System (ADS)

Liu, Meng-Wei; Chang, Hao-Jung; Lee, Shu-sheng; Lee, Chih-Kung

2016-03-01

Tuberculosis is a highly contagious disease such that global latent patient can be as high as one third of the world population. Currently, latent tuberculosis was diagnosed by stimulating the T cells to produce the biomarker of tuberculosis, i.e., interferon-γ. In this paper, we developed a paraboloidal mirror enabled surface plasmon resonance (SPR) interferometer that has the potential to also integrate ellipsometry to analyze the antibody and antigen reactions. To examine the feasibility of developing a platform for cross calibrating the performance and detection limit of various bio-detection techniques, electrochemical impedance spectroscopy (EIS) method was also implemented onto a biochip that can be incorporated into this newly developed platform. The microfluidic channel of the biochip was functionalized by coating the interferon-γ antibody so as to enhance the detection specificity. To facilitate the processing steps needed for using the biochip to detect various antigen of vastly different concentrations, a kinetic mount was also developed to guarantee the biochip re-positioning accuracy whenever the biochip was removed and placed back for another round of detection. With EIS being utilized, SPR was also adopted to observe the real-time signals on the computer in order to analyze the success of each biochip processing steps such as functionalization, wash, etc. Finally, the EIS results and the optical signals obtained from the newly developed optical detection platform was cross-calibrated. Preliminary experimental results demonstrate the accuracy and performance of SPR and EIS measurement done at the newly integrated platform.

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

NASA Astrophysics Data System (ADS)

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

2014-07-01

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

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

An artificial test substrate for evaluating electron microscopic immunocytochemical labeling reactions.

PubMed

Gagne, G D; Miller, M F

1987-08-01

We describe an artificial substrate system for optimization of labeling parameters in electron microscope immunocytochemical studies. The system involves use of blocks of glutaraldehyde-polymerized BSA into which a desired antigen is incorporated by a simple soaking procedure. The resulting antigen-impregnated artificial substrate can then be fixed and embedded identically to a piece of tissue. The BSA substrate can also be dried and then sectioned for immunolabeling with or without chemical fixation and without exposing the antigen to dehydrating agents and embedding resins. The effects of various fixation and embedding procedures can thus be evaluated separately. Other parameters affecting immunocytochemical labeling, such as antibody and conjugate concentration, can also be evaluated. We used this system, along with immunogold labeling, to determine quantitatively the optimal fixation and embedding conditions for labeling of hepatitis B surface antigen (HbsAg), human IgG, and horseradish peroxidase. Using unfixed and unembedded HBsAg, we were able to detect antigen concentrations below 20 micrograms/ml. We have shown that it is not possible to label HBsAg within resin-embedded cells using conventional aldehyde fixation protocols and polyclonal antibodies.

Humoral immune response against two surface antigens of Chlamydia pecorum in vaccinated and naturally infected sheep.

PubMed

Bommana, Sankhya; Walker, Evelyn; Desclozeaux, Marion; Timms, Peter; Polkinghorne, Adam

2017-01-01

Chlamydia pecorum is a globally recognised livestock pathogen due to the significant clinical and economic impact it poses to livestock producers. Routine serological diagnosis is through a complement fixation test (CFT), which is often criticised for cross-reactivity, poor sensitivity and specificity. Although serology remains the preferred method in veterinary diagnostic laboratories, serological assays based on surface antigens of C. pecorum have not been established until now. In this study, we evaluated the use of two chlamydial recombinant protein antigens (PmpG and MOMP-G) by a direct IgG ELISA method for detection of ovine anti-chlamydial antibodies. Using the Pepscan method we then identified B cell epitopes across PmpG and MOMP-G proteins, in lambs with (a) naturally occurring asymptomatic C. pecorum infections (b) C. pecorum-associated polyarthritis and (c) recombinant PmpG and MOMP-G vaccine. Plasma IgG antibodies to PmpG in natural infection of lambs were detected earlier in infection than CFT and served as an acute phase marker. Antibodies to MOMP-G IgG were significantly heightened in lambs with C. pecorum-associated polyarthritis. PmpG and MOMP-G specific B-cell epitope mapping revealed epitope responses in immunised lambs cluster with some of the epitope responses in naturally infected lambs. B-cell epitope mapping further revealed that lambs with polyarthritis recognised several unique PmpG (50% frequency, peptide 8, 25, 40, 41 and 50) and MOMP (50% frequency, peptide 50) epitopes in comparison to asymptomatic infections. The findings of this study will have implications towards improved serodiagnosis of C. pecorum infections in livestock and inform the downstream development of alternative peptide-based antigens for future C. pecorum vaccine studies.

Red blood cell microparticles and blood group antigens: an analysis by flow cytometry

PubMed Central

Canellini, Giorgia; Rubin, Olivier; Delobel, Julien; Crettaz, David; Lion, Niels; Tissot, Jean-Daniel

2012-01-01

Background The storage of blood induces the formation of erythrocytes-derived microparticles. Their pathogenic role in blood transfusion is not known so far, especially the risk to trigger alloantibody production in the recipient. This work aims to study the expression of clinically significant blood group antigens on the surface of red blood cells microparticles. Material and methods Red blood cells contained in erythrocyte concentrates were stained with specific antibodies directed against blood group antigens and routinely used in immunohematology practice. After inducing erythrocytes vesiculation with calcium ionophore, the presence of blood group antigens was analysed by flow cytometry. Results The expression of several blood group antigens from the RH, KEL, JK, FY, MNS, LE and LU systems was detected on erythrocyte microparticles. The presence of M (MNS1), N (MNS2) and s (MNS4) antigens could not be demonstrated by flow cytometry, despite that glycophorin A and B were identified on microparticles using anti-CD235a and anti-MNS3. Discussion We conclude that blood group antigens are localized on erythrocytes-derived microparticles and probably keep their immunogenicity because of their capacity to bind specific antibody. Selective segregation process during vesiculation or their ability to elicit an immune response in vivo has to be tested by further studies. PMID:22890266

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets.

PubMed

Rhoden, John J; Dyas, Gregory L; Wroblewski, Victor J

2016-05-20

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Highly sensitive immunosensing of prostate specific antigen using poly cysteine caped by graphene quantum dots and gold nanoparticle: A novel signal amplification strategy.

PubMed

Malekzad, Hediyeh; Hasanzadeh, Mohammad; Shadjou, Nasrin; Jouyban, Abolghasem

2017-12-01

A mediator-free electrochemical immunosensor for quantitation of prostate specific antigen (PSA) based on dual signal amplification strategy was fabricated. In this work, PSA-antibody (anti-PSA) was immobilized onto a green and biocompatible nanocomposite containing poly l-cysteine (P-Cys) as conductive matrix and graphene quantum dots (GQDs)/gold nanoparticles (GNPs) as dual signal amplification elements. Therefore, a novel multilayer film based on P-Cys, GQDs, and GNPs was exploited to develop a highly sensitive amperometric immunosensor for detection of PSA. Fully electrochemical methodology was used to prepare a new transducer on a gold surface which provided a high surface area to immobilize a high amount of the anti-PSA. Importantly, GNPs prepared by soft template synthesized method lead to compact morphology was achieved. The surface morphology of electrode surface was characterized by high-resolution field emission scanning electron microscope (FE-SEM) and energy dispersive spectroscopy (EDX). Chemical compositions of the gold nanoparticles were analysed by an EDX. The immunosensor was employed for the detection of PSA in physiological pH. Under optimized condition the calibration curve for PSA concentration was linear up to 2-9pgmL -1 with lower limit of quantification of 1.8pgmL -1 . Copyright © 2017 Elsevier B.V. All rights reserved.

Antibody response to Ehrlichia risticii and antibody reactivity to the component antigens in horses with induced Potomac horse fever.

PubMed

Dutta, S K; Mattingly, B L; Shankarappa, B

1989-10-01

The antibody response and the antibody reactivity to component antigens of Ehrlichia risticii were studied in horses with induced Potomac horse fever. These horses had no detectable antibodies to E. risticii in their preinoculation (PrI) sera by indirect fluorescent-antibody assay and enzyme-linked immunosorbent assay (ELISA). All the horses exhibited typical disease features following experimental infection and responded with specific antibodies, as measured by ELISA and indirect fluorescent-antibody assay. A primary antibody response was detected in 70% of the horses, while a secondary-type antibody response was detected in 30% of the horses by ELISA. In the primary antibody response, a distinct titer was observed at 2 weeks postinoculation (PI), when the immunoglobulin M (IgM)/IgG ratio was 2 to 5, and the overall antibody titer peaked at 6 to 8 weeks PI. The secondary-type antibody response exhibited a characteristic titer at 1 week PI, the IgM and IgG titers were about equal at 2 weeks PI, and the overall antibody titer peaked at 6 weeks PI. A transient depression in the IgG response at 4 weeks PI was observed in both response types. The antibody was maintained at a high titer for over a year in all horses. Western immunoblot reactivity showed that the antisera collected from these infected horses at 4 to 5 weeks PI recognized some or all of the six major E. risticii component antigens (70, 55, 51, 44, 33, and 28 kilodaltons), all of which were apparent surface components. The 6- to 8-week PI antisera recognized up to 16 component antigens, including 9 major antigens (110, 86, 70, 55, 51, 49, 44, 33, and 28 kilodaltons). However, the PrI sera of these horses showed reactivity at various intensities with one to seven of the component antigens. There was no apparent correlation between this reactivity pattern and the subsequent antibody response types.

Reverse enzyme-linked immunosorbent assay using monoclonal antibodies against SAG1-related sequence, SAG2A, and p97 antigens from Toxoplasma gondii to detect specific immunoglobulin G (IgG), IgM, and IgA antibodies in human sera.

PubMed

Carvalho, Fernando R; Silva, Deise A O; Cunha-Júnior, Jair P; Souza, Maria A; Oliveira, Taísa C; Béla, Samantha R; Faria, Gabriele G; Lopes, Carolina S; Mineo, José R

2008-08-01

The present study aimed to evaluate the performance of three monoclonal antibodies (MAbs) in reverse enzyme-linked immunosorbent assays (ELISAs) for detecting immunoglobulin G (IgG), IgM, and IgA antibodies against Toxoplasma gondii in 175 serum samples from patients at different stages of T. gondii infection, as defined by both serological and clinical criteria, as follows: recent (n = 45), transient (n = 40), and chronic (n = 55) infection as well as seronegative subjects (n = 35). The results were compared with those obtained by indirect ELISA using soluble Toxoplasma total antigen (STAg). Our data demonstrated that MAb A3A4 recognizes a conformational epitope in SAG1-related-sequence (SRS) antigens, while A4D12 and 1B8 recognize linear epitopes defined as SAG2A surface antigen and p97 cytoplasmatic antigen, respectively. Reverse ELISA for IgG with A3A4 or A4D12 MAbs was highly correlated with indirect ELISA for anti-STAg IgG, whereas only A4D12 reverse ELISA showed high correlation with indirect ELISA for IgM and IgA isotypes. To our knowledge, this is the first report analyzing the performance of a reverse ELISA for simultaneous detection of IgG, IgM, and IgA isotypes active toward native SAG2A, SRS, and p97 molecules from STAg, using a panel of human sera from patients with recent and chronic toxoplasmosis. Thus, reverse ELISA based on the capture of native SAG2A and SRS antigens of STAg by MAbs could be an additional approach for strengthening the helpfulness of serological tests assessing the stage of infection, particularly in combination with highly sensitive and specific assays that are frequently used nowadays for diagnosis of toxoplasmosis during pregnancy or congenital infection in newborns.

Aberrant lymphoid antigen expression in acute myeloid leukemia in Saudi Arabia.

PubMed

El-Sissy, Azza H; El-Mashari, May A; Bassuni, Wafaa Y; El-Swaayed, Aziza F

2006-09-01

Immunophenotyping improves both accuracy and reproducibility of acute leukemia classification and is considered particularly useful for identifying aberrant lineage association of acute leukemia, biphenotypic and bilineal acute leukemia, as well as monitoring minimal residual disease. Some immunophenotypes correlate with cytogenetic abnormalities and prognosis. Is to determine aberrant lymphoid antigen expression in Saudi acute myeloid leukemia (AML), correlate them with FAB subtypes, evaluate early surface markers CD7 and CD56, and to investigate the role of cytoplasmic CD79a (a B cell marker that is assigned a high score of 2.0 in the WHO classification). Thirty four newly diagnosed AML cases were included in this study, 47% showed aberrant lymphoid antigen expression. CD9 was the most frequently expressed lymphoid antigen (29.4%) followed by CD7 & CD19 (11.8%), CD4 (8.8%) and CD22 (2.9%). CD9 was expressed in 3/6 (50%) of M3 cases, CD7 was expressed in 11.8% and was mostly confined to FAB M1 and M2 and associated with immature antigens CD34, HLA-DR and TdT. CD56 was expressed in 7/34 (20.6%) cases, three of these cases (42.9%) belonged to the monocytic group. CD56 was also detected in 2 cases with 11q23 rearrangement. CD56 was expressed in 2/7 (28.6%) M2 cases, and was associated with t (8;21) (q22;q22) together with CD19. Co-expression of CD56 and CD7 was detected in 2.9% of the cases. CD79a was expressed in one case together with CD19, diagnosed as acute biphenotypic leukemia, and was associated with t(8;21) (q22;q22). Minimal residual disease in AML is very difficult to trace, detection of aberrant expression of lymphoid antigens will make it easier. The high score given to CD79a by EGIL is questionable based on cytogenetic classification.

Engineering antigens for in situ erythrocyte binding induces T-cell deletion.

PubMed

Kontos, Stephan; Kourtis, Iraklis C; Dane, Karen Y; Hubbell, Jeffrey A

2013-01-02

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

Detection of CEA in human serum using surface-enhanced Raman spectroscopy coupled with antibody-modified Au and γ-Fe₂O₃@Au nanoparticles.

PubMed

Lin, Yan; Xu, Guanhong; Wei, Fangdi; Zhang, Aixia; Yang, Jing; Hu, Qin

2016-03-20

In this present work, a rapid and simple method to detect carcinoembryonic antigen (CEA) was developed by using surface-enhanced Raman spectroscopy (SERS) coupled with antibody-modified Au and γ-Fe2O3@Au nanoparticles. First, Au@Raman reporter and γ-Fe2O3@Au were prepared, and then modified with CEA antibody. When CEA was present, the immuno-Au@Raman reporter and immuno-γ-Fe2O3@Au formed a complex through antibody-antigen-antibody interaction. The selective and sensitive detection of CEA could be achieved by SERS after magnetic separation. Under the optimal conditions, a linear relationship was observed between the Raman peak intensity and the concentration of CEA in the range of 1-50 ng mL(-1) with an excellent correlation coefficient of 0.9942. The limit of detection based on two times ratio of signal to noise was 0.1 ng/mL. The recoveries of CEA standard solution spiked with human serum samples were in the range of 88.5-105.9% with the relative standard deviations less than 17.4%. The method built was applied to the detection of CEA in human serum, and the relative deviations of the analysis results between the present method and electrochemiluminescence immunoassay were all less than 16.6%. The proposed method is practical and has a potential for clinic test of CEA. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of electrochemical immunosensors based on different serum antibody immobilization methods for detection of Japanese encephalitis virus

NASA Astrophysics Data System (ADS)

Tran, Quang Huy; Hanh Nguyen, Thi Hong; Mai, Anh Tuan; Thuy Nguyen, Thi; Khue Vu, Quang; Nga Phan, Thi

2012-03-01

This paper describes the development of electrochemical immunosensors based on human serum antibodies with different immobilization methods for detection of Japanese encephalitis virus (JEV). Human serum containing anti-JEV antibodies was used to immobilize onto the surface of silanized interdigitated electrodes by four methods: direct adsorption (APTES-serum), covalent binding with a cross linker of glutaraldehyde (APTES-GA-serum), covalent binding with a cross linker of glutaraldehyde combined with anti-human IgG (APTES-GA-anti-HIgG-serum) and covalent binding with a cross linker of glutaraldehyde combined with a bioaffinity of protein A (APTES-GA-PrA-serum). Atomic force microscopy was used to verify surface characteristics of the interdigitated electrodes before and after treatment with serum antibodies. The output signal of the immunosensors was measured by the change of conductivity resulting from the specific binding of JEV antigens and serum antibodies immobilized on the electrodes, with the help of horseradish peroxidase (HRP)-labeled secondary antibody against JEV. The results showed that the APTES-GA-PrA-serum method provided the highest signal of the electrochemical immunosensor for detection of JEV antigens, with the linear range from 25 ng ml-1 to 1 μg ml-1, and the limit of detection was about 10 ng ml-1. This study shows a potential development of novel electrochemical immunosensors applied for virus detection in clinical samples in case of possible outbreaks.

Ultrasensitive electrochemical immunoassay for surface array protein, a Bacillus anthracis biomarker using Au-Pd nanocrystals loaded on boron-nitride nanosheets as catalytic labels.

PubMed

Sharma, Mukesh Kumar; Narayanan, J; Pardasani, Deepak; Srivastava, Divesh N; Upadhyay, Sanjay; Goel, Ajay Kumar

2016-06-15

Bacillus anthracis, the causative agent of anthrax, is a well known bioterrorism agent. The determination of surface array protein (Sap), a unique biomarker for B. anthracis can offer an opportunity for specific detection of B. anthracis in culture broth. In this study, we designed a new catalytic bionanolabel and fabricated a novel electrochemical immunosensor for ultrasensitive detection of B. anthracis Sap antigen. Bimetallic gold-palladium nanoparticles were in-situ grown on poly (diallyldimethylammonium chloride) functionalized boron nitride nanosheets (Au-Pd NPs@BNNSs) and conjugated with the mouse anti-B. anthracis Sap antibodies (Ab2); named Au-Pd NPs@BNNSs/Ab2. The resulting Au-Pd NPs@BNNSs/Ab2 bionanolabel demonstrated high catalytic activity towards reduction of 4-nitrophenol. The sensitivity of the electrochemical immunosensor along with redox cycling of 4-aminophenol to 4-quinoneimine was improved to a great extent. Under optimal conditions, the proposed immunosensor exhibited a wide working range from 5 pg/mL to 100 ng/mL with a minimum detection limit of 1 pg/mL B. anthracis Sap antigen. The practical applicability of the immunosensor was demonstrated by specific detection of Sap secreted by the B. anthracis in culture broth just after 1h of growth. These labels open a new direction for the ultrasensitive detection of different biological warfare agents and their markers in different matrices. Copyright © 2016 Elsevier B.V. All rights reserved.

Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

PubMed

Hoane, Jessica S; Morrow, Jennifer K; Saville, William J; Dubey, J P; Granstrom, David E; Howe, Daniel K

2005-09-01

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

Antibodies to Plasmodium falciparum Antigens Predict a Higher Risk of Malaria But Protection From Symptoms Once Parasitemic

PubMed Central

Hubbard, Alan; Njama-Meya, Denise; Narum, David L.; Lanar, David E.; Dutta, Sheetij; Rosenthal, Philip J.; Dorsey, Grant; John, Chandy C.

2011-01-01

(See the article by Bejon et al, on pages 9–18, and Bousema et al, on pages 1–3.) Background. Associations between antibody responses to Plasmodium falciparum antigens and protection against symptomatic malaria have been difficult to ascertain, in part because antibodies are potential markers of both exposure to P. falciparum and protection against disease. Methods. We measured IgG responses to P. falciparum circumsporozoite protein, liver-stage antigen 1, apical-membrane antigen 1 (AMA-1), and merozoite surface proteins (MSP) 1 and 3, in children in Kampala, Uganda, and measured incidence of malaria before and after antibody measurement. Results. Stronger responses to all 5 antigens were associated with an increased risk of clinical malaria (P < .01) because of confounding with prior exposure to P. falciparum. However, with use of another assessment, risk of clinical malaria once parasitemic, stronger responses to AMA-1, MSP-1, and MSP-3 were associated with protection (odds ratios, 0.34, 0.36, and 0.31, respectively, per 10-fold increase; P < .01). Analyses assessing antibodies in combination suggested that any protective effect of antibodies was overestimated by associations between individual responses and protection. Conclusions. Using the risk of symptomatic malaria once parasitemic as an outcome may improve detection of associations between immune responses and protection from disease. Immunoepidemiology studies designed to detect mechanisms of immune protection should integrate prior exposure into the analysis and evaluate multiple immune responses. PMID:21628654

Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

PubMed

Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G; Lai, Michelle; D'Alessio, Joseph A; Theunissen, Jan-Willem

2015-01-01

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

The Association of High Prevalence of Trophozoites in Peripheral Blood with Lower Antibody Response to P. falciparum Infected Erythrocytes among Asymptomatic Children in Sudan.

PubMed

Mohamed, Sara N; Hassan, Dina A; El Hussein, Abdelrahim M; Osman, Ihssan M; Ibrahim, Muntasir E; Mohamed, Hiba S; Nour, Bakri Y; Abdulhadi, Nasreldin H

2016-01-01

Background. The most prominent variant surface antigens (VSAs) of Plasmodium falciparum are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which serves as a parasite-sequestering ligand to endothelial cells. In this study we have examined the antibody reactivity of autologous plasma from symptomatic and asymptomatic malaria infected children against the infected erythrocytes' surface antigens using flow cytometry. Methods. Ethidium-bromide-labelled erythrocytic mature forms of P. falciparum parasites obtained from symptomatic and asymptomatic children were sequentially incubated with autologous plasma and fluorescein isothiocyanate-conjugated (FITC) antihuman IgG. Plasma antibody reactivity was detected by flow cytometry. Results. Asymptomatic children had more prevalence of trophozoites in peripheral blood (66%) compared to symptomatic children (16%), p = 0.002. The mean percentage of infected RBCs reacting with autologous sera was 89.78 among symptomatic children compared to 79.62 among asymptomatic children (p = 0.09). Moreover, the mean fluorescence intensity (MFI) in the asymptomatic was significantly higher compared to symptomatic children (p value = 0.040). Conclusion. Variant surface antigens on Plasmodium falciparum infected RBCs from symptomatic malaria children tend to be better recognized by IgG antibodies. This may suggest a role of some IgG antibodies in severity of malaria.

Novel mucosal vaccines generated by genetic conjugation of heterologous proteins to pneumolysin (PLY) from Streptococcus pneumoniae.

PubMed

Douce, Gill; Ross, Kirsty; Cowan, Graeme; Ma, Jiangtao; Mitchell, Tim J

2010-04-19

Induction of immunity at mucosal surfaces is thought to be an essential feature in the protection of the host against the many pathogens that gain access through these surfaces. Here we describe how strong local and systemic immune responses can be generated when proteins are genetically conjugated to pneumolysin (PLY) from Streptococcus pneumoniae. Using green fluorescent protein (eGFP) and PsaA from S. pneumoniae, we have shown that genetic fusion (eGFPPLY and PsaAPLY) is essential to ensure high levels of antigen specific IgG and IgA in the serum and at mucosal surfaces. This form of vaccination is highly effective with antigen specific antibodies detected after a single dose of nanogram quantities of the conjugated proteins. In addition, generation of a non-toxic variant (eGFPDelta6PLY) indicated that while the toxic activity of PLY was not essential for adjuvanticity, it contributed to the magnitude of the response generated. Whilst vaccination with the PsaAPLY fusion proteins did not protect the animals from challenge, these studies confirm the utility of pneumolysin to act as a novel mucosal adjuvant to substantially increase the local and systemic humoral response to genetically fused protein antigens. Copyright 2010 Elsevier Ltd. All rights reserved.

Cell-mediated immunity to herpes simplex virus: recognition of type-specific and type-common surface antigens by cytotoxic T cell populations.

PubMed Central

Eberle, R; Russell, R G; Rouse, B T

1981-01-01

In this communication, we examine the specificity of anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL). Serological studies of the two related HSV serotypes (HSV-1 and HSV-2) have revealed both type-specific and cross-reactive antigenic determinants in the viral envelope and on the surface of infected cells. By analysis of cytotoxicity of CTL, generated in vitro by restimulation of splenocytes from mice primed with one or the other HSV serotype, the recognition of both type-specific and cross-reactive determinants on infected target cells by anti-HSV CTL was detectable. Thus, effector cells generated by priming and restimulating with the same virus recognized both type-specific and cross-reactive determinants on target cells infected with the homologous virus, but only cross-reactive determinants on target cells infected with the heterologous HSV serotype. CTL generated by restimulation with the heterologous virus were capable of recognizing only the cross-reactive determinants on either HSV-1- or HSV-2-infected target cells. These results indicate that two subpopulations of CTL exist in a population of anti-HSV immune spleen cells--those which recognize type-specific determinants and those specific for cross-reactive antigenic determinants present on the surface of HSV infected cells. The type-specific subset of anti-HSV CTL was shown to recognize the gC glycoprotein of HSV-1 infected target cells. In addition to the gC glycoprotein, at least one other type-specific surface antigen was also recognized by anti-HSV CTL in addition to the cross-reactive determinants recognized by anti-HSV CTL. PMID:6277790

Construction of novel electrochemical immunosensor for detection of prostate specific antigen using ferrocene-PAMAM dendrimers.

PubMed

Çevik, Emre; Bahar, Özlem; Şenel, Mehmet; Abasıyanık, M Fatih

2016-12-15

In this study, an immunosensor was designed to utilize for the detection of prostate specific antigen (PSA) based on three different generations (G1, G2 and G3) of ferrocene (Fc) cored polyamidiamine dendrimers (Fc-PAMAM) gold (Au) electrode. The self-assembled monolayer principle (SAM) was used to fabricate the sensitive, selective and disposable immunosensor electrodes. In electrode fabrication cysteamine (Cys) was the first agent covalently linked on the Au electrode surface. Immobilized redox center (ferrocene) cored PAMAM dendrimers served as a layer for the further binding of biological components. The monoclonal antibody of PSA (anti-PSA) was covalently immobilized on dendrimers which were attached onto the modified Au surface (Au/Cys/Fc-PAMAMs/anti-PSA). PSA levels were quantitatively analyzed by using electrochemical differential pulse voltammetry (DPV) whose lowest detection limit was calculated as 0.001ngmL(-1). The Au/Cys/FcPAMAM/anti-PSA immunosensor showed excellent performance for PSA at the pulse amplitude; 50mV and the scan rate; 10mV/s in a wide linear concentration range of 0.01ng-100ngmL(-1). Analytical performance and specificity assays were carried out using human serum and different proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2015-04-01

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

Sweat Allergy.

PubMed

Hiragun, Takaaki; Hide, Michihiro

2016-01-01

For many years, sweat has been recognized as an exacerbation factor in all age groups of atopic dermatitis (AD) and a trigger of cholinergic urticaria (CholU). Recently, we reported the improvement of AD symptoms by spray with tannic acid, which suppresses basophil histamine release by semipurified sweat antigens in vitro, and showering that removes antigens in sweat from the skin surface. We finally identified MGL_1304 secreted by Malassezia globosa as a major histamine-releasing antigen in human sweat. MGL_1304 is detected as a 17-kDa protein in sweat and exhibits almost the highest histamine-release ability from basophils of patients with AD and CholU among antigens derived from Malassezia species. Moreover, serum levels of anti-MGL_1304 IgE of patients with AD and CholU were significantly higher than those of normal controls. Desensitization therapy using autologous sweat or MGL_1304 purified from culture of M. globosa or its cognates might be beneficial for patients with intractable CholU due to sweat allergy. © 2016 S. Karger AG, Basel.

Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

PubMed

Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

2016-07-01

Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand. Copyright © 2016 Elsevier B.V. All rights reserved.

Case report: diffuse splenic metastasis of occult breast cancer with incompatible blood group antigenic determinants.

PubMed

Baranyay, Ferenc

2009-01-01

Cancer cells with immunogenic properties having altered protein glycosilation, modified blood group substances have been widely studied [Kannagi R, Miyake M, Zenita KM, Itai S, Hiraiwa N, Shigeta K, et al. Cancer-associated carbohydrate antigens: modified blood group substances and oncodevelopmental antigens on tumor cells. Gann Monogr Cancer Res 1988; 34: p. 15-28; Hakomori S. Antigen structure and genetic basis of histo-blood groups A, B and O their changes associated with human cancer. Biochem Biophys Acta 1999; 1473: p. 247-266; Brooks SA, Carter TM, Royle L, Harvey DJ, Fry SA, Kinch C, et al. Altered glycosilation of proteins in cancer: what is the potential for new anti-tumour strategies. Anticancer Agents Med Chem 2008; 8: p. 2-21]. In the study reported here, a 78-year-old female patient was admitted to the hospital with circulatory failure. At autopsy, the spleen (weight: 420 g) was extremely firm with a diffusely blackberry-colored cut surface. There were no signs of carcinomatous process at autopsy. By histology, the spleen showed diffuse metastatic carcinomatous infiltration. Using immunohistochemistry, an antibody to breast carcinoma antigen (BioGenex) labelled metastatic cells of the spleen and bone marrow. The patient was blood group O. Labelling for binding of lectins with and without blood group antigen specificity and monoclonal antibodies was carried out. The B blood group specific Banderiaea simplicifolia agglutinin I and an anti-B blood group monoclonal antibody labelled all the metastatic cells of spleen and bone marrow intensely. There was no detection of blood group A antigen by either binding of Dolichos biflorus agglutinin or anti-blood group A monoclonal antibodies. These observations raise the possibility that the detected incompatible B blood group antigen determinants on the metastatic cells were immunogenic. The surviving carcinoma cells may have found a place of refuge from immune surveillance in the spleen and in the bone marrow, where the complement-mediated tumor cell lysis immune rejection was not effective.

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2012 CFR

2012-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2013 CFR

2013-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center for... used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen. [40 FR...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2014 CFR

2014-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

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

NASA Astrophysics Data System (ADS)

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

2018-03-01

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

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

Serum antibodies to outer membrane proteins (OMPs) of Moraxella (Branhamella) catarrhalis in patients with bronchiectasis: identification of OMP B1 as an important antigen.

PubMed Central

Sethi, S; Hill, S L; Murphy, T F

1995-01-01

Moraxella (Branhamella) catarrhalis is a common cause of lower respiratory tract infections in adults and of otitis media in children. Little is known about the human immune response to this bacterium. In this study, immunoblot assays were performed to detect serum immunoglobulin G antibodies directed at purified outer membrane of M. catarrhalis. Twelve serum samples, two each from six patients with bronchiectasis who were persistently colonized with this organism, were tested with their homologous M. catarrhalis sputum isolates. In all the sera, the most prominent and consistent antibody response was to a minor 84-kDa outer membrane protein, OMP B1. Immunoblot adsorption assays show that these antibodies recognize surface exposed epitopes on OMP B1. Further analysis of human serum antibodies eluted from the surface of intact bacterial cells shows that these surface-exposed epitopes on OMP B1 are heterogeneous among strains of M. catarrhalis. OMP B1 is therefore an important OMP antigen on the surface of M. catarrhalis for the human immune response to infection by this bacterium. PMID:7890418

Application of immunoassay for detection of Helicobacter pylori antigens in the dental plaque.

PubMed

Leszczyńska, K; Namiot, D B; Namiot, Z; Leszczyńska, J K; Jakoniuk, P; Kemona, A

2009-01-01

The aim of this study was to determine the viability of the commercial test currently used for detection of H. pylori antigens in the stool for detection of H. pylori antigens in dental plaque. A total of 164 dyspeptic patients entered the study; 95 H. pylori infected (positive result of at least 4 of 5 diagnostic tests: Campylobacter-like organisms test (CLO test), histology, culture, stool antigens, serology) and 69 noninfected (negative results of 4 diagnostic tests: CLO test, histology, culture, stool antigens). Dental plaque was collected from natural teeth of the patients and incubated in microaerophilic conditions for 72 hours before immunoassay. Experimental findings included that optimal dental plaque weight to perform the examination was over 2 mg and that preliminary incubation increased significantly the number of positive results (p<0.002). It was also found that H. pylori antigens in the dental plaque were positive in 81.2% of infected and only 17.7% of non-infected subjects (p<0.001), while the reproducibility of results was 95%. The immunoassay for detection of H. pylori antigens in the stool may be used, after minor adaptations (specifically pre-incubation in microaerophilic conditions) for H. pylori antigen detection in dental plaque.

[Effect of Acetonitrile and n-hexane on the Immunoassay of Environmental Representative Pollutants].

PubMed

Lou, Xue-ning; Zhou, Li-ping; Song, Dan; Yang, Rong; Long, Feng

2016-01-15

Based on indirect competitive immunoassay mechanism, bisphenol A (BPA) was detected by the evanescent wave all-fiber immunosensor previously developed with the detection limit of 0.2 microg x L(-1) and the linear detection range of 0.3-33.4 microg x L(-1). The effects of two commonly used organic solvents, including acetonitrile and n-hexane, on the immunosensing assay of BPA were investigated. The influence mechanism of organic solvents on immunosensing assay was discussed. The experimental results showed that the effect of n-hexane on immunosensing assay was negligible even at a high concentration of up to 10%, whereas the effect of acetonitrile on the immunosensing assay was relatively great. BPA could be detected in solutions containing a low concentration of acetonitrile. However, the specific binding reaction between antibody and antigen in homogeneous solution was completely inhibited by high concentrations of acetonitrile, and the quantitative analysis of BPA was not achieved. This might result from the changes of antibody conformation or binding capability between antibody and antigen because acetonitrile replaced a part of the water molecules on the antibody surface.

A sensitive label-free electrochemical immunosensor for detection of cytokeratin 19 fragment antigen 21-1 based on 3D graphene with gold nanopaticle modified electrode.

PubMed

Zeng, Yan; Bao, Jing; Zhao, Yanan; Huo, Danqun; Chen, Mei; Yang, Mei; Fa, Huanbao; Hou, Changjun

2018-02-01

Previous studies have confirmed that cytokeratin 19 fragment antigen 21-1 (CYFRA 21-1) serves as a powerful biomarker in non-small cell lung cancer (NSCLC). Herein, we report for the first time a label-free electrochemical immunosensor for sensitive and selective detection of tumor marker CYFRA21-1. In this work, three-dimensional graphene @ gold nanoparticles (3D-G@Au) nanocomposite was modified on the glassy carbon electrode (GCE) surface to enhance the conductivity of immunosensor. The anti-CYFRA21-1 captured and fixed on the modified GCE through the cross-linking of chitosan (CS), glutaraldehyde (GA) and anti-CYFRA21-1. The differential pulse voltammetry (DPV) peak current change due to the specific interaction between anti-CYFRA21-1 and CYFRA21-1 on the modified electrode surface was utilized to detect CYFRA21-1. Under optimized conditions, the proposed electrochemical immunosensor was employed to detect CYFRA21-1 and exhibited a wide linear range of 0.25-800ngmL -1 and low detection limit of 100pgmL -1 (S/N = 3). Moreover, the recovery rates of serum samples were in the range from 95.2% to 108.7% and the developed immunosensor also shows a good correlation (less than 6.6%) with enzyme-linked immunosorbent assay (ELISA) in the detection of clinical serum samples. Therefore, it is expected that the proposed immunosensor based on a 3D-G@Au has great potential in clinical medical diagnosis of CYFRA21-1. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

PubMed

Crowdus, Carolyn A; Marsh, Antoinette E; Saville, Willliam J; Lindsay, David S; Dubey, J P; Granstrom, David E; Howe, Daniel K

2008-11-25

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen.

PubMed

Guo, Jinjin; Han, Xiaowei; Wang, Junchun; Zhao, Junqing; Guo, Zilin; Zhang, Yuzhong

2015-12-15

In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP-Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP-Au NRs nanocomposites and the labeling of secondary antibody (Ab2) were performed by one-pot assembly of HRP and Ab2 on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab1) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen-antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1-100 ng ml(-1), and the limit of detection was 30 pg ml(-1) (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Antigen vehiculization particles based on the Z protein of Junin virus.

PubMed

Borio, Cristina S; Bilen, Marcos F; Argüelles, Marcelo H; Goñi, Sandra E; Iserte, Javier A; Glikmann, Graciela; Lozano, Mario E

2012-11-02

Arenavirus matrix protein Z plays an important role in virus budding and is able to generate enveloped virus-like-particles (VLPs) in absence of any other viral proteins. In these VLPs, Z protein is associated to the plasma membrane inner surface by its myristoyl residue. Budding induction and vesicle formation properties can be exploited to generate enveloped VLPs platform. These structures can be designed to carry specific antigen in the inner side or on the surface of VLPs.Vaccines based on VLPs are a highly effective type of subunit vaccines that mimic the overall structure of virus particles in absence of viral nucleic acid, being noninfectious.In this work we assayed the capacity of Junin Z protein to produce VLPs carrying the green fluorescent protein (eGFP), as a model antigen. In this report the Junin Z protein ability to produce VLPs from 293T cells and its capacity to deliver a specific antigen (eGFP) fused to Z was evaluated. Confocal microscopy showed a particular membrane bending in cells expressing Z and a spot welded distribution in the cytoplasm. VLPs were detected by TEM (transmission electron microscopy) and were purified from cell supernatant. The proteinase protection assay demonstrated the VLPs integrity and the absence of degradation of the fused antigen, thus indicating its internal localization. Finally, immunization of mice with purified VLPs produced high titres of anti-eGFP antibodies compared to the controls. It was proved that VLPs can be generated from cells transfected with a fusion Junin virus Z-eGFP protein in absence of any other viral protein, and the capacity of Z protein to support fusions at the C-terminal, without impairing its budding activity, allowing vehiculization of specific antigens into VLPs.

Analysis of peroxidase-negative acute unclassifiable leukemias by monoclonal antibodies. 1. Acute myelogenous leukemia and acute myelomonocytic leukemia.

PubMed

Imamura, N; Tanaka, R; Kajihara, H; Kuramoto, A

1988-11-01

In this study, pretreatment peripheral and/or bone marrow blasts from 12 patients with acute unclassifiable leukemia (AUL) expressing the myeloid-related cell-surface antigen (CD 11) were isolated for further analysis. Despite a lack of myeloperoxidase (MPO) activity, 1 patient's blasts contained cytoplasmic Auer rods. The circulating blasts from another patient expressed MPO while maintaining the same surface phenotype during 20 months of clinical follow-up. In addition, the blasts from 3 cases demonstrated both myelomonocytic and monocyte-specific surface antigens, whereas the remaining 9 cases completely lacked any monocyte-specific antigen detectable by monoclonal antibodies, Mo2, My4 and Leu M3 (CD 14). The first case eventually was diagnosed as acute myelomonocytic leukemia and the second as acute myelogenous leukemia by means of immunophenotypic analysis using flow cytometry (FACS IV). In addition, the presence of MPO protein was identified in the cytoplasm of blast cells from 5 patients with AUL by means of a cytoplasmic immunofluorescence test using a monoclonal antibody (MA1). Our study indicates that non-T, non-B AUL expressing OKM1 (CD 11) antigens include acute leukemias which are unequivocally identifiable as being of either myeloid or myelomonocytic origin. However, further investigations, including immunophenotypic and cytoplasmic analysis, ultrastructural cytochemistry and gene analysis with molecular probes (tests applicable to normal myeloid cells), are necessary in order to determine the actual origin of blasts and to recognize the differentiation stages of the various types of leukemic cells from patients with undifferentiated forms of leukemia.

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.

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum.

PubMed

Lyon, J A; Haynes, J D; Diggs, C L; Chulay, J D; Pratt-Rossiter, J M

1986-03-15

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.

Gold nanostructure-integrated silica-on-silicon waveguide for the detection of antibiotics in milk and milk products

NASA Astrophysics Data System (ADS)

Ozhikandathil, Jayan; Badilescu, Simona; Packirisamy, Muthukumaran

2012-10-01

Antibiotics are extensively used in veterinary medicine for the treatment of infectious diseases. The use of antibiotics for the treatment of animals used for food production raised the concern of the public and a rapid screening method became necessary. A novel approach of detection of antibiotics in milk is reported in this work by using an immunoassay format and the Localized Surface Plasmon Resonance property of gold. An antibiotic from the penicillin family that is, ampicillin is used for testing. Gold nanostructures deposited on a glass substrate by a novel convective assembly method were heat-treated to form a nanoisland morphology. The Au nanostructures were functionalized and the corresponding antibody was absorbed from a solution. Solutions with known concentrations of antigen (antibiotics) were subsequently added and the spectral changes were monitored step by step. The Au LSPR band corresponding to the nano-island structure was found to be suitable for the detection of the antibody antigen interaction. The detection of the ampicillin was successfully demonstrated with the gold nano-islands deposited on glass substrate. This process was subsequently adapted for the integration of gold nanostructures on the silica-on-silicon waveguide for the purpose of detecting antibiotics.

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.

Specific binding of magnetic nanoparticle probes to platelets in whole blood detected by magnetorelaxometry

NASA Astrophysics Data System (ADS)

Eberbeck, Dietmar; Wiekhorst, Frank; Steinhoff, Uwe; Schwarz, Kay Oliver; Kummrow, Andreas; Kammel, Martin; Neukammer, Jörg; Trahms, Lutz

2009-05-01

The binding of monoclonal antibodies labelled with magnetic nanoparticles to CD61 surface proteins expressed by platelets in whole blood samples was measured by magnetorelaxometry. This technique is sensitive to immobilization of the magnetic labels upon binding. Control experiments with previous saturation of the epitopes on the platelet surfaces demonstrated the specificity of the binding. The kinetics of the antibody antigen reaction is accessible with a temporal resolution of 12 s. The minimal detectable platelet concentration is about 2000 μL -1 (sample volume 150 μL). The proportionality of the magnetic relaxation amplitude to the number of bound labels allows a quantification of the antibody binding capacity.

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

PubMed Central

Gentz, R; Certa, U; Takacs, B; Matile, H; Döbeli, H; Pink, R; Mackay, M; Bone, N; Scaife, J G

1988-01-01

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene. Images PMID:2452082

An integrated micro-manipulation and biosensing platform built in glass-based LTPS TFT technology

NASA Astrophysics Data System (ADS)

Chen, Lei-Guang; Wu, Dong-Yi; S-C Lu, Michael

2012-09-01

The glass-based low-temperature polycrystalline-silicon (LTPS) thin-film transistor (TFT) process, widely known for making liquid crystal displays, is utilized in this work to realize a fully integrated, microbead-based micro-manipulation and biosensing platform. The operation utilizes arrays of microelectrodes made of transparent iridium tin oxide (ITO) to move the immobilized polystyrene microbeads to the sensor surface by dielectrophoresis (DEP). Detection of remaining microbeads after a specific antigen/antibody reaction is accomplished by photo-detectors under the transparent electrodes. It was found that microbeads can be driven successfully by the 30 × 30 µm2 microelectrodes separated by 10 µm with no more than 6 Vp-p, which is compatible with the operating range of thin-film transistors. Microbeads immobilized with antimouse immunoglobulin (IgG) and prostate-specific antigen (PSA) antibody were successfully detected after specific binding, illustrating the potential of LTPS TFT microarrays for more versatile biosensing applications.

Point-of-need detection using surface-based biosensors with an examination of protein immobilization and development of magnetic labels

NASA Astrophysics Data System (ADS)

Lim, China Ye-Ling

Over the past decade, our research group has worked on developing surface-based immunoassays to detect disease biomarkers. Our immunoassay platforms use a gold surface coated with an N-hydroxysuccinimide (NHS)-based monolayer and a layer of antibodies to capture a target antigen. Readout is achieved by surface-enhanced Raman scattering (SERS) or giant magnetoresistance (GMR) after labeling of the captured antigen with Raman dye-modified gold nanoparticles or magnetic particles, which are also coated with antibodies. Both of these platforms enable the low-level detection of numerous biomarkers and have the potential for translation into a point-of-need (PON) (i.e., rapid, easy to use, and field deployable) test. As part of an effort to develop a PON test, this dissertation includes investigations of: (1) SERS-based detection of botulinum neurotoxins (BoNTs), (2) protein immobilization procedures, and (3) magnetic microcapsules (MMCs) for use with GMR detection. First, a SERS-based immunoassay for bioterrorism agents, botulinum neurotoxins A (BoNT-A) and B (BoNT-B) with picomolar (or lower) detection limits for BoNT-A and BoNT-B in buffer and serum is described. These results not only demonstrate sufficient detection of these markers at levels important to homeland security and human health monitoring, but also the potential to translate this methodology to a PON test. Next, the reactivity of NHS ester-terminated monolayers, a common approach in protein immobilization chemistry, is investigated to assess the competition of the purported amidization reaction to that of hydrolysis. Results of kinetic studies on hydrolysis and aminolysis under relevant assay conditions show the rate of hydrolysis is 300x faster than that of aminolysis. These results indicate that it is highly unlikely that proteins are covalently linked to the surface and suggest that the protein layer is adsorbed via hydrophobic, hydrogen bonding, and electrostatic interactions. The last section examines the development of an MMC-based label. With marked improvement in both stability and magnetization over commercially-available magnetic nanoparticles, these MMCs show potential for the eventual enhanced function as a label in a GMR-based immunoassay. With these results, this dissertation aims to set the stage for the rational development of assays that will facilitate a paradigm shift towards PON tests.

Ultrasensitive detection in optically dense physiological media: applications to fast reliable biological assays

NASA Astrophysics Data System (ADS)

Matveeva, Evgenia G.; Gryczynski, Ignacy; Berndt, Klaus W.; Lakowicz, Joseph R.; Goldys, Ewa; Gryczynski, Zygmunt

2006-02-01

We present a novel approach for performing fluorescence immunoassay in serum and whole blood using fluorescently labeled anti-rabbit IgG. This approach, which is based on Surface Plasmon-Coupled Emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bio-affinity surface. Effective coupling range for SPCE is only couple of hundred nanometers from the metallic surface. Excited fluorophores outside the coupling layer do not contribute to SPCE, and their free-space emission is not transmitted through the opaque metallic film into the glass substrate. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal is discussed. The kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately 2- and 3-fold, respectively (compared to buffer), resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Both glass and plastic slides can be used for SPCE-based assays. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood, without any need for washing steps.

Presence of IgT-C and I-A subregion-encoded determinants on distinct chains of monoclonal antigen-specific augmenting factor derived from a T cell hybridoma

PubMed Central

1983-01-01

Monoclonal antibodies specific for mouse T cell alloantigens, Tindd and Tsud, linked to the Igh-1 locus on chromosome 12, were used to directly define the antigen-binding molecule produced by a cloned hybridoma. The T cell hybridoma, FL10, was established from antigen-binding T cells of A/J mice. FL10 produces an antigen-specific augmenting T cell factor (TaF) that bears a unique I region-controlled determinant (I-A) and has antigen-binding capacity. The Tindd, but not the Tsud, determinant was detected on the surface of FL10. The presence of both Tindd and I-A subregion-controlled determinants on FL10-derived TaF was directly demonstrated by the adsorption of TaF with immunoadsorbents prepared with monoclonal antibodies. The Igh-1-linked T cell alloantigen, Tsud, was not found on TaF. Further experiments indicated that Tindd is present on the antigen-binding polypeptide chain and not on the second chain bearing the I-A determinant. Despite the presence of the Tindd determinant on hybridoma-derived TaF, augmentation induced by TaF was restricted by the H-2 type of the responding mice and not by the Igh-1 allotype. PMID:6189953

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

NASA Technical Reports Server (NTRS)

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

2014-01-01

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

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

DOEpatents

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

2014-04-01

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

First field trial of an immunoradiometric assay for the detection of malaria sporozoites in mosquitoes

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

Collins, F.H.; Zavala, F.; Graves, P.M.

1984-07-01

An immunoradiometric assay (IRMA) using a monoclonal antibody to the major surface protein of Plasmodium falciparum sporozoites was used to assess the P. falciparum sporozoite rate in a West African population of Anopheles gambiae (s.1.). Unlike current dissection techniques, the IRMA could detect sporozoite antigen in dried as well as fresh mosquitoes. In a controlled comparison, the sensitivity of the IRMA was comparable that of the dissection technique. Additionally, the IRMA was species specific and quantitative. Sensitivity of the assay was sufficient to detect sporozoite infections resulting from the development of a single oocyst.

Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

PubMed Central

Cram, Erik D.; Simmons, Ryan S.; Palmer, Amy L.; Hildebrand, William H.; Rockey, Daniel D.

2015-01-01

The direct major histocompatibility complex (MHC) class I antigen presentation pathway ensures intracellular peptides are displayed at the cellular surface for recognition of infected or transformed cells by CD8+ cytotoxic T lymphocytes. Chlamydia spp. are obligate intracellular bacteria and, as such, should be targeted by CD8+ T cells. It is likely that Chlamydia spp. have evolved mechanisms to avoid the CD8+ killer T cell responses by interfering with MHC class I antigen presentation. Using a model system of self-peptide presentation which allows for posttranslational control of the model protein's stability, we tested the ability of various Chlamydia species to alter direct MHC class I antigen presentation. Infection of the JY lymphoblastoid cell line limited the accumulation of a model host protein and increased presentation of the model-protein-derived peptides. Enhanced self-peptide presentation was detected only when presentation was restricted to defective ribosomal products, or DRiPs, and total MHC class I levels remained unaltered. Skewed antigen presentation was dependent on a bacterial synthesized component, as evidenced by reversal of the observed phenotype upon preventing bacterial transcription, translation, and the inhibition of bacterial lipooligosaccharide synthesis. These data suggest that Chlamydia spp. have evolved to alter the host antigen presentation machinery to favor presentation of defective and rapidly degraded forms of self-antigen, possibly as a mechanism to diminish the presentation of peptides derived from bacterial proteins. PMID:26597986

Immunohistochemical techniques and their applications in the histopathology of the respiratory system.

PubMed Central

Linnoila, I; Petrusz, P

1984-01-01

Subsequent to the first report in the 1940s on incubation of tissue sections with fluorescein-conjugated antibodies for localization of antigens, a great number of modifications were introduced to improve the validity of immunohistochemistry which has become a growingly popular tool. The use of immunoenzymatic techniques eliminates the need for expensive fluorescence microscopy equipment, the lack of permanency of preparations and the lack of electron density required in ultrastructural localization of antigens. Regardless of the technique, it is also important to choose a correct fixation which allows the proper preservation of antigens and morphology and the penetration of antibodies through the entire thickness of the preparation. A variety of immunohistochemical techniques have been applied to study several components of the lung, such as collagen, surface active material, lung specific antigens, and enzymes and the detection of tumor markers, immunoglobulins and infectious agents in the respiratory system which is reviewed. The large surface area and the multiplicity of cell types provided by the respiratory tract epithelium of humans for exposure to microbial as well as toxic substances in the environment make this organ system very vulnerable but a good early indicator of adverse health effects. Immunohistochemistry provides valuable information complementary to the immunochemical and biochemical characterization of this barrier. Images FIGURE 2. FIGURE 3. FIGURE 3. FIGURE 4. FIGURE 4. FIGURE 5. PMID:6090113

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.

Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

PubMed

Peretti, Leandro E; Gonzalez, Verónica D G; Marcipar, Iván S; Gugliotta, Luis M

2014-08-01

The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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.

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 Label-Free Detection of Biomolecules Using Micromechanical Biosensors

NASA Astrophysics Data System (ADS)

Meisam, Omidi; A. Malakoutian, M.; Mohammadmehdi, Choolaei; Oroojalian, F.; Haghiralsadat, F.; Yazdian, F.

2013-06-01

A Microcantilevers resonator is used to detect a protein biomarker called prostate specific antigen (PSA), which is associated with prostate cancer. Different concentrations of PSA in a buffer solution are detected as a function of deflection of the beams. For this purpose, we use a surface micromachined, antibody-coated polycrystalline silicon micromechanical cantilever beam. Cantilevers have mass sensitivities of the order of 10-17 g/Hz, which result from their small mass. This matter allows them to detect an immobilized antibody monolayer corresponding to a mass of about 70 fg. With these devices, concentrations as low as 150 fg/mL, or 4.5 fM, could be detected from the realistic samples.

Side-polished fiber immunosensor based on surface plasmon resonance for detection of Legionella pneumophila

NASA Astrophysics Data System (ADS)

Tsao, Yu-Chia; Yang, Yi-Wen; Tsai, Woo-Hu; Yan, Tsong-Rong

2008-02-01

Side-polished fiber immunosensor based on surface plasmon resonance (SPR) onto self-assembled protein A layer was proposed for the detection of Legionella pneumophila. A self-assembled protein A layer on gold (Au) surface was fabricated by adsorbing a mixture of 11-mercaptoundecanoic acid (MUA) and activated by N-Ethyl-N'-(3-dimethylaminopropyl) carbodiimide/ N-Hydroxysuccinimide (EDC/NHS). The formation of self-assembled protein A and gold layer on side-polished surface and the binding of antibody and antigen in series were confirmed by SPR response on spectrum. The binding protein A layer can improve the sensitivity, which indirectly supports the configurations that antibody layer is immobilized on the binding protein A layer with a well-ordered orientation. The surface morphology analyses of self-assembled protein A layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein A were demonstrated by SPR dip shifts on optical spectrum analyzer. The SPR fiber immunosensor for detection of L. pneumophila was developed and the detection limit was 10 CFU/ml with the SPR dip shift in wavelength from 1070 to 1105nm. The current fabrication technique of a SPR immunosensor using optical fiber for the detection of Legionella pneumophila could be applied to construct other biosensor.

Development of Raman Spectroscopy as a Clinical Diagnostic Tool

NASA Astrophysics Data System (ADS)

Borel, Santa

Raman spectroscopy is the collection of inelastically scattered light in which the spectra contain biochemical information of the probed cells or tissue. This work presents both targeted and untargeted ways that the technique can be exploited in biological samples. First, surface enhanced Raman scattering (SERS) gold nanoparticles conjugated to targeting antibodies were shown to be successful for multiplexed detection of overexpressed surface antigens in lung cancer cell lines. Further work will need to optimize the conjugation technique to preserve the strong binding affinity of the antibodies. Second, untargeted Raman microspectroscopy combined with multivariate statistical analysis was able to successfully differentiate mouse ovarian surface epithelial (MOSE) cells and spontaneously transformed ovarian surface epithelial (STOSE) cells with high accuracy. The differences between the two groups were associated with increased nucleic acid content in the STOSE cells. This shows potential for single cell detection of ovarian cancer.

Immunological properties of Micrococcus lysodeikticus membranes.

PubMed

Fukui, Y; Nachbar, M S; Salton, M R

1971-01-01

Membranes of Micrococcus lysodeikticus possess antigens which are distinct from other cellular components such as cytoplasm, ribosomes, and cell walls. Only a few (two to three) components are found when dissociated membranes are examined by immunodiffusion and immunoelectrophoresis techniques. Membranes treated with 0.3% sodium dodecyl sulfate, 0.3% Triton X-100, trypsin, phospholipase A or C, or by sonic oscillation at pH 9.0, all showed the same pattern (three major bands) when examined against membrane antisera by immunoelectrophoresis. Immunological analysis of fractions isolated by sucrose gradient centrifugation or by polyacrylamide gel electrophoresis suggests that individual components cross-react. Antibodies to adenosine triphosphatase (EC 3.6.1.3) and fast-moving component are not removed by absorption with protoplasts. Removal of antibody to one of the membrane antigens by protoplast absorption indicated a surface location. Glutaraldehyde fixation of protoplasts resulted in the loss of membrane antigens detectable by immunodiffusion.

Comparison between immunofluorescence and immunomagnetic techniques of cytometry

NASA Astrophysics Data System (ADS)

Tchikov, V.; Schütze, S.; Krönke, M.

1999-04-01

Magnetophoresis and fluorescence activated cell sorting were used for evaluation of immunochemical properties of magnetic particles and fluorescent probes. The HLA-Bw6 antigen on surfaces of REH cells was detected with a primary monoclonal antibody and a secondary antibody coupled with fluorescent molecules or magnetic particles. Magnetophoresis can find applications in biology and medicine for measuring percentages of cell subpopulations.

Aggregated silver nanoparticles based surface-enhanced Raman scattering enzyme-linked immunosorbent assay for ultrasensitive detection of protein biomarkers and small molecules.

PubMed

Liang, Jiajie; Liu, Hongwu; Huang, Caihong; Yao, Cuize; Fu, Qiangqiang; Li, Xiuqing; Cao, Donglin; Luo, Zhi; Tang, Yong

2015-06-02

Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac.

A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

PubMed

Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

2016-11-01

Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

Evaluation of a Commercial Latex Agglutination Test Kit for Cryptococcal Antigen

PubMed Central

Kaufman, Leo; Cowart, Glenda; Blumer, Sharon; Stine, Amy; Wood, Ross

1974-01-01

Two dozen Crypto-LA kits for detecting Cryptococcus neoformans capsular polysaccharide antigens were evaluated. Ten kits proved reliable for detecting and titering antigen in clinical materials. Fourteen kits were found to be inadequate. PMID:4596394

Preventing vaccinia virus class-I epitopes presentation by HSV-ICP47 enhances the immunogenicity of a TAP-independent cancer vaccine epitope.

PubMed

Raafat, Nermin; Sadowski-Cron, Charlotte; Mengus, Chantal; Heberer, Michael; Spagnoli, Giulio C; Zajac, Paul

2012-09-01

Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class-I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class-I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV-US12) or together with endoplasmic reticulum (ER)-targeted Melan-A/MART-1(27-35) model tumor epitope (rVV-MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV-US12, display a decreased ability to present TAP dependent MHC class-I restricted viral antigens to CD8+ T-cells. While HLA class-I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class-II are unaffected. Characterization of rVV-MUS12 infected cells demonstrates that over-expression of a TAP-independent peptide, partially compensates for ICP47 induced surface MHC class-I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus-specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV-MUS12, as compared to those stimulated by rVV-MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector-specific responsiveness. Copyright © 2011 UICC.

Tomato bushy stunt virus (TBSV), a versatile platform for polyvalent display of antigenic epitopes and vaccine design.

PubMed

Kumar, Shantanu; Ochoa, Wendy; Singh, Pratik; Hsu, Catherine; Schneemann, Anette; Manchester, Marianne; Olson, Mark; Reddy, Vijay

2009-05-25

Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NDelta52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and image reconstruction of the chimeric VLPs at 22 A resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T=1, T=3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.

Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function.

PubMed

Geng, Jie; Altman, John D; Krishnakumar, Sujatha; Raghavan, Malini

2018-05-09

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8 + T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8 + T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8 + T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8 + T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8 + T cell activation, mediated by the binding of empty HLA-I to CD8. © 2018, Geng et al.

Evaluation of surface antigen TF1.17 in feline Tritrichomonas foetus isolates.

PubMed

Gould, E N; Corbeil, L B; Kania, S A; Tolbert, M K

2017-09-15

Tritrichomonas foetus (T. foetus) is a flagellated protozoa that infects the distal ileum and proximal colon of domestic cats, as well as the urogenital tract of cattle. Feline trichomonosis is recognized as a prevalent cause of chronic diarrhea in cats worldwide. The suspected route of transmission is fecal-oral, with cats in densely crowded environments at highest risk for infection. Thus, the recommended strategy for minimizing spread of infection is to identify and isolate T. foetus-positive cats from the general population. Rapid identification of infected cats can be challenging due to the inability to accurately and quickly detect the organism in samples at point of care facilities. Thus, identification of targets for use in development of a novel diagnostic test, as well as a vaccine or therapy for T. foetus infection is a significant area of research. Despite a difference in organ tropism between T. foetus genotypes, evidence exists for conserved virulence factors between feline and bovine T. foetus. The bovine T. foetus surface antigen, TF1.17, is an adhesin that is conserved across isolates. Vaccination with the purified antigen results in amelioration of cytopathogenicity and more rapid clearance of infection in cattle. We previously showed that three feline isolates of T. foetus were positive for TF1.17 antigen so we further hypothesized that TF1.17 is conserved across feline T. foetus isolates and that this antigen would represent an attractive target for development of a novel diagnostic test or therapy for feline trichomonosis. In these studies, we used monoclonal antibodies previously generated against 1.15 and 1.17 epitopes of the bovine T. foetus TF1.17 antigen, to evaluate for the presence and role of TF1.17 in the cytopathogenicity of feline T. foetus. A previously validated in vitro co-culture approach was used to model feline T. foetus infection. Immunoblotting, immunofluorescence assays, and flow cytometric analysis confirmed the presence and surface localization of antigen TF1.17 across all feline T. foetus isolates tested. Antigen TF1.17 was notably absent in the presumably nonpathogenic intestinal trichomonad, Pentatrichomonas hominis, a parasite that can be confused microscopically with T. foetus. Similar to bovine trichomoniasis, TF1.17 was found to promote T. foetus adhesion to the intestinal epithelium. These results support further characterization and development of the TF1.17 antigen as a possible target for the diagnosis and prevention of feline T. foetus infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrochemical Quartz Crystal Nanobalance (EQCN) Based Biosensor for Sensitive Detection of Antibiotic Residues in Milk.

PubMed

Bhand, Sunil; Mishra, Geetesh K

2017-01-01

An electrochemical quartz crystal nanobalance (EQCN), which provides real-time analysis of dynamic surface events, is a valuable tool for analyzing biomolecular interactions. EQCN biosensors are based on mass-sensitive measurements that can detect small mass changes caused by chemical binding to small piezoelectric crystals. Among the various biosensors, the piezoelectric biosensor is considered one of the most sensitive analytical techniques, capable of detecting antigens at picogram levels. EQCN is an effective monitoring technique for regulation of the antibiotics below the maximum residual limit (MRL). The analysis of antibiotic residues requires high sensitivity, rapidity, reliability and cost effectiveness. For analytical purposes the general approach is to take advantage of the piezoelectric effect by immobilizing a biosensing layer on top of the piezoelectric crystal. The sensing layer usually comprises a biological material such as an antibody, enzymes, or aptamers having high specificity and selectivity for the target molecule to be detected. The biosensing layer is usually functionalized using surface chemistry modifications. When these bio-functionalized quartz crystals are exposed to a particular substance of interest (e.g., a substrate, inhibitor, antigen or protein), binding interaction occurs. This causes a frequency or mass change that can be used to determine the amount of material interacted or bound. EQCN biosensors can easily be automated by using a flow injection analysis (FIA) setup coupled through automated pumps and injection valves. Such FIA-EQCN biosensors have great potential for the detection of different analytes such as antibiotic residues in various matrices such as water, waste water, and milk.

Target-triggered signal turn-on detection of prostate specific antigen based on metal-enhanced fluorescence of Ag@SiO2@SiO2-RuBpy composite nanoparticles

NASA Astrophysics Data System (ADS)

Deng, Yun-Liang; Xu, Dang-Dang; Pang, Dai-Wen; Tang, Hong-Wu

2017-02-01

A three-layer core-shell nanostructure consisting of a silver core, a silica spacer, and a fluorescent dye RuBpy-doped outer silica layer was fabricated, and the optimal metal-enhanced fluorescence (MEF) distance was explored through adjusting the thickness of the silica spacer. The results show that the optimal distance is ˜10.4 nm with the maximum fluorescence enhancement factor 2.12. Then a new target-triggered MEF ‘turn-on’ strategy based on the optimized composite nanoparticles was successfully constructed for quantitative detection of prostate specific antigen (PSA), by using RuBpy as the energy donor and BHQ-2 as the acceptor. The hybridization of the complementary DNA of PSA-aptamer immobilized on the surface of the MEF nanoparticles with PSA-aptamer modified with BHQ-2, brought BHQ-2 in close proximity to RuBpy-doped silica shell and resulted in the decrease of fluorescence. In the presence of target PSA molecules, the BHQ-PSA aptamer is dissociated from the surface of the nanoparticles with the fluorescence switched on. Therefore, the assay of PSA was achieved by measuring the varying fluorescence intensity. The results show that PSA can be detected in the range of 1-100 ng ml-1 with a detection limit of 0.20 ng ml-1 (6.1 pM), which is 6.7-fold increase of that using hollow RuBpy-doped silica nanoparticles. Moreover, satisfactory results were obtained when PSA was detected in 1% serum.

Immunoelectrophoretic study of cell surface antigens from different Streptococcus mutans serotypes and Streptococcus sanguis.

PubMed

Ogier, J A; Klein, J P; Niddam, R; Frank, R M

1985-06-01

Antigens prepared from culture supernatants or whole cells of several cariogenic strains were examined by immunoelectrophoresis for their crossed antigenicity, with reference to Streptococcus mutans OMZ175, serotype f. Crossed immunoelectrophoresis revealed a crossreactivity between soluble extracellular and wall associated antigens of six strains of Streptococcus mutans and one strain of Streptococcus sanguis. Protease destroyed the immunoreactivity of crossreactive antigens. One of them was shown to be localized on the bacterial surface.

SYNTHESIS, INTRACELLULAR DISTRIBUTION, AND SECRETION OF IMMUNOGLOBULIN AND H-2 ANTIGEN IN MURINE SPLENOCYTES

PubMed Central

Wernet, Dorothee; Vitetta, Ellen S.; Uhr, Jonathan W.; Boyse, Edward A.

1973-01-01

A/J spleen cells were labeled with [3H]leucine and at intervals thereafter were homogenized and separated into microsomes and cell sap. Ig and H-2 antigens were assayed in the cell fractions and cell supernatants using immunoprecipitation. In addition, cells labeled by enzymatic radioiodination were incubated to determine the rates of release of Ig and H-2 antigens from the surface. The results indicate that the majority of Ig and H-2 antigens remain membrane bound throughout their intracellular life. In contrast to Ig, H-2 antigens are neither secreted nor shed from the cell surface. It is suggested that Ig is a peripheral protein of the cell membrane, whereas H-2 antigens are integral ones. The release of Ig on a fragment of plasma membrane could occur at fixed cell surface areas that contain no H-2 antigens or from which they have migrated before release. PMID:4200648

HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells.

PubMed

Varma, Sandeep R; Sundaram, R; Gopumadhavan, S; Vidyashankar, Satyakumar; Patki, Pralhad S

2013-01-01

HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000  μ g/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380  μ g/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections.

Correlation of the cell surface antigens with stage and grade in cancer of the bladder.

PubMed

Emmott, R C; Javadpour, N; Bergman, S M; Soares, T

1979-01-01

We examined 76 bladder tumors of various stages and grades for the presence of the ABO (H) cell surface antigen, using the specific red cell adherence technique. Of the grade I lesions studied 70 per cent were positive for the cell surface antigen and none of the 26 grade III tumors retained the antigens. When correlated with clinical stage the tumors showed no antigens for those of stages B1 to D, while 12 of 16 stage A lesions were positive for the antigen. When stage A lesions were studied and the findings were correlated with recurrence and metastasis/invasion rates the cell surface antigen was present on the initial tumor in only 1 lesion that recurred at an invasive stage. The findings of this study show that the specific red cell adherence technique may be valuable for predicting malignant potential in low grade, low stage cancer of the bladder. If supported by further investigation this technique may offer the capability of selecting low grade, low stage bladder tumors that are destined to invade or metastasize while they are at curable stages.

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.

Roles for SH2 and SH3 domains in Lyn kinase association with activated FcepsilonRI in RBL mast cells revealed by patterned surface analysis.

PubMed

Hammond, Stephanie; Wagenknecht-Wiesner, Alice; Veatch, Sarah L; Holowka, David; Baird, Barbara

2009-10-01

In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcepsilonRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcepsilonRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells.

Roles of oral bacteria in cardiovascular diseases--from molecular mechanisms to clinical cases: Cell-surface structures of novel serotype k Streptococcus mutans strains and their correlation to virulence.

PubMed

Nakano, Kazuhiko; Nomura, Ryota; Matsumoto, Michiyo; Ooshima, Takashi

2010-01-01

Streptococcus mutans is generally known as a pathogen of dental caries, and it is also considered to cause bacteremia and infective endocarditis (IE). S. mutans was previously classified into 3 serotypes, c, e, and f, due to the different chemical compositions of the serotype-specific polysaccharides, which are composed of a rhamnose backbone and glucose side chains. We recently designated non-c/e/f serotype S. mutans strains as novel serotype k, which is characterized by a drastic reduction in the amount of the glucose side chain. A common biological feature of novel serotype-k strains is a lower level of cariogenicity due to alterations of several major cell surface protein antigens. As for virulence in blood, these strains survive in blood for a longer duration due to lower antigenicity, while the detection rate of all strains carrying the gene encoding collagen-binding adhesin has been shown to be high. Furthermore, molecular biological analyses of infected heart valve specimens obtained from IE patients revealed a high detection rate of serotype-k S. mutans. Together, these findings suggest that serotype-k S. mutans strains show low cariogenicity but high virulence in blood as compared to the other serotypes, due to alterations of several cell surface structures.

An electrochemical immunosensing method for detecting melanoma cells.

PubMed

Seenivasan, Rajesh; Maddodi, Nityanand; Setaluri, Vijaysaradhi; Gunasekaran, Sundaram

2015-06-15

An electrochemical immunosensing method was developed to detect melanoma cells based on the affinity between cell surface melanocortin 1 receptor (MC1R) antigen and anti-MC1R antibody (MC1R-Ab). The MC1R-Abs were immobilized in amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite modified on working electrode surface of screen-printed electrode (SPE). Cyclic voltammetry was employed, with the help of redox mediator ([Fe(CN)6](3-)), to measure the change in anodic oxidation peak current arising due to the specific interaction between MC1R antigens and MC1R-Abs when the target melanoma cells are present in the sample. Various factors affecting the sensor performance, such as the amount of MC1R-Abs loaded, incubation time with the target melanoma cells, the presence of interfering non-melanoma cells, were tested and optimized over different expected melanoma cell loads in the range of 50-7500 cells/2.5 mL. The immunosensor is highly sensitive (20 cells/mL), specific, and reproducible, and the antibody-loaded electrode in ready-to-use stage is stable over two weeks. Thus, in conjunction with a microfluidic lab-on-a-chip device our electrochemical immunosensing approach may be suitable for highly sensitive, selective, and rapid detection of circulating tumor cells (CTCs) in blood samples. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Optofluidic ring resonator sensor for sensitive label-free detection of breast cancer antigen CA15-3 in human serum

NASA Astrophysics Data System (ADS)

Zhu, Hongying; Dale, Paul S.; Fan, Xudong

2009-05-01

Breast cancer is the most frequently diagnosed malignancy in women worldwide. Because of its great impact on society, a lot of research funding has been used to develop novel detection tools for aiding breast cancer diagnosis and prognosis. In this work, we demonstrated a simple, fast, and sensitive detection of circulating breast cancer biomarker CA15-3 with opto-fluidic ring resonator (OFRR) sensors. The OFRR sensor employs a thin-walled capillary with wall thickness less than 4 μm. The circular cross section of the capillary forms the optical ring resonator, in which the light circulates in the form of whispering gallery modes (WGMs). The capillary wall is thin enough that the evanescent field of the WGM extends into the capillary core and responds to refractive index changes in the capillary core or close to its interior surface. The WGM spectral position will change when the biomolecules bind to the surface, yielding quantitative and kinetic information about the biomolecule interaction. Here, the direct immunoassay method was employed for the detection of CA15-3 antigen without any signal amplification steps. The sensor performance in both PBS buffer and human serum were investigated, respectively. The experimental detection limit was 5 units/mL in PBS buffer and 30 units/mL for CA15-3 spiked in serum, both of which satisfied clinical diagnosis requirements. The potential use of the OFRR as the point-of-care device for breast cancer detection was tested by measuring the CA15-3 level in blood samples collected from stage IV breast cancer patients and the results were compared with standard clinical test.

Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens

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

Tomavo, S.; Schwarz, R.T.; Dubremetz, J.F.

1989-10-01

The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with {sup 3}H-fatty acids, ({sup 3}H)ethanolamine, and ({sup 3}H)carbohydrates. Treatment of {sup 3}H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment.

Microspot-based ELISA in microfluidics: chemiluminescence and colorimetry detection using integrated thin-film hydrogenated amorphous silicon photodiodes.

PubMed

Novo, Pedro; Prazeres, Duarte Miguel França; Chu, Virginia; Conde, João Pedro

2011-12-07

Microfluidic technology has the potential to decrease the time of analysis and the quantity of sample and reactants required in immunoassays, together with the potential of achieving high sensitivity, multiplexing, and portability. A lab-on-a-chip system was developed and optimized using optical and fluorescence microscopy. Primary antibodies are adsorbed onto the walls of a PDMS-based microchannel via microspotting. This probe antibody is then recognised using secondary FITC or HRP labelled antibodies responsible for providing fluorescence or chemiluminescent and colorimetric signals, respectively. The system incorporated a micron-sized thin-film hydrogenated amorphous silicon photodiode microfabricated on a glass substrate. The primary antibody spots in the PDMS-based microfluidic were precisely aligned with the photodiodes for the direct detection of the antibody-antigen molecular recognition reactions using chemiluminescence and colorimetry. The immunoassay takes ~30 min from assay to the integrated detection. The conditions for probe antibody microspotting and for the flow-through ELISA analysis in the microfluidic format with integrated detection were defined using antibody solutions with concentrations in the nM-μM range. Sequential colorimetric or chemiluminescence detection of specific antibody-antigen molecular recognition was quantitatively detected using the photodiode. Primary antibody surface densities down to 0.182 pmol cm(-2) were detected. Multiplex detection using different microspotted primary antibodies was demonstrated.

Successful treatment of HCV/HBV/HDV-coinfection with pegylated interferon and ribavirin

PubMed Central

Hartl, Janine; Ott, Claudia; Kirchner, Gabriele; Salzberger, Bernd; Wiest, Reiner

2012-01-01

Dual and triple infections with hepatitis virus C (HCV), B (HBV) and D (HDV) frequently lead to severe liver damage. Hereby we describe a 38-year-old Caucasian male coinfected with HCV (genotype 3a), HBV [positive hepatitis B surface antigen (HbsAg) and antibody to hepatitis B core antigen; negative hepatitis B e antigen (HbeAg) and antibody to hepatitis B e antigen (anti-HBe)] and HDV. Laboratory diagnostics revealed increased liver enzymes and histological examination of the liver showed signs of fibrosis with moderate inflammation. On therapy with pegIFN-α2b and ribavirin HCV-RNA was undetectable at week 8. After week 24 the antiviral therapy was stopped because of a HBs-seroconversion, the loss of HbeAg and the detection of anti-HBe. Furthermore the HCV-RNA was negative. Six months after successful treatment of the triple-infection, HCV- and HDV-RNA and HbsAg remained negative and the liver enzymes had been completely normalized. In conclusion, pegylated-interferon plus ribavirin may be an effective therapy for HCV, HBV and HDV-coinfected patients. PMID:24765463

Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

PubMed Central

2012-01-01

Background The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR. Results A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose–response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was <6 % for repeatability and <2 % for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed. Conclusions The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to encapsulate multiple reporters per liposome also helps overcome the effect of polymerase inhibitors present in biological specimens. Finally, the biotin-labeled liposome detection reagent can be coupled through a NeutrAvidin bridge to a multitude of biotin-labeled probes, making ILPCR a highly generic assay system. PMID:22726242

EMMPRIN (CD147) is induced by C/EBPβ and is differentially expressed in ALK+ and ALK- anaplastic large-cell lymphoma.

PubMed

Schmidt, Janine; Bonzheim, Irina; Steinhilber, Julia; Montes-Mojarro, Ivonne A; Ortiz-Hidalgo, Carlos; Klapper, Wolfram; Fend, Falko; Quintanilla-Martínez, Leticia

2017-09-01

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPβ, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPβ for the unusual phenotype of ALK+ ALCL cells, C/EBPβ was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK- cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPβ knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK- ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPβ knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK- ALCL, suggesting an additional role of C/EBPβ in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPβ plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as T-cell activation and proliferation suggest its involvement in the pathogenesis of ALCL.

UV-light-assisted functionalization for sensing of light molecules

NASA Astrophysics Data System (ADS)

Funari, Riccardo; Della Ventura, Bartolomeo; Ambrosio, Antonio; Lettieri, Stefano; Maddalena, Pasqualino; Altucci, Carlo; Velotta, Raffaele

2013-05-01

An antibody immobilization technique based on the formation of thiol groups after UV irradiation of the proteins is shown to be able to orient upside antibodies on a gold electrode of a Quartz Crystal Microbalance (QCM). This greatly affects the aptitude of antibodies in recognizing small antigens thereby increasing the sensitivity of the QCM. The capability of such a procedure to orient antibodies is confirmed by the Atomic Force Microscopy (AFM) of the surface that shows different statistical distributions for the height of the detected peaks, whether the irradiation is performed or not. In particular, the distributions are Gaussian with a standard deviation smaller when irradiated antibodies are used compared to that obtained with no treated antibodies. The standard deviation reduction is explained in terms of higher order induced on the host surface resulting from the trend of irradiated antibodies to be anchored upside on the surface with their antigen binding sites free to catch recognized analytes. As a result the sensitivity of the realized biosensor is increased by even more than one order of magnitude.

Production of a Recombinant Antibody Specific for i Blood Group Antigen, a Mesenchymal Stem Cell Marker

PubMed Central

Suila, Heli; Tiitinen, Sari; Natunen, Suvi; Laukkanen, Marja-Leena; Kotovuori, Annika; Reinman, Mirka; Satomaa, Tero; Alfthan, Kaija; Laitinen, Saara; Takkinen, Kristiina; Räbinä, Jarkko; Valmu, Leena

2013-01-01

Abstract Multipotent mesenchymal stem/stromal cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. Panels of functional and phenotypical markers are currently used in characterization of different therapeutic stem cell populations from various sources. The i antigen (linear poly-N-acetyllactosamine) from the Ii blood group system has been suggested as a marker for MSCs derived from umbilical cord blood (UCB). However, there are currently no commercially available antibodies recognizing the i antigen. In the present study, we describe the use of antibody phage display technology to produce recombinant antibodies recognizing a structure from the surface of mesenchymal stem cells. We constructed IgM phage display libraries from the lymphocytes of a donor with an elevated serum anti-i titer. Antibody phage display technology is not dependent on immunization and thus allows the generation of antibodies against poorly immunogenic molecules, such as carbohydrates. Agglutination assays utilizing i antigen–positive red blood cells (RBCs) from UCB revealed six promising single-chain variable fragment (scFv) antibodies, three of which recognized epitopes from the surface of UCB-MSCs in flow cytometric assays. The amino acid sequence of the VH gene segment of B12.2 scFv was highly similar to the VH4.21 gene segment required to encode anti-i specificities. Further characterization of binding properties revealed that the binding of B12.2 hyperphage was inhibited by soluble linear lactosamine oligosaccharide. Based on these findings, we suggest that the B12.2 scFv we have generated is a prominent anti-i antibody that recognizes i antigen on the surface of both UCB-MSCs and RBCs. This binder can thus be utilized in UCB-MSC detection and isolation as well as in blood group serology. PMID:24083089

CMOS image sensor for detection of interferon gamma protein interaction as a point-of-care approach.

PubMed

Marimuthu, Mohana; Kandasamy, Karthikeyan; Ahn, Chang Geun; Sung, Gun Yong; Kim, Min-Gon; Kim, Sanghyo

2011-09-01

Complementary metal oxide semiconductor (CMOS)-based image sensors have received increased attention owing to the possibility of incorporating them into portable diagnostic devices. The present research examined the efficiency and sensitivity of a CMOS image sensor for the detection of antigen-antibody interactions involving interferon gamma protein without the aid of expensive instruments. The highest detection sensitivity of about 1 fg/ml primary antibody was achieved simply by a transmission mechanism. When photons are prevented from hitting the sensor surface, a reduction in digital output occurs in which the number of photons hitting the sensor surface is approximately proportional to the digital number. Nanoscale variation in substrate thickness after protein binding can be detected with high sensitivity by the CMOS image sensor. Therefore, this technique can be easily applied to smartphones or any clinical diagnostic devices for the detection of several biological entities, with high impact on the development of point-of-care applications.

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.

Nanoporous Anodic Alumina Surface Modification by Electrostatic, Covalent, and Immune Complexation Binding Investigated by Capillary Filling.

PubMed

Eckstein, Chris; Acosta, Laura K; Pol, Laura; Xifré-Pérez, Elisabet; Pallares, Josep; Ferré-Borrull, Josep; Marsal, Lluis F

2018-03-28

The fluid imbibition-coupled laser interferometry (FICLI) technique has been applied to detect and quantify surface changes and pore dimension variations in nanoporous anodic alumina (NAA) structures. FICLI is a noninvasive optical technique that permits the determination of the NAA average pore radius with high accuracy. In this work, the technique is applied after each step of different surface modification paths of the NAA pores: (i) electrostatic immobilization of bovine serum albumin (BSA), (ii) covalent attachment of streptavidin via (3-aminipropyl)-triethoxysilane and glutaraldehyde grafting, and (iii) immune complexation. Results show that BSA attachment can be detected as a reduction in estimated radius from FICLI with high accuracy and reproducibility. In the case of the covalent attachment of streptavidin, FICLI is able to recognize a multilayer formation of the silane and the protein. For immune complexation, the technique is able to detect different antibody-antigen bindings and distinguish different dynamics among different immune species.

Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA.

PubMed

Xiang, Kuan-Hui; Michailidis, Eleftherios; Ding, Hai; Peng, Ya-Qin; Su, Ming-Ze; Li, Yao; Liu, Xue-En; Dao Thi, Viet Loan; Wu, Xian-Fang; Schneider, William M; Rice, Charles M; Zhuang, Hui; Li, Tong

2017-02-01

As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69∗), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69∗, and sG145R by co-expression of wild-type HBsAg. The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements. The hepatitis B surface antigen (HBsAg) is a major viral protein of the hepatitis B virus (HBV) secreted into patient blood serum and its quantification value serves as an important marker for the evaluation of chronic HBV infection and antiviral response. We found a few new amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These different substitutions might impair virion secretion, change the ability of HBsAg to bind to antibodies, or impact HBV replication. These could all result in decreased detectable levels of serum HBsAg. The factors affecting circulating HBsAg level and HBsAg detection are varied and caution is needed when interpreting clinical significance of serum HBsAg levels. Clinical trial number: NCT01088009. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis.

PubMed

Bien, Christian G; Vincent, Angela; Barnett, Michael H; Becker, Albert J; Blümcke, Ingmar; Graus, Francesc; Jellinger, Kurt A; Reuss, David E; Ribalta, Teresa; Schlegel, Jürgen; Sutton, Ian; Lassmann, Hans; Bauer, Jan

2012-05-01

Classical paraneoplastic encephalitis syndromes with 'onconeural' antibodies directed to intracellular antigens, and the recently described paraneoplastic or non-paraneoplastic encephalitides and antibodies against both neural surface antigens (voltage-gated potassium channel-complexes, N-methyl-d-aspartate receptors) and intracellular antigens (glutamic acid decarboxylase-65), constitute an increasingly recognized group of immune-mediated brain diseases. Evidence for specific immune mechanisms, however, is scarce. Here, we report qualitative and quantitative immunopathology in brain tissue (biopsy or autopsy material) of 17 cases with encephalitis and antibodies to either intracellular (Hu, Ma2, glutamic acid decarboxylase) or surface antigenic targets (voltage-gated potassium channel-complex or N-methyl-d-aspartate receptors). We hypothesized that the encephalitides with antibodies against intracellular antigens (intracellular antigen-onconeural and intracellular antigen-glutamic acid decarboxylase groups) would show neurodegeneration mediated by T cell cytotoxicity and the encephalitides with antibodies against surface antigens would be antibody-mediated and would show less T cell involvement. We found a higher CD8/CD3 ratio and more frequent appositions of granzyme-B(+) cytotoxic T cells to neurons, with associated neuronal loss, in the intracellular antigen-onconeural group (anti-Hu and anti-Ma2 cases) compared to the patients with surface antigens (anti-N-methyl-d-aspartate receptors and anti-voltage-gated potassium channel complex cases). One of the glutamic acid decarboxylase antibody encephalitis cases (intracellular antigen-glutamic acid decarboxylase group) showed multiple appositions of GrB-positive T cells to neurons. Generally, however, the glutamic acid decarboxylase antibody cases showed less intense inflammation and also had relatively low CD8/CD3 ratios compared with the intracellular antigen-onconeural cases. Conversely, we found complement C9neo deposition on neurons associated with acute neuronal cell death in the surface antigen group only, specifically in the voltage-gated potassium channel-complex antibody patients. N-methyl-d-aspartate receptors-antibody cases showed no evidence of antibody and complement-mediated tissue injury and were distinguished from all other encephalitides by the absence of clear neuronal pathology and a low density of inflammatory cells. Although tissue samples varied in location and in the stage of disease, our findings strongly support a central role for T cell-mediated neuronal cytotoxicity in encephalitides with antibodies against intracellular antigens. In voltage-gated potassium channel-complex encephalitis, a subset of the surface antigen antibody encephalitides, an antibody- and complement-mediated immune response appears to be responsible for neuronal loss and cerebral atrophy; the apparent absence of these mechanisms in N-methyl-d-aspartate receptors antibody encephalitis is intriguing and requires further study.

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

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.

Characterisation of monoclonal antibodies to common protein epitopes on the cell surface of Streptococcus mutans and Streptococcus sobrinus.

PubMed

Smith, R; Lehner, T

1989-09-01

Three monoclonal antibodies (MAb) were prepared against a cell surface antigen which cross-react between Streptococcus mutans (serotypes c, e and f) and Streptococcus sobrinus (serotypes d and g). Two of the MAb also recognise a determinant on the surface of Streptococcus cricetus (serotype a). The common antigen shared between S. mutans and S. sobrinus was demonstrated by Western blotting to be about 200 kD in size. This antigen is shared not only by the cell surfaces of serotypes a, c, d, e, f and g, but also by the major cell surface antigen of S. mutans of 185 kD and another of 150 kD. These MAb identify all but one mutans type of streptococci and can be utilised as analytical reagents.

The Klebsiella pneumoniae O Antigen Contributes to Bacteremia and Lethality during Murine Pneumonia

PubMed Central

Shankar-Sinha, Sunita; Valencia, Gabriel A.; Janes, Brian K.; Rosenberg, Jessica K.; Whitfield, Chris; Bender, Robert A.; Standiford, Ted J.; Younger, John G.

2004-01-01

Bacterial surface carbohydrates are important pathogenic factors in gram-negative pneumonia infections. Among these factors, O antigen has been reported to protect pathogens against complement-mediated killing. To examine further the role of O antigen, we insertionally inactivated the gene encoding a galactosyltransferase necessary for serotype O1 O-antigen synthesis (wbbO) from Klebsiella pneumoniae 43816. Analysis of the mutant lipopolysaccharide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the absence of O antigen. In vitro, there were no detectable differences between wild-type K. pneumoniae and the O-antigen-deficient mutant in regard to avid binding by murine complement C3 or resistance to serum- or whole-blood-mediated killing. Nevertheless, the 72-h 50% lethal dose of the wild-type strain was 30-fold greater than that of the mutant (2 × 103 versus 6 × 104 CFU) after intratracheal injection in ICR strain mice. Despite being less lethal, the mutant organism exhibited comparable intrapulmonary proliferation at 24 h compared to the level of the wild type. Whole-lung chemokine expression (CCL3 and CXCL2) and bronchoalveolar inflammatory cell content were also similar between the two infections. However, whereas the wild-type organism produced bacteremia within 24 h of infection in every instance, bacteremia was not seen in mutant-infected mice. These results suggest that during murine pneumonia caused by K. pneumoniae, O antigen contributes to lethality by increasing the propensity for bacteremia and not by significantly changing the early course of intrapulmonary infection. PMID:14977947

Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP).

PubMed

Schmitz, H

1981-01-01

An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.

Expression of PCV2 antigen in the ovarian tissues of gilts.

PubMed

Tummaruk, Padet; Pearodwong, Pachara

2016-03-01

The present study was performed to determine the expression of porcine circovirus type 2 (PCV2) antigen in the ovarian tissue of naturally infected gilts. Ovarian tissues were obtained from 11 culled gilts. The ovarian tissues sections were divided into two groups according to PCV2 DNA detection using PCR. PCV2 antigen was assessed in the paraffin embedded ovarian tissue sections by immunohistochemistry. A total of 2,131 ovarian follicles (i.e., 1,437 primordial, 133 primary, 353 secondary and 208 antral follicles), 66 atretic follicles and 131 corpora lutea were evaluated. It was found that PCV2 antigen was detected in 280 ovarian follicles (i.e., 239 primordial follicles, 12 primary follicles, 10 secondary follicles and 19 antral follicles), 1 atretic follicles and 3 corpora lutea (P<0.05). PCV2 antigen was detected in primordial follicles more often than in secondary follicles, atretic follicles and corpora lutea (P<0.05). The detection of PCV2 antigen was found mainly in oocytes. PCV2 antigen was found in both PCV2 DNA positive and negative ovarian tissues. It can be concluded that PCV2 antigen is expressed in all types of the ovarian follicles and corpora lutea. Further studies should be carried out to determine the influence of PCV2 on porcine ovarian function and oocyte quality.

Frequency and reactivity of antigen-specific T cells were concurrently measured through the combination of artificial antigen-presenting cell, MACS and ELISPOT.

PubMed

Shen, Chuanlai; Xu, Tao; Wu, You; Li, Xiaoe; Xia, Lingzhi; Wang, Wei; Shahzad, Khawar Ali; Zhang, Lei; Wan, Xin; Qiu, Jie

2017-11-27

Conventional peptide-major histocompatibility complex (pMHC) multimer staining, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay cannot concurrently determine the frequency and reactivity of antigen-specific T cells (AST) in a single assay. In this report, pMHC multimer, magnetic-activated cell sorting (MACS), and ELISPOT techniques have been integrated into a micro well by coupling pMHC multimers onto cell-sized magnetic beads to characterize AST cell populations in a 96-well microplate which pre-coated with cytokine-capture antibodies. This method, termed AAPC-microplate, allows the enumeration and local cytokine production of AST cells in a single assay without using flow cytometry or fluorescence intensity scanning, thus will be widely applicable. Here, ovalbumin 257-264 -specific CD8 + T cells from OT-1 T cell receptor (TCR) transgenic mice were measured. The methodological accuracy, specificity, reproducibility, and sensitivity in enumerating AST cells compared well with conventional pMHC multimer staining. Furthermore, the AAPC-microplate was applied to detect the frequency and reactivity of Hepatitis B virus (HBV) core antigen 18-27 - and surface antigen 183-191 -specific CD8 + T cells for the patients, and was compared with conventional method. This method without the need of high-end instruments may facilitate the routine analysis of patient-specific cellular immune response pattern to a given antigen in translational studies.

Nanotechnology for the detection and kill of circulating tumor cells

NASA Astrophysics Data System (ADS)

Gao, Yang; Yuan, Zhou

2014-09-01

Circulating tumor cells (CTCs) represent a surrogate biomarker of hematogenous metastases and thus could be considered as a `liquid biopsy' which reveals metastasis in action. But it is absolutely a challenge to detect CTCs due to their extreme rarity. At present, the most common principle is to take advantage of the epithelial surface markers of CTCs which attach to a specific antibody. Antibody-magnetic nanobeads combine with the epithelial surface markers, and then the compound is processed by washing, separation, and detection. However, a proportion of CTC antigen expressions are down-regulated or lost in the process of epithelial-mesenchymal transition (EMT), and thus, this part of CTCs cannot be detected by classical detection methods such as CellSearch. To resolve this problem, some multiple-marker CTC detections have been developed rapidly. Additionally, nanotechnology is a promising approach to kill CTCs with high efficiency. Implantable nanotubes coated with apoptosis-promoting molecules improve the disease-free survival and overall survival. The review introduces some novel CTC detection techniques and therapeutic methods by virtue of nanotechnology to provide a better knowledge of the progress about CTC study.

Binding of antibodies to the extractable nuclear antigens SS-A/Ro and SS-B/La is induced on the surface of human keratinocytes by ultraviolet light (UVL): Implications for the pathogenesis of photosensitive cutaneous lupus

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

Furukawa, F.; Kashihara-Sawami, M.; Lyons, M.B.

1990-01-01

Autoantibodies to the non-histone nucleoprotein antigens SS-A/Ro, SS-B/La, and RNP are highly associated with photosensitive cutaneous lupus erythematosus (LE). In order to better understand the potential mechanisms of ultraviolet (UV) light on photosensitivity in patients with cutaneous LE, we designed immunopathologic in vitro and in vivo experiments to evaluate the effects of UV on the binding of such autoantibodies to the surface of human keratinocytes, one major target of immunologic damage in photosensitive LE. Short-term 2% paraformaldehyde fixation of suspensions of cultured human keratinocytes previously incubated with monospecific antiserum probes enabled the detection of ENA expression on the cell surfacemore » by flow-cytometry analysis. UVB light (280-320 nm) induced the binding of monospecific antibody probes for SS-A/Ro and SS-B/La on keratinocytes in a dose-dependent pattern with maximal induction observed at the dose of 200 mJ/cm2 UVB. Binding of SS-A/Ro, SS-B/La, and RNP antibody was augmented strongly, but binding of anti-Sm was very weak. In contrast, UVA (320-400 nm) light had no effect on the induction of binding of these antibody probes. Identical results were seen by standard immunofluorescence techniques. Hydroxyurea-treated keratinocytes showed similar induction of those antigens by UVB irradiation, which suggested that ENA expression on cultured keratinocytes by UVB were cell-cycle independent. Tunicamycin, an inhibitor of glycosylation of proteins, reduced UVB light effect on the SS-A/Ro and SS-B/La antigen's expression. These in vitro FACS analyses revealed that ENA augmentation on the keratinocyte cell surface was dose dependent, UVB dependent, glycosylation dependent, and cell-cycle independent. In vivo ENA augmentation on the keratinocyte surface was examined in suction blister epidermal roofs.« less

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.

Detection of Soluble Antigen and DNA of Trypanosoma cruzi in Urine Is Independent of Renal Injury in the Guinea Pig Model

PubMed Central

Castro-Sesquen, Yagahira E.; Gilman, Robert H.; Yauri, Verónica; Cok, Jaime; Angulo, Noelia; Escalante, Hermes; Bern, Caryn

2013-01-01

The diagnosis of Chagas disease in humans is generally limited to the detection of specific antibodies. Detection of T. cruzi antigens in urine has been reported previously, but is not used in the diagnosis. In this study, soluble T. cruzi antigens and DNA were detected in urine samples and were associated with kidney injury and systemic detection of the parasite. We used 72 guinea pigs infected with T. cruzi Y strain and 18 non-infected guinea pigs. Blood, kidney, heart and urine samples were collected during the acute phase and chronic phase. Urine samples were concentrated by ultrafiltration. Antigens were detected by Western Blot using a polyclonal antibody against trypomastigote excretory-secretory antigen (TESA). T. cruzi DNA was detected by PCR using primers 121/122 and TcZ1/TcZ2. Levels of T. cruzi DNA in blood, heart and kidney were determined by quantitative PCR. T. cruzi antigens (75 kDa, 80 kDa, 120 kDa, 150 kDa) were detected in the acute phase (67.5%) and the chronic phase (45%). Parasite DNA in urine was detected only in the acute phase (45%). Kidney injury was characterized by high levels of proteinuria, kidney injury molecule-1 (KIM-1) and urea, and some histopathological changes such as inflammation, necrosis, fibrosis and scarce parasites. The detection of antigens and DNA in urine was associated with the presence of parasite DNA in blood and heart and with high levels of parasite DNA in blood, but not with the presence of parasite in kidney or kidney injury. These results suggest that the detection of T. cruzi in urine could be improved to be a valuable method for the diagnosis of Chagas disease, particularly in congenital Chagas disease and in immunocompromised patients. PMID:23520515

Bench-Top Antigen Detection Technique that Utilizes Nanofiltration and Fluorescent Dyes which Emit and Absorb Light in the Near Infrared

NASA Technical Reports Server (NTRS)

Varaljay-Spence, Vanessa A.; Scardelletti, Maximilian C.

2007-01-01

This article discusses the development of a bench-top technique to detect antigens in fluids. The technique involves the use of near infrared NIR fluorescent dyes conjugated to antibodies, centrifugation, nanofilters, and spectrometry. The system used to detect the antigens utilizes a spectrometer, fiber optic cables, NIR laser, and laptop computer thus making it portable and ideally suited for desk top analysis. Using IgM as an antigen and the secondary antibody, anti-IgM conjugated to the near infrared dye, IRDye (trademark) 800, for detection, we show that nanofiltration can efficiently and specifically separate antibody-antigen complexes in solution and that the complexes can be detected by a spectrometer and software using NIR laser excitation at 778 nm and NIR dye offset emission at 804 nm. The peak power detected at 778 nm for the excitation emission and at 804 nm for the offset emission is 879 pW (-60.06 dBm) and 35.7 pW (-74.5 dBm), respectively.

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

PubMed

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

2013-01-01

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

Rapid and ultrasensitive flexible palladium nano-thin film biosensing electrode development for cancer antigen HER2 detection

NASA Astrophysics Data System (ADS)

Huang, Yun-Tzu; Chang, Chia-Yu; Chen, Wei; Su, Chien-Hao; Hsu, Guo-Cheng; Chang, Chia-Ching

HER2 (human epidermal growth factor receptor 2) is one of the significant surface antigens of breast cancer Trace amount of HER2 protein in human serum is highly correlated to the tumor progression in breast cancers especially in the cases of recurrence. Therefore, HER2 detection of human serum is significant for early detection of cancer recurrence. Conventional HER2 detection approaches may not be sensitive enough or contain highly false positive rate or time consuming for accurate detection. Therefore, a rapid, highly sensitive and specific sensing is highly desired. By using HER2 specific binding peptide functionalized palladium thin film electrochemical electrode the HER2 protein concentration can be determined at sub-nanogram level by electrochemical impedance spectroscopy (EIS) within 10 mins. The Pd nano-film is sputtered on the flexible plastics substrate and reduces the cost of this electrode. Due to the low cost of the electrode, it is designed as a disposable biosensing probe which may reduce the concern of human sample contamination. The self-management after breast cancer operation may be feasible in the near future. Keywords: Electrochemical impedance spectroscopy(EIS), breast cancer, biosensor Corresponding author: ccchang01@faculty.nctu.edu.tw; Cheeshin Technology Co. Collaboration.

On the importance of controlling film architecture in detecting prostate specific antigen

NASA Astrophysics Data System (ADS)

Graça, Juliana Santos; Miyazaki, Celina Massumi; Shimizu, Flavio Makoto; Volpati, Diogo; Mejía-Salazar, J. R.; Oliveira, Osvaldo N., Jr.; Ferreira, Marystela

2018-03-01

Immunosensors made with nanostructured films are promising for detecting cancer biomarkers, even at early stages of the disease, but this requires control of film architecture to preserve the biological activity of immobilized antibodies. In this study, we used electrochemical impedance spectroscopy (EIS) to detect Prostate Specific Antigen (PSA) with immunosensors produced with layer-by-layer (LbL) films containing anti-PSA antibodies in two distinct film architectures. The antibodies were either adsorbed from solutions in which they were free, or from solutions where they were incorporated into liposomes of dipalmitoyl phosphatidyl glycerol (DPPG). Incorporation into DPPG liposomes was confirmed with surface plasmon resonance experiments, while the importance of electrostatic interactions on the electrical response was highlighted using the Finite Difference Time-Domain Method (FDTD). The sensitivity of both architectures was sufficient to detect the threshold value to diagnose prostate cancer (ca. 4 ng mL-1). In contrast to expectation, the sensor with the antibodies incorporated into DPPG liposomes had lower sensitivity, though the range of concentrations amenable to detection increased, according to the fitting of the EIS data using the Langmuir-Freundlich adsorption model. The performance of the two film architectures was compared qualitatively by plotting the data with a multidimensional projection technique, which constitutes a generic approach for optimizing immunosensors and other types of sensors.

Detection of pathogens using luminescent CdSe/ZnS dendron nanocrystals and a porous membrane immunofilter.

PubMed

Liu, Yongcheng; Brandon, Robert; Cate, Michael; Peng, Xiaogang; Stony, Robert; Johnson, Michael

2007-11-15

A biosensor system for detection of pathogens was developed by using CdSe/ZnS core/shell dendron nanocrystals with high efficiency and stability as fluorescence labels and a flowing chamber with a microporous immunofilter. The antibody-immobilized immunofilter captured the targeted pathogens, Escherichia coli O157:H7 as an example for bacteria and hepatitis B being a model system for viruses. The CdSe/ZnS core/shell dendron nanocrystals were conjugated with the corresponding antibodies and then passed through the microporous membrane where they attached to the membrane-antigen-antibody. The efficient and stable photoluminescence (PL) of the CdSe/ZnS nanocrystals on the formed "sandwich" structure complexes (membrane-antigen-antibody conjugated with the nanocrystals) was used as the detection means. The effects of the pore size of the membranes, buffer pH, and assay time on the detection of E. coli O157:H7 were investigated and optimized. The detectable level of this new system was as low as 2.3 CFU/mL for E. coli O157:H7 and 5 ng/mL for hepatitis B surface Ag (HBsAg). The assay time was shortened to 30 min without any enrichment and incubation.

Enzyme-Linked Immunosorbent Assays for Detection of Equine Antibodies Specific to Sarcocystis neurona Surface Antigens†

PubMed Central

Hoane, Jessica S.; Morrow, Jennifer K.; Saville, William J.; Dubey, J. P.; Granstrom, David E.; Howe, Daniel K.

2005-01-01

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM. PMID:16148170

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

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.

Novel reassortant H9N2 viruses in pigeons and evidence for antigenic diversity of H9N2 viruses isolated from quails in Egypt.

PubMed

Kandeil, Ahmed; El-Shesheny, Rabeh; Maatouq, Asmaa; Moatasim, Yassmin; Cai, Zhipeng; McKenzie, Pamela; Webby, Richard; Kayali, Ghazi; Ali, Mohamed A

2017-04-01

The endemicity of avian influenza viruses (AIVs) among Egyptian poultry represents a public health risk. Co-circulation of low pathogenic AIV H9N2 subtype with highly pathogenic AIV H5N1 subtype in Egyptian farms provides a possibility to generate novel reassortant viruses. Here, the genetic characteristics of surface glycoproteins of 59 Egyptian H9N2 viruses, isolated between 2013 and 2015, were analysed. To elucidate the potential of genetic reassortment, 10 H9N2 isolates were selected based on different avian hosts (chickens, ducks, pigeons and quails) and phylogenetic analyses of their full genome sequences were conducted. Additionally, we performed antigenic analysis to further investigate the antigenic evolution of H9N2 viruses isolated during 2011-2015. Different viral characteristics including receptor-binding affinity and drug resistance of representative Egyptian H9N2 viruses were further investigated. The surface glycoproteins of current Egyptian H9N2 viruses were closely related to viruses of the G1-like lineage isolated from Egypt. Several genetic markers that enhance virulence in poultry and transmission to humans were detected. Analysis of the full genome of 10 H9N2 isolates indicated that two pigeon isolates inherited five internal genes from Eurasian AIVs circulating in wild birds. Antigenic conservation of different Egyptian H9N2 isolates from chickens, pigeons and ducks was observed, whereas quail isolates showed antigenic drift. The Egyptian H9N2 viruses preferentially bound to the human-like receptor rather than to the avian-like receptor. Our results suggest that the endemic H9N2 viruses in Egypt contain elements that may favour avian-to-human transmission and thus represent a public health risk.

Detection of viral antigens in renal tissue of glomerulonephritis patients without serological evidence of hepatitis B virus and hepatitis C virus infection.

PubMed

Kong, Dan; Wu, Di; Wang, Tianzhen; Li, Tianzhu; Xu, Shengjie; Chen, Fulai; Jin, Xiaoming; Lou, Ge

2013-07-01

Glomerulonephritis is an important extrahepatic manifestation of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. HBV and HCV infection may be occult, and they are often overlooked by both patients and doctors. The aim of this study was to assess the importance of HBV and HCV infection in glomerulonephritis patients with undetectable HBV surface antigen (HBsAg) and HCV antibody in serum. The HBsAg, the HBV core antigen (HBcAg), and the HCV antigen were detected using immunohistochemistry in frozen renal tissues of 500 glomerulonephritis patients without serological evidence of HBV and HCV infection. Electron microscopy was used to trace the virus particles, and clinicopathological features were also reviewed. HBsAg or HBcAg was positive in nine out of 500 cases (9/500, 1.8%). Three cases were HBsAg-positive and another six cases were HBcAg-positive. The HCV antigen was found in eight cases (8/500, 1.6%). There was one case of HBV and HCV co-infection (1/500, 0.2%). Under electron microscopy, virus particles were found in the base membrane and cytoplasm of endotheliocytes in the glomerulus. The most common clinical manifestation was nephrotic syndrome (9/18), followed by nephritic syndrome (7/18). Membranous nephropathy was the most common pathological diagnosis (5/18), followed by mesangioproliferative glomerulonephritis (4/18) and IgA nephropathy (4/18). Occult HBV and HCV infection might be implicated in HBV- or HCV-associated glomerulonephritis. More attention should be focused on the underlying cause. Copyright © 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins

NASA Technical Reports Server (NTRS)

Schatten, G.; Schatten, H.; Simerly, C.; Maul, G. G.; Chaly, N.

1985-01-01

Nuclear structural changes during fertilization and embryogenesis in mice and sea urchins are traced using four antibodies. The oocytes from virgin female mice, morulae and blastocytes from mated females, and gametes from the sea urchin Lytechnius variegatis are studied using mouse monoclonal antibodies to nuclear lamin A/C, monoclonal antibody to P1, human autoimmune antibodies to lamin A/C, and to lamin B. The mouse fertilization data reveal no lamins on the oocyte; however, lamins are present on the pronuclei, and chromosomes are found on the oocytes and pronuclei. It is detected that on the sea urchin sperm the lamins are reduced to acrosomal and centriolar fossae and peripheral antigens are around the sperm nucleus. The mouse sperm bind lamin antibodies regionally and do not contain antigens. Lamins and antigens are observed on both pronuclei and chromosomes during sea urchin fertilization. Mouse embryogenesis reveals that lamin A/C is not recognized at morula and blastocyst stages; however, lamin B stains are retained. In sea urchin embryogenesis lamin recognition is lost at the blastrula, gastrula, and plutei stages. It is noted that nuclear lamins lost during spermatogenesis are restored at fertilization and peripheral antigens are associated with the surface of chromosomes during meiosis and mitosis and with the periphery of the pronuclei and nuclei during interphase.

Characterization of occult hepatitis B virus infection among HIV positive patients in Cameroon.

PubMed

Gachara, George; Magoro, Tshifhiwa; Mavhandu, Lufuno; Lum, Emmaculate; Kimbi, Helen K; Ndip, Roland N; Bessong, Pascal O

2017-03-08

Occult hepatitis B infection (OBI) among HIV positive patients varies widely in different geographic regions. We undertook a study to determine the prevalence of occult hepatitis B infection among HIV infected individuals visiting a health facility in South West Cameroon and characterized occult HBV strains based on sequence analyses. Plasma samples (n = 337), which previously tested negative for hepatitis B surface antigen (HBsAg), were screened for antibodies against hepatitis B core (anti-HBc) and surface (anti-HBs) antigens followed by DNA extraction. A 366 bp region covering the overlapping surface/polymerase gene of HBV was then amplified in a nested PCR and the amplicons sequenced using Sanger sequencing. The resulting sequences were then analyzed for genotypes and for escape and drug resistance mutations. Twenty samples were HBV DNA positive and were classified as OBI giving a prevalence of 5.9%. Out of these, 9 (45%) were anti-HBs positive, while 10 (52.6%) were anti-HBc positive. Additionally, 2 had dual anti-HBs and anti-HBc reactivity, while 6 had no detectable HBV antibodies. Out of the ten samples that were successfully sequenced, nine were classified as genotype E and one as genotype A. Three sequences possessed mutations associated with lamivudine resistance. We detected a number of mutations within the major hydrophilic region of the surface gene where most immune escape mutations occur. Findings from this study show the presence of hepatitis B in patients without any of the HBV serological markers. Further prospective studies are required to determine the risk factors and markers of OBI.

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.

Amplified cathodic electrochemiluminescence of luminol based on Pd and Pt nanoparticles and glucose oxidase decorated graphene as trace label for ultrasensitive detection of protein.

PubMed

Cao, Yaling; Yuan, Ruo; Chai, Yaqin; Liu, Huijing; Liao, Yuhong; Zhuo, Ying

2013-09-15

An ultrasensitive electrochemiluminescence (ECL) immunosensor was constructed for ultrasensitive detection of carcinoembryonic antigen (CEA) based on an amplified cathodic ECL of luminol at low potential. Firstly, Au nanoparticles (AuNPs) were electrodeposited onto single walled carbon nanotube-graphene composites (CNTs-Gra) coated glass carbon electrode (GCE) with enhanced surface area and good biocompatibility to capture primary antibody (Ab1) and then bind the antigen analytes. Secondly, Pd and Pt nanoparticles (Pd&PtNPs) decorated reduced graphene oxide (Pd&PtNPs@rGO) and glucose oxidase (GOD) labeled secondary antibody (Pd&PtNPs@ rGO-GOD-Ab2) could be captured onto the electrode surface by a sandwich immunoassay protocol to generate amplified cathodic ECL signals of luminol in the presence of glucose. The Pd&PtNPs@rGO composites and loaded GOD promoted luminol cathodic ECL response by efficiently catalyzing glucose to in-situ produce amount of hydrogen peroxide (H2O2) working as a coreactant of luminol. Then in turn Pd&PtNPs catalyzed H2O2 to generate various reactive oxygen species (ROSs), which accelerated the cathodic ECL reaction of luminol, enhanced the cathodic ECL intensity of luminol and improved the sensitivity of the immunosensor. The as-proposed ECL immunosensor exhibited sensitive response on the detection of CEA ranging from 0.0001 ng mL(-1) to 160 ng mL(-1) with a detection limit of 0.03 pg mL(-1) (S/N=3). Moreover, the stability, specificity, lifetime and reproducibility tests demonstrated the feasibility of the developed immunoassay, which can be further extended to the detection of other disease biomarkers. Copyright © 2013 Elsevier B.V. All rights reserved.

Top-down nanofabrication of silicon nanoribbon field effect transistor (Si-NR FET) for carcinoembryonic antigen detection.

PubMed

Bao, Zengtao; Sun, Jialin; Zhao, Xiaoqian; Li, Zengyao; Cui, Songkui; Meng, Qingyang; Zhang, Ye; Wang, Tong; Jiang, Yanfeng

2017-01-01

Sensitive and quantitative detection of tumor markers is highly required in the clinic for cancer diagnosis and consequent treatment. A field-effect transistor-based (FET-based) nanobiosensor emerges with characteristics of being label-free, real-time, having high sensitivity, and providing direct electrical readout for detection of biomarkers. In this paper, a top-down approach is proposed and implemented to fulfill a novel silicon nano-ribbon FET, which acts as biomarker sensor for future clinical application. Compared with the bottom-up approach, a top-down fabrication approach can confine width and length of the silicon FET precisely to control its electrical properties. The silicon nanoribbon (Si-NR) transistor is fabricated on a Silicon-on-Insulator (SOI) substrate by a top-down approach with complementary metal oxide semiconductor (CMOS)-compatible technology. After the preparation, the surface of Si-NR is functionalized with 3-aminopropyltriethoxysilane (APTES). Glutaraldehyde is utilized to bind the amino terminals of APTES and antibody on the surface. Finally, a microfluidic channel is integrated on the top of the device, acting as a flowing channel for the carcinoembryonic antigen (CEA) solution. The Si-NR FET is 120 nm in width and 25 nm in height, with ambipolar electrical characteristics. A logarithmic relationship between the changing ratio of the current and the CEA concentration is measured in the range of 0.1-100 ng/mL. The sensitivity of detection is measured as 10 pg/mL. The top-down fabricated biochip shows feasibility in direct detecting of CEA with the benefits of real-time, low cost, and high sensitivity as a promising biosensor for tumor early diagnosis.

A Polymer/Oil Based Nanovaccine as a Single-Dose Immunization Approach

PubMed Central

Vicente, Sara; Diaz-Freitas, Belen; Peleteiro, Mercedes; Sanchez, Alejandro; Pascual, David W.; Gonzalez-Fernandez, Africa; Alonso, Maria J.

2013-01-01

The recognized necessity for new antigen delivery carriers with the capacity to boost, modulate and prolong neutralizing immune responses prompted our approach, in which we describe a multifunctional nanocarrier consisting of an oily nanocontainer protected by a polymeric shell made of chitosan (CS), named CS nanocapsules (CSNC). The CS shell can associate the antigen on its surface, whereas the oily core might provide additional immunostimulating properties. In this first characterization of the system, we intended to study the influence of different antigen organizations on the nanocarrier's surface (using the recombinant hepatitis B surface antigen –rHBsAg– as a model antigen) on their long-term immunopotentiating effect, without any additional immunostimulant. Thus, two prototypes of antigen-loaded CSNC (CSNC+ and CSNC−), exhibiting similar particle size (200 nm) and high antigen association efficiency (>80%), were developed with different surface composition (polymer/antigen ratios) and surface charge (positive/negative, respectively). The biological evaluation of these nanovaccines evidenced the superiority of the CSNC+ as compared to CSNC- and alum-rHBsAg in terms of neutralizing antibody responses, following intramuscular vaccination. Moreover, a single dose of CSNC+ led to similar IgG levels to the positive control. The IgG1/IgG2a ratio suggested a mixed Th1/Th2 response elicited by CSNC+, in contrast to the typical Th2-biased response of alum. Finally, CSNC+ could be freeze-dried without altering its physicochemical properties and adjuvant effect in vivo. In conclusion, the evaluation of CSNC+ confirms its interesting features for enhancing, prolonging and modulating the type of immune response against subunit antigens, such as rHBsAg. PMID:23614052

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

PubMed

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

1988-06-01

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

Ultrasensitive Detection of Low-Abundance Surface-Marker Protein using Isothermal Rolling Circle Amplification in Microfluidic Nano-Liter Platform

PubMed Central

Konry, Tania; Yarmush, Joel M.; Irimia, Daniel

2011-01-01

With advances in immunology and cancer biology, there is an unmet need for increasingly sensitive systems to monitor the expression of specific cell markers for the development of new diagnostic and therapeutic tools. To address this challenge, we have applied a highly sensitive labeling method that translates antigen-antibody recognition processes into DNA detection event that can be greatly amplified via isothermal Rolling Circle Amplification (RCA). By merging the single-molecule detection power of RCA reaction with microfluidic technology we were able to demonstrate that identification of specific protein markers can be achieved on tumor cell surface in miniaturized nano-liter reaction droplets. Furthermore, this combined approach of signal amplification in a microfluidic format could extend the utility of existing methods by reducing sample and reagent consumption and enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer. PMID:21294269

Fabrication of microfluidic integrated biosensor

NASA Astrophysics Data System (ADS)

Adam, Tijjani; Dhahi, Th S.; Mohammed, Mohammed; Hashim, U.; Noriman, N. Z.; Dahham, Omar S.

2017-09-01

An event of miniaturizing for sensor systems to carry out biological diagnostics are gaining wade spread acceptance. The system may contain several different sensor units for the detection of specific analyte, the analyte to be detected might be any kind of biological molecules (DNA, mRNA or proteins) or chemical substances. In most cases, the detection is based on receptor-ligand binding like DNA hybridization or antibody-antigen interaction, achieving this on a nanostructure. DNA or protein must be attached to certain locations within the structure. Critical for this is to have a robust binding chemistry to the surface in the microstructure. Here we successfully designed and fabricated microfluidics element for passive fluid delivery into polysilicon Nanowire sensing domain, we further demonstrated a very simple and effective way of integrating the two devices to give full functionalities of laboratory on a single chip. The sensing element was successfully surface modified and tested on real biomedical clinical sample for evaluation and validation.

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...

Equine infectious anemia virus-infected dendritic cells retain antigen presentation capability

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

Rivera, Julie A.; McGuire, Travis C.

2005-05-10

To determine if equine monocyte-derived dendritic cells (DC) were susceptible to equine infectious anemia virus (EIAV) infection, ex vivo-generated DC were infected with virus in vitro. EIAV antigen was detected by immunofluorescence 3 days post-infection with maximum antigen being detected on day 4, whereas there was no antigen detected in DC incubated with the same amount of heat-inactivated EIAV. No cytolytic activity was observed after EIAV{sub WSU5} infection of DC. These monocyte-derived DC were more effective than macrophages and B cells in stimulating allogenic T lymphocytes. Both infected macrophages and DC stimulated similar levels of memory CTL responses in mixturesmore » of CD8+ and CD4+ cells as detected with {sup 51}Cr-release assays indicating that EIAV infection of DC did not alter antigen presentation. However, EIAV-infected DC were more effective than infected macrophages when used to stimulate memory CTL in isolated CD8+ cells. The maintenance of antigen processing and presenting function by EIAV-infected DC in vitro suggests that this function is maintained during in vivo infection.« less

Advantages of bioconjugated silica-coated nanoparticles as an innovative diagnosis for human toxoplasmosis.

PubMed

Aly, Ibrahim; Taher, Eman E; El Nain, Gehan; El Sayed, Hoda; Mohammed, Faten A; Hamad, Rabab S; Bayoumy, Elsayed M

2018-01-01

Nanotechnology is a promising arena for generating new applications in Medicine. To successfully functionalised nanoparticles for a given biomedical application, a wide range of chemical, physical and biological factors have to be taken into account. Silica-coated nanoparticles, (SiO2NP) exhibit substantial diagnostic activity owing to their large surface to volume ratios and crystallographic surface structure. This work aimed to evaluate the advantage of bioconjugation of SiO2NP with PAb against Toxoplasma lyzate antigen (TLA) as an innovative diagnostic method for human toxoplasmosis. This cross-sectional study included 120 individuals, divided into Group I: 70 patients suspected for Toxoplasma gondii based on the presence of clinical manifestation. Group II: 30 patients harboring other parasites than T. gondii Group III: 20 apparently healthy individuals free from toxoplasmosis and other parasitic infections served as negative control. Detection of circulating Toxoplasma antigen was performed by Sandwich ELISA and Nano-sandwich ELISA on sera and pooled urine of human samples. Using Sandwich ELISA, 10 out of 70 suspected Toxoplasma-infected human serum samples showed false negative and 8 out of 30 of other parasites groups were false positive giving 85.7% sensitivity and 84.0% specificity, while the sensitivity and specificity were 78.6% and 70% respectively in urine samples. Using Nano-Sandwich ELISA, 7 out of 70 suspected Toxoplasma-infected human samples showed false negative results and the sensitivity of the assay was 90.0%, while 4 out of 30 of other parasites groups were false positive giving 92.0% specificity, while the sensitivity and specificity were 82.6% and 80% respectively in urine samples. In conclusion, our data demonstrated that loading SiO2 nanoparticles with pAb increased the sensitivity and specificity of Nano-sandwich ELISA for detection of T.gondii antigens in serum and urine samples, thus active (early) and light infections could be easily detected. Copyright © 2017 Elsevier B.V. All rights reserved.

Silicon photonic resonator for label-free bio-sensing application

NASA Astrophysics Data System (ADS)

Udomsom, Suruk; Mankong, Ukrit; Theera-Umpon, Nipon; Ittipratheep, Nattapol; Umezawa, Toshimasa; Matsumoto, Atsushi; Yamamoto, Naokatsu

2018-03-01

In medical diagnostics there is an increasing demand for biosensors that can specifically detect biological analytes in a fluid. Especially label-free sensing, consistings of a transducer with biorecognition molecules immobilized on its surface without relying on fluorescent dye. In this paper we study the design and fabrication of a silicon nanowire photonic ring resonator and its feasibility as a biosensor. We have simulated and fabricated racetrack ring resonators which have a few tenths of micrometer gap, up to 0.5 μm between the input / output waveguides and the resonators. It is found that the devices can be designed with large Q factors. Sensitivity to biomaterial detection has been simulated for antibody (goat anti-mouse IgG) - antigen (mouse IgG) using 3-dimensional Finite Difference Time Domain technique. The simulated results show that the ring resonator has a response 15 nm resonance shift per refractive index unit. Antibody coating method is also discussed in this paper which can be applied to other antibody-antigen types.

Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

PubMed Central

MacKay, P; Pasek, M; Magazin, M; Kovacic, R T; Allet, B; Stahl, S; Gilbert, W; Schaller, H; Bruce, S A; Murray, K

1981-01-01

Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals. PMID:6170067

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

Tomato bushy stunt virus (TBSV), a versatile platform for polyvalent display of antigenic epitopes and vaccine design

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

Kumar, Shantanu; Ochoa, Wendy; Singh, Pratik

2009-05-25

Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NDELTA52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and imagemore » reconstruction of the chimeric VLPs at 22 A resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T = 1, T = 3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.« less

Miniature silicon electronic biological assay chip and applications for rapid battlefield diagnostics

NASA Astrophysics Data System (ADS)

Cunningham, Brian T.; Regan, Robert A.; Clapp, Christopher; Hildebrant, Eric; Weinberg, Marc S.; Williams, John

1999-07-01

Assessing the medical condition of battlefield personnel requires the development of rapid, portable biological diagnostic assays for a wide variety of antigens and enzymes. Ideally, such an assay would be inexpensive, small, and require no added reagents while maintaining the sensitivity and accuracy of laboratory-based assays. In this work, a microelectromechanical (MEMS) based biological assay sensor is presented which is expected to meet the above requirements. The sensor is a thin silicon membrane resonator (SMR) which registers a decrease in resonant frequency when mass is adsorbed onto its surface. By coating the sensor surface with a monolayer of antibody, for example, we have detected the corresponding antigen with a detection resolution of 0.25 ng/ml in phosphate buffer solution. Micromachining techniques are being used to integrate many (64 elements on the first test chip) identical SMR sensors into a single silicon chip which would be capable of simultaneously performing a wide variety of biomedical assays. The sensors require only a small printed circuit board and 8V power supply to operate and provide a readout. The presentation will describe the operation of the SMR sensor, the fabrication of the sensor array, and initial test results using commercially available animal immunoglobulins in laboratory-prepared test solutions.

Antibodies to dendritic neuronal surface antigens in opsoclonus myoclonus ataxia syndrome

PubMed Central

Panzer, Jessica A.; Anand, Ronan; Dalmau, Josep; Lynch, David R.

2015-01-01

Opsoclonus myoclonus ataxia syndrome (OMAS) is an autoimmune disorder characterized by rapid, random, conjugate eye movements (opsoclonus), myoclonus, and ataxia. Given these symptoms, autoantibodies targeting the cerebellum or brainstem could mediate the disease or be markers of autoimmunity. In a subset of patients with OMAS, we identified such autoantibodies, which bind to non-synaptic puncta on the surface of live cultured cerebellar and brainstem neuronal dendrites. These findings implicate autoimmunity to a neuronal surface antigen in the pathophysiology of OMAS. Identification of the targeted antigen(s) could elucidate the mechanisms underlying OMAS and provide a biomarker for diagnosis and response to therapy. PMID:26298330

Surface antigens contribute differently to the pathophysiological features in serotype K1 and K2 Klebsiella pneumoniae strains isolated from liver abscesses.

PubMed

Yeh, Kuo-Ming; Chiu, Sheng-Kung; Lin, Chii-Lan; Huang, Li-Yueh; Tsai, Yu-Kuo; Chang, Jen-Chang; Lin, Jung-Chung; Chang, Feng-Yee; Siu, Leung-Kei

2016-01-01

The virulence role of surface antigens in a single serotype of Klebsiella pneumoniae strain have been studied, but little is known about whether their contribution will vary with serotype. To investigate the role of K and O antigen in hyper-virulent strains, we constructed O and K antigen deficient mutants from serotype K1 STL43 and K2 TSGH strains from patients with liver abscess, and characterized their virulence in according to the abscess formation and resistance to neutrophil phagocytosis, serum, and bacterial clearance in liver. Both of K1 and K2-antigen mutants lost their wildtype resistance to neutrophil phagocytosis and hepatic clearance, and failed to cause abscess formation. K2-antigen mutant became serum susceptible while K1-antigen mutant maintained its resistance to serum killing. The amount of glucuronic acid, indicating the amount of capsular polysaccharide (CPS, K antigen), was inversed proportional to the rate of phagocytosis. O-antigen mutant of serotype K1 strains had significantly more amount of CPS, and more resistant to neutrophil phagocytosis than its wildtype counterpart. O-antigen mutants of serotype K1 and K2 strains lost their wildtype serum resistance, and kept resistant to neutrophil phagocytosis. While both mutants lacked the same O1 antigen, O-antigen mutant of serotype K1 became susceptible to liver clearance and cause mild abscess formation, but its serotype K2 counterpart maintained these wildtype virulence. We conclude that the contribution of surface antigens to virulence of K. pneumoniae strains varies with serotypes.

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

PubMed

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

2017-11-01

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

Detection of hepatitis A viral antigen by radioimmunoassay

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

Hollinger, F.B.; Bradley, D.W.; Maynard, J.E.

1975-11-01

With coded samples, the effectiveness and specificity of a micro-SPIRA procedure for rapidly and quantitatively detecting type A hepatitis-associated antigen in large numbers of specimens from infected liver, stool, or serum has been demonstrated. Samples which were judged to be negative by IEM were found to contain significant levels of HAV antigen by this immunoradiometric technique. The detection of significant levels of HAV antigen in infected chimpanzees supports epidemiologic evidence of viremia during the acute stage of the disease. The results of this study suggest that the diagnosis of type A hepatitis by a convention serologic procedure may now bemore » at hand. (auth)« less

Comparison of a stool antigen detection kit and PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar infections in asymptomatic cyst passers in Iran.

PubMed

Solaymani-Mohammadi, Shahram; Rezaian, Mostafa; Babaei, Zahra; Rajabpour, Azam; Meamar, Ahmad R; Pourbabai, Ahmad A; Petri, William A

2006-06-01

The present study was conducted to compare stool antigen detection with PCR for the diagnosis of Entamoeba sp. infection in asymptomatic cyst passers from Iran. Entamoeba dispar and, in one case, E. moshkovskii were the Entamoeba spp. found in the amebic cyst passers. There was a 100% correlation between the results from the TechLab E. histolytica II stool antigen kit and those from nested PCR. We concluded that E. dispar is much more common in asymptomatic cyst passers in Iran and that antigen detection and PCR are comparable diagnostic modalities.

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.

Agglutination Assays of the Plasmodium falciparum-Infected Erythrocyte.

PubMed

Tan, Joshua; Bull, Peter C

2015-01-01

The agglutination assay is used to determine the ability of antibodies to recognize parasite variant antigens on the surface of Plasmodium falciparum-infected erythrocytes. In this technique, infected erythrocytes are selectively labelled with a DNA-binding fluorescent dye and mixed with antibodies of interest to allow antibody-surface antigen binding. Recognition of surface antigens by the antibodies can result in the formation of agglutinates containing multiple parasite-infected erythrocytes. These can be viewed and quantified using a fluorescence microscope.

Expanding Cancer Detection Using Molecular Imprinting for a Novel Point-of-Care Diagnostic Device

NASA Astrophysics Data System (ADS)

Yu, Yingjie; Rafailovich, Miriam; Wang, Yantian; Kang, Yeona; Zhang, Lingxi; Rigas, Basil; Division of Gastroenterology, School of Medicine Team

2013-03-01

We propose the use of a potentiometric biosensor that incorporates the efficient and specific molecular imprinting (MI) method with a self-assembled monolayer (SAM). We first tested the biosensor using carcinoembryonic antigen, CEA, a biomarker associated with pancreatic cancer. No change in detection efficiency was observed, indicating that the sensor is able to discriminate for the template analyte even in concentrated solution of similar substances. In addition, we use biosensor to discriminate normal fibrinogen and damaged fibrinogen, which is critical for the detection of bleeding disorder. Computer simulations of the protein structure were performed in order to estimate the changes in morphology and determine the sensitivity of the biosensor to conformational changes in the proteins. We found that even small changes in PH can generate rotation of the surface functional groups. Yet, the results show that only when the detection and imprinting conditions are similar, robust signals occurs. Hence we concluded that both morphology and surface chemistry play a role in the recognition.

Expanding Cancer Detection Using Molecular Imprinting for a Novel Point-of-Care Diagnostic Device

NASA Astrophysics Data System (ADS)

Yu, Yingjie; Rafailovich, Miriam; Wang, Yantian; Ranjbaran, Alina; Wang, Tom; Nam, David

2012-02-01

We propose the use of a potentiometric biosensor that incorporates the efficient and specific molecular imprinting (MI) method with a self-assembled monolayer (SAM). We first tested the biosensor using carcinoembryonic antigen, CEA, a biomarker associated with pancreatic cancer. No change in detection efficiency was observed when detection was performed in the presence of 100% serum albumin, indicating that the sensor is able to discriminate for the template analyte even in concentrated solution of similar substances. Computer simulations of the protein structure were performed in order to estimate the changes in morphology and determine the sensitivity of the biosensor to conformational changes in the proteins. We found that even small changes in PH can generate rotation of the surface functional groups, without significant change in the morphology. Yet, the results show that only when the detection and imprinting conditions are similar, robust signals occurs. Hence we concluded that both morphology and surface chemistry play a role in the recognition.

A nonfouling voltammetric immunosensor for the carcinoembryonic antigen based on the use of polyaniline nanowires wrapped with hyaluronic acid.

PubMed

Wang, Jiasheng; Hui, Ni

2018-06-16

A non-fouling electrochemical immunosensor is described for determination of the tumor biomarker carcinoembryonic antigen (CEA). It is based on the use of composite wires made by chemical grafting of hyaluronic acid onto polyaniline nanowires. The modified nanowires possess excellent antifouling property both in single protein solutions and in dilute serum samples. The current of immunoelectrode exhibits a linear response in the 0.01 pg mL -1 to 10,000 pg mL -1 CEA concentration range and 0.0075 pg mL -1 detection limit. This work demonstrates that coating an electrode with hyaluronic acid can largely reduce unspecific adsorption of proteins on the electrode surface. Graphical abstract Schematic of a nonfouling electrochemical immunosensor for the carcinoembryonic antigen. It is based on novel composite wires made through the chemical grafting of easily available hyaluronic acid (HA) onto polyaniline (PANI) nanowires. The HA/PANI demonstrated excellent antifouling property both in single protein solutions and human serum samples.

Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach.

PubMed

Encinas, Paloma; Rodriguez-Milla, Miguel A; Novoa, Beatriz; Estepa, Amparo; Figueras, Antonio; Coll, Julio

2010-09-27

Despite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV). Two-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36). The genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

Validity of rapid antigen detection testing in group A beta-hemolytic streptococcal tonsillopharyngitis.

PubMed

Küçük, Oznur; Biçer, Suat; Giray, Tuba; Cöl, Defne; Erdağ, Gülay Ciler; Gürol, Yeşim; Kaspar, Ciğdem E; Vitrinel, Ayça

2014-02-01

To evaluate the utility of rapid antigen detection testing (RADT) for the diagnosis of group A beta-hemolytic streptococcal tonsillopharyngitis in children, and to detect the sensitivity and specificity of rapid antigen detection of group A beta-hemolytic streptococci from throat specimen compared with throat culture. Rapid antigen detection and throat culture results for group A beta-hemolytic streptococci from outpatients attending university hospital between 1st January 2011 and 31st of December 2011 were evaluated retrospectively. The antigen test negative-throat culture positive patients were investigated for streptococcal carriage. For this purpose, the throat culture results taken from these patients were reviewed after treatment. Eight hundred and ninetytwo children were included in the studywith a mean age of 5.34 y. There were 639 and 253 children in two groups with age of 0-6 and 7-17 y, RADT sensitivity and specificity were found to be 59.5 % and 97.2 %, respectively. The positive predictive value was 87.1 %, whereas negative predictive value was 88.4 %. After treatment of 74 patients with throat culture positive and antigen test negative. Group A beta-hemolytic streptococci were isolated in 12 of them (16.2 %) and accepted as a carrier. The low sensitivity of the RADT may be related to streptococcal carriage in some patients. The throat culture should be repeated after treatment to detect streptococcal carriage.

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

Evaluation of monoclonal antibody-based sandwich direct ELISA (MSD-ELISA) for antigen detection of foot-and-mouth disease virus using clinical samples.

PubMed

Morioka, Kazuki; Fukai, Katsuhiko; Sakamoto, Kenichi; Yoshida, Kazuo; Kanno, Toru

2014-01-01

A monoclonal antibody-based sandwich direct ELISA (MSD-ELISA) method was previously developed for foot-and-mouth disease (FMD) viral antigen detection. Here we evaluated the sensitivity and specificity of two FMD viral antigen detection MSD-ELISAs and compared them with conventional indirect sandwich (IS)-ELISA. The MSD-ELISAs were able to detect the antigen in saliva samples of experimentally-infected pigs for a longer term compared to the IS-ELISA. We also used 178 RT-PCR-positive field samples from cattle and pigs affected by the 2010 type-O FMD outbreak in Japan, and we found that the sensitivities of both MSD-ELISAs were about 7 times higher than that of the IS-ELISA against each sample (P<0.01). In terms of the FMD-positive farm detection rate, the sensitivities of the MSD-ELISAs were about 6 times higher than that of the IS-ELISA against each farm (P<0.01). Although it is necessary to conduct further validation study using the other virus strains, MSD-ELISAs could be appropriate as a method to replace IS-ELISA for FMD antigen detection.

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

PubMed

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

2017-10-24

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

Detection of proliferating cell nuclear antigens and interleukin-2 beta receptor molecules on mitogen- and antigen-stimulated lymphocytes.

PubMed Central

Hesketh, J; Dobbelaere, D; Griffin, J F; Buchan, G

1993-01-01

The expression of interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) were compared for their usefulness as markers of lymphocyte activation. Heterologous polyclonal (anti-bovine IL-2R) and monoclonal (anti-human PCNA) antibodies were used to detect the expression of these molecules on activated deer lymphocytes. Both molecules were co-expressed on blast cells which had been activated with mitogen [concanavalin A (Con A)]. There was detectable up-regulation of IL-2R expression in response to antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] stimulation while PCNA expression mimicked lymphocyte transformation (LT) reactivity. PCNA expression was found to more accurately reflect both antigen- and mitogen-activated lymphocyte activation, as estimated by LT activity. The expression of PCNA was used to identify antigen reactive cells from animals exposed to M. bovis. A very low percentage (1.1 +/- 0.4%) of peripheral blood lymphocytes from non-infected animals could be stimulated to express PCNA by in vitro culture with antigen (PPD). Within the infected group both diseased and healthy, 'in-contact', animals expressed significantly higher levels of PCNA upon antigen stimulation. PMID:8104884

Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation.

PubMed

Courtney, Adam H; Puffer, Erik B; Pontrello, Jason K; Yang, Zhi-Qiang; Kiessling, Laura L

2009-02-24

CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling.

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

[Research advances of genomic GYP coding MNS blood group antigens].

PubMed

Liu, Chang-Li; Zhao, Wei-Jun

2012-02-01

The MNS blood group system includes more than 40 antigens, and the M, N, S and s antigens are the most significant ones in the system. The antigenic determinants of M and N antigens lie on the top of GPA on the surface of red blood cells, while the antigenic determinants of S and s antigens lie on the top of GPB on the surface of red blood cells. The GYPA gene coding GPA and the GYPB gene coding GPB locate at the longarm of chromosome 4 and display 95% homologus sequence, meanwhile both genes locate closely to GYPE gene that did not express product. These three genes formed "GYPA-GYPB-GYPE" structure called GYP genome. This review focuses on the molecular basis of genomic GYP and the variety of GYP genome in the expression of diversity MNS blood group antigens. The molecular basis of Miltenberger hybrid glycophorin polymorphism is specifically expounded.

Use of Sera from Humans and Dolphins with Lacaziosis and Sera from Experimentally Infected Mice for Western Blot Analyses of Lacazia loboi Antigens▿

PubMed Central

Mendoza, Leonel; Belone, Andréa F. F.; Vilela, Raquel; Rehtanz, Manuela; Bossart, Gregory D.; Reif, John S.; Fair, Patricia A.; Durden, Wendy N.; St. Leger, Judy; Travassos, Luiz R.; Rosa, Patricia S.

2008-01-01

Antibodies in the sera of patients with lacaziosis recognized an ∼193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera. PMID:17959822

Cross-reactivity among antigens of different air-borne fungi detected by ELISA using five monoclonal antibodies against Penicillium notatum.

PubMed

Shen, H D; Lin, W L; Chen, R J; Han, S H

1990-10-01

Cross-reactivity among antigens of 12 genera of air-borne fungi, 13 species of Penicillium, and 5 species of Aspergillus was studied by ELISA using five monoclonal antibodies (MoAbs) against Penicillium notatum. Epitopes recognized by all the five MoAbs were susceptible to treatment of mild periodate oxidation and may therefore be associated with carbohydrates. Furthermore, our results showed that there is cross-reactivity among antigens of Penicillium, Aspergillus, and Eurotium species. By using these MoAbs, cross reactivity was not detected between antigens of Penicillium notatum and antigens of Fusarium solani, Alternaria porri, Cladosporium cladosporoides, Curvularia species, Nigrospora species, Aureobasidium pullulans, Wallemia species, Rhizopus arrhizus, and Candida albicans. Cross-reactivity among antigens of 11 species of Penicillium and 5 species of Aspergillus could be detected by ELISA using one of the five MoAbs (MoAb P15). The fact that there may be cross-reactivity among antigens of closely related fungi species should be considered in the diagnosis and treatment of mold allergic diseases.

Plasmodium falciparum Malaria in the Peruvian Amazon, a Region of Low Transmission, Is Associated with Immunologic Memory

PubMed Central

Clark, Eva H.; Silva, Claudia J.; Weiss, Greta E.; Li, Shanping; Padilla, Carlos; Crompton, Peter D.; Hernandez, Jean N.

2012-01-01

The development of clinical immunity to Plasmodium falciparum malaria is thought to require years of parasite exposure, a delay often attributed to difficulties in developing protective antibody levels. In this study, we evaluated several P. falciparum vaccine candidate antigens, including apical membrane antigen 1 (AMA-1), circumsporozoite protein (CSP), erythrocyte binding antigen 175 (EBA-175), and the 19-kDa region of merozoite surface protein 1 (MSP119). After observing a more robust antibody response to MSP119, we evaluated the magnitude and longevity of IgG responses specific to this antigen in Peruvian adults and children before, during, and after P. falciparum infection. In this low-transmission region, even one reported prior infection was sufficient to produce a positive anti-MSP119 IgG response for >5 months in the absence of reinfection. We also observed an expansion of the total plasmablast (CD19+ CD27+ CD38high) population in the majority of individuals shortly after infection and detected MSP1-specific memory B cells in a subset of individuals at various postinfection time points. This evidence supports our hypothesis that effective antimalaria humoral immunity can develop in low-transmission regions. PMID:22252876

Plasmodium falciparum malaria in the Peruvian Amazon, a region of low transmission, is associated with immunologic memory.

PubMed

Clark, Eva H; Silva, Claudia J; Weiss, Greta E; Li, Shanping; Padilla, Carlos; Crompton, Peter D; Hernandez, Jean N; Branch, OraLee H

2012-04-01

The development of clinical immunity to Plasmodium falciparum malaria is thought to require years of parasite exposure, a delay often attributed to difficulties in developing protective antibody levels. In this study, we evaluated several P. falciparum vaccine candidate antigens, including apical membrane antigen 1 (AMA-1), circumsporozoite protein (CSP), erythrocyte binding antigen 175 (EBA-175), and the 19-kDa region of merozoite surface protein 1 (MSP1(19)). After observing a more robust antibody response to MSP1(19), we evaluated the magnitude and longevity of IgG responses specific to this antigen in Peruvian adults and children before, during, and after P. falciparum infection. In this low-transmission region, even one reported prior infection was sufficient to produce a positive anti-MSP1(19) IgG response for >5 months in the absence of reinfection. We also observed an expansion of the total plasmablast (CD19(+) CD27(+) CD38(high)) population in the majority of individuals shortly after infection and detected MSP1-specific memory B cells in a subset of individuals at various postinfection time points. This evidence supports our hypothesis that effective antimalaria humoral immunity can develop in low-transmission regions.

Clinical and laboratory features of patients with an inherited deficiency of neutrophil membrane complement receptor type 3 (CR3) and the related membrane antigens LFA-1 and p150,95.

PubMed

Ross, G D

1986-03-01

Over the last 3 years a group of more than 20 patients has been described worldwide who have a similar history of recurrent bacterial infections and an inherited deficiency of three related leukocyte membrane surface antigens known as CR3, LFA-1 (lymphocyte function-associated antigen type 1), and p150,95 (function unknown). These antigens share a common beta-chain structure linked noncovalently to one of three distinct alpha-chain types. It is believed that the patients with this disease have a reduced or absent ability to synthesize the common beta subunit of the antigen family, resulting in absent or reduced expression of all three antigen family members on different leukocyte types. Neutrophils have a reduced phagocytic and respiratory burst response to bacteria and yeast as well as a reduced ability to adhere to various substrates and migrate into sites of infection. In vitro functional studies of normal neutrophils, monocytes, and lymphocytes treated with monoclonal antibodies to the individual alpha and beta chains of these antigens suggest that most of the clinical features of the patients may be due to the neutrophil and monocyte deficiency of CR3. Although natural killer-cell activity is diminished or absent, no immune deficiency of the patients' lymphocytes attributable to the absence of LFA-1 has been detected. Diagnosis of this disease has been facilitated by the commercial availability of monoclonal antibodies specific for the alpha chains of CR3 and p150,95.

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

A nanoforest structure for practical surface-enhanced Raman scattering substrates

NASA Astrophysics Data System (ADS)

Seol, Myeong-Lok; Choi, Sung-Jin; Baek, David J.; Park, Tae Jung; Ahn, Jae-Hyuk; Lee, Sang Yup; Choi, Yang-Kyu

2012-03-01

A nanoforest structure for surface-enhanced Raman scattering (SERS) active substrates is fabricated and analyzed. The detailed morphology of the resulting structure can be easily controlled by modifying the process parameters such as initial gold layer thickness and etching time. The applicability of the nanoforest substrate as a label-free SERS immunosensor is demonstrated using influenza A virus subtype H1N1. Selective binding of the H1N1 surface antigen and the anti-H1 antibody is directly detected by the SERS signal differences. Simple fabrication and high throughput with strong in-plane hot-spots imply that the nanoforest structure can be a practical sensing component of a chip-based SERS sensing system.

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

ZnO thin film transistor immunosensor with high sensitivity and selectivity

NASA Astrophysics Data System (ADS)

Reyes, Pavel Ivanoff; Ku, Chieh-Jen; Duan, Ziqing; Lu, Yicheng; Solanki, Aniruddh; Lee, Ki-Bum

2011-04-01

A zinc oxide thin film transistor-based immunosensor (ZnO-bioTFT) is presented. The back-gate TFT has an on-off ratio of 108 and a threshold voltage of 4.25 V. The ZnO channel surface is biofunctionalized with primary monoclonal antibodies that selectively bind with epidermal growth factor receptor (EGFR). Detection of the antibody-antigen reaction is achieved through channel carrier modulation via pseudo double-gating field effect caused by the biochemical reaction. The sensitivity of 10 fM detection of pure EGFR proteins is achieved. The ZnO-bioTFT immunosensor also enables selectively detecting 10 fM of EGFR in a 5 mg/ml goat serum solution containing various other proteins.

Piezoresistive measurement of Swine H1N1 Hemagglutinin peptide binding with microcantilever arrays

NASA Astrophysics Data System (ADS)

Bajwa, N.; Maldonado, C. J.; Thundat, T.; Passian, A.

2014-03-01

Effective detection of Swine H1N1 Hemagglutinin peptide is crucial as it could be used as a positive control to screen for highly infectious flu strains such as Swine-Origin Influenza A (H1N1). Piezoresistive microcantilever arrays present a pathway towards highly sensitive and label-free detection of biomolecules by transducing the antigen-antibody binding into change in resistivity via induced surface stress variation. We demonstrate a mechanical transduction of Swine H1N1 Hemagglutinin peptide binding and suggest the employed technique may offer a potential platform for detection of the H1N1 virus, which could be clinically used to diagnose and provide subsequent relief.

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.

Immunotherapy for B-Cell Neoplasms using T Cells expressing Chimeric Antigen Receptors

PubMed Central

Boulassel, Mohamed-Rachid; Galal, Ahmed

2012-01-01

Immunotherapy with T cells expressing chimeric antigen receptors (CAR) is being evaluated as a potential treatment for B-cell neoplasms. In recent clinical trials it has shown promising results. As the number of potential candidate antigens expands, the choice of suitable target antigens becomes more challenging to design studies and to assess optimal efficacy of CAR. Careful evaluation of candidate target antigens is required to ensure that T cells expressing CAR will preferentially kill malignant cells with a minimal toxicity against normal tissues. B cells express specific surface antigens that can theoretically act as targets for CAR design. Although many of these antigens can stimulate effective cellular immune responses in vivo, their implementation in clinical settings remains a challenge. Only targeted B-cell antigens CD19 and CD20 have been tested in clinical trials. This article reviews exploitable B cell surface antigens for CAR design and examines obstacles that could interfere with the identification of potentially useful cellular targets. PMID:23269948

Detection of influenza antigenic variants directly from clinical samples using polyclonal antibody based proximity ligation assays

PubMed Central

Martin, Brigitte E.; Jia, Kun; Sun, Hailiang; Ye, Jianqiang; Hall, Crystal; Ware, Daphne; Wan, Xiu-Feng

2016-01-01

Identification of antigenic variants is the key to a successful influenza vaccination program. The empirical serological methods to determine influenza antigenic properties require viral propagation. Here a novel quantitative PCR-based antigenic characterization method using polyclonal antibody and proximity ligation assays, or so-called polyPLA, was developed and validated. This method can detect a viral titer that is less than 1000 TCID50/mL. Not only can this method differentiate between different HA subtypes of influenza viruses but also effectively identify antigenic drift events within the same HA subtype of influenza viruses. Applications in H3N2 seasonal influenza data showed that the results from this novel method are consistent with those from the conventional serological assays. This method is not limited to the detection of antigenic variants in influenza but also other pathogens. It has the potential to be applied through a large-scale platform in disease surveillance requiring minimal biosafety and directly using clinical samples. PMID:25546251

Detection of varicella-zoster virus antigens in lesional skin of zosteriform lichen planus but not in that of linear lichen planus.

PubMed

Mizukawa, Y; Horie, C; Yamazaki, Y; Shiohara, T

2012-01-01

Distinctions between 'linear lichen planus' (LP) and 'zosteriform LP' are difficult to determine solely based on clinical findings. The aim of this study is to determine whether the presence of the varicella-zoster virus (VZV) antigens could be used to differentiate the zosteriform LP from the linear LP. We immunohistochemically investigated the presence of in vivo localization of VZV antigens in 8 LP lesions (zosteriform LP: n = 5, linear LP: n = 3). We describe 2 cases of zosteriform LP without apparent prior episodes of herpes zoster, in whom VZV antigens were detected in the eccrine epithelium. Further analysis showed that VZV antigens were exclusively detected in the eccrine epithelium in the zosteriform LP lesions, but not in the linear LP lesions. Etiological differences exist between zosteriform LP and linear LP. The presence of VZV antigens in lesional skin of the former indicates a possible triggering role of this virus in the pathogenesis of this variant. Copyright © 2012 S. Karger AG, Basel.

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires.

PubMed

DeKosky, Brandon J; Lungu, Oana I; Park, Daechan; Johnson, Erik L; Charab, Wissam; Chrysostomou, Constantine; Kuroda, Daisuke; Ellington, Andrew D; Ippolito, Gregory C; Gray, Jeffrey J; Georgiou, George

2016-05-10

Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19(+)CD20(+)CD27(-) IgM-naive B cells, >120,000 antibody clusters from CD19(+)CD20(+)CD27(+) antigen-experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ-Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease.

Immunohistochemical detection and correlation between MHC antigen and cell-mediated immune system in recurrent glioma by APAAP method.

PubMed

Miyagi, K; Ingram, M; Techy, G B; Jacques, D B; Freshwater, D B; Sheldon, H

1990-09-01

As part of an on-going clinical trial of immunotherapy for recurrent malignant gliomas, using alkaline phosphatase-anti-alkaline phosphatase method with monoclonal antibodies, we investigated the correlation between expression of the major histocompatibility complex (MHC) and the subpopulation of tumor-infiltrating lymphocytes (TILs) in 38 glioma specimens (20 grade IV, 11 grade III, and 7 grade II) from 33 patients. Thirty specimens (78.9%) were positive to class I MHC antigen and 20 (52.6%) were positive to class II MHC antigen. The correlations between class I MHC antigen expression and the number of infiltrating T8 (p less than 0.01), and also between class II MHC antigen expression and the number of infiltrating T4 (p less than 0.05) were significant. We conclude that TILs are the result of immunoreaction (host-defense mechanism). 31.6% of specimens had perivascular infiltration of T cells. The main infiltrating lymphocyte subset in moderate to marked perivascular cuffing was T4. Our results may indicate that lack of MHC antigen on the glioma cell surface has a share in the poor immunogenicity in glioma-bearing patients. In addition, considering the effector/target ratio, the number of infiltrating lymphocytes against glioma cells was too small, so the immunological intervention seems to be essential in glioma therapy. Previous radiation therapy and chemotherapy, including steroid therapy, did not influence lymphocyte and macrophage infiltration.

Nature of immobilization surface affects antibody specificity to placental alkaline phosphatase.

PubMed

Kumar, Mukesh; Khan, Imran; Sinha, Subrata

2015-01-01

Retention of native conformation of immobilized protein is essential for various applications including selection and detection of specific recombinant antibodies (scFvs). Placental alkaline phosphatase (PAP), an onco-fetal antigen expressed on the surface of several tumors, was immobilized on supermagnetic particles for selection of recombinant antibodies from a human phage display antibody library. The isolated antibodies were found to be cross-reactive to either of the isozymes of alkaline phosphatase, i.e., bone alkaline phosphatase (BAP) or intestinal alkaline phosphatase (IAP) and could not be used for tumor targeting. A specific anti-PAP monoclonal antibody H17E2 was tested for retention of specificity under these conditions. Binding of the antibody to magnetic beads conjugated IAP and BAP along with PAP and the ability of the two isozymes to inhibit its binding to PAP depicted the loss of isozyme specificity of the antibody. However, the antibody retained its specificity to PAP immobilized on polyvinyl chloride (PVC) surface. Enzyme activity was observed on both surfaces. This demonstrates that nature of immobilization may affect antigen-antibody binding in subtle ways, resulting in alteration of conformation of the epitopes. This may have consequences for determining the specificity of antibody binding for proteins that share a high degree of homology.

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

PubMed

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

2017-11-09

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

Deletion and anergy of polyclonal B cells specific for ubiquitous membrane-bound self-antigen

PubMed Central

Taylor, Justin J.; Martinez, Ryan J.; Titcombe, Philip J.; Barsness, Laura O.; Thomas, Stephanie R.; Zhang, Na; Katzman, Shoshana D.; Jenkins, Marc K.

2012-01-01

B cell tolerance to self-antigen is critical to preventing antibody-mediated autoimmunity. Previous work using B cell antigen receptor transgenic animals suggested that self-antigen–specific B cells are either deleted from the repertoire, enter a state of diminished function termed anergy, or are ignorant to the presence of self-antigen. These mechanisms have not been assessed in a normal polyclonal repertoire because of an inability to detect rare antigen-specific B cells. Using a novel detection and enrichment strategy to assess polyclonal self-antigen–specific B cells, we find no evidence of deletion or anergy of cells specific for antigen not bound to membrane, and tolerance to these types of antigens appears to be largely maintained by the absence of T cell help. In contrast, a combination of deleting cells expressing receptors with high affinity for antigen with anergy of the undeleted lower affinity cells maintains tolerance to ubiquitous membrane-bound self-antigens. PMID:23071255

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining1

PubMed Central

Behbehani, Gregory K.; Thom, Colin; Zunder, Eli R.; Finck, Rachel; Gaudilliere, Brice; Fragiadakis, Gabriela K.; Fantl, Wendy J.; Nolan, Garry P.

2015-01-01

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression. PMID:25274027

Mapping antigenic motifs in the trypomastigote small surface antigen from Trypanosoma cruzi.

PubMed

Balouz, Virginia; Cámara, María de Los Milagros; Cánepa, Gaspar E; Carmona, Santiago J; Volcovich, Romina; Gonzalez, Nicolás; Altcheh, Jaime; Agüero, Fernán; Buscaglia, Carlos A

2015-03-01

The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mapping Antigenic Motifs in the Trypomastigote Small Surface Antigen from Trypanosoma cruzi

PubMed Central

Balouz, Virginia; Cámara, María de los Milagros; Cánepa, Gaspar E.; Carmona, Santiago J.; Volcovich, Romina; Gonzalez, Nicolás; Altcheh, Jaime; Agüero, Fernán

2015-01-01

The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease. PMID:25589551

Immuno-analysis of microparticles: probing at the limits of detection

PubMed Central

Latham, Sharissa L.; Tiberti, Natalia; Gokoolparsadh, Naveena; Holdaway, Karen; Olivier Couraud, Pierre; Grau, Georges E. R.; Combes, Valery

2015-01-01

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30–40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data. PMID:26553743

A photoelectrochemical immunosensor for detection of α-fetoprotein based on Au-ZnO flower-rod heterostructures

NASA Astrophysics Data System (ADS)

Han, Zhizhong; Luo, Min; Chen, Li; Chen, Jinghua; Li, Chunyan

2017-04-01

In this work, a novel label free photoelectrochemical (PEC) immunosensor has been developed for the detection of α-fetoprotein (AFP). The immunosensor was based on Au-ZnO flower-rods (FRs) heterostructure, where Au nanoparticles (NPs) were firstly electrodeposited by cyclic voltammetry methods. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Mott-Schottky plot (MS), UV-vis diffuse reflectance spectrum and fluorescence emission spectrum were used for the characterizations of Au-ZnO FRs. The results demonstrated that Au NPs not only obviously enhanced the visible light absorption of ZnO FRs due to surface plasmon resonance (SPR) but also improved the separation of photo-generated electron-hole pairs. Therefore, the photocurrent of Au-ZnO FRs was increased under simulated sunlight. The photocurrent was reduced after the specific antibody-antigen immune reaction. And the photocurrent decrement was linear with the logarithm of AFP antigen concentration in the range from 0.005 ng mL-1 to 50 ng mL-1 with a low detection limit of 0.56 pg mL-1 (S/N = 3). The PEC immunosensor also exhibited high anti-interference property and acceptable stability. This work would provide a promising photoelectrochemical strategy for the detection of other proteins in clinical diagnosis.

Abbott prism: a multichannel heterogeneous chemiluminescence immunoassay analyzer.

PubMed

Khalil, O S; Zurek, T F; Tryba, J; Hanna, C F; Hollar, R; Pepe, C; Genger, K; Brentz, C; Murphy, B; Abunimeh, N

1991-09-01

We describe a multichannel heterogeneous immunoassay analyzer in which a sample is split between disposable reaction trays in a group of linear tracks. The system's pipettor uses noninvasive sensing of the sample volume and disposable pipet tips. Each assay track has (a) a conveyor belt for moving reaction trays to predetermined functional stations, (b) temperature-controlled tunnels, (c) noncontact transfer of the reaction mixture between incubation and detection wells, and (d) single-photon counting to detect a chemiluminescence (CL) signal from the captured immunochemical product. A novel disposable reaction tray, with separate reaction and detection wells and self-contained fluid removal, is used in conjunction with the transfer device on the track to produce a carryover-free system. The linear immunoassay track has nine predetermined positions for performing individual assay steps. Assay step sequence and timing is selected by changing the location of the assay modules between these predetermined positions. The assay methodology, a combination of microparticle capture and direct detection of a CL signal on a porous matrix, offers excellent sensitivity, specificity, and ease of automation. Immunoassay configurations have been tested for hepatitis B surface antigen and for antibodies to hepatitis B core antigen, hepatitis C virus, human immunodeficiency virus I and II, and human T-cell leukemia virus I and II.

An ultrasensitive electrochemical immunosensor for the detection of prostate-specific antigen based on conductivity nanocomposite with halloysite nanotubes.

PubMed

Li, Yueyuan; Khan, Malik Saddam; Tian, Lihui; Liu, Li; Hu, Lihua; Fan, Dawei; Cao, Wei; Wei, Qin

2017-05-01

A sensitive label-free amperometric electrochemical immunosensor for detection of prostate-specific antigen (PSA) was proposed in this work. The nanocomposite of halloysite nanotubes with polypyrrole shell and palladium nanoparticles (HNTs@PPy-Pd) was used as a novel signal label. The HNTs with adequate hydroxyl groups are economically available raw materials. PPy, as an electrically conducting polymer material, can be absorbed to the surface of HNTs by in situ oxidative polymerization of the pyrrole monomer and form a shell on the HNTs. The shell of PPy could not only improve the conductivity of the nanocomposite but also absorb large amounts of Pd nanoparticles (NPs). The Pd NPs with high electrocatalytic activity toward the reduction of H 2 O 2 and the HNTs@PPy-Pd nanocomposite as the analytical signal label could improve the sensitivity of the immunosensor. Under optimal conditions, the immunosensor showed a low detection limit (0.03 pg/mL) and a wide linear range (0.0001 to 25 ng/mL) of PSA. Moreover, its merits such as good selectivity, acceptable reproducibility, and stability indicate that the fabricated immunosensor has a promising application potential in clinical diagnosis. Graphical Abstract A new label-free amperometric electrochemical immunosensor based on HNTs@PPy-Pd nanocomposite for quantitative detection of PSA.

Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor

PubMed Central

Baca, Justin T.; Severns, Virginia; Lovato, Debbie; Branch, Darren W.; Larson, Richard S.

2015-01-01

Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 104 PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodology has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions. PMID:25875186

The bovine immune response to Brucella abortus I. A water soluble antigen precipitated by sera of some naturally infected cattle.

PubMed Central

Stemshorn, B; Nielsen, K

1977-01-01

Selected sera from cattle naturally infected with Brucella abortus precipitate water soluble antigens extracted by sonication from B. abortus. One of these antigens resembles antigen E (Baughn and Freeman) as it is excluded from Sephadex G-200 gels, migrates anodally when electrophoresed at pH 8.6, resists heating at 100 degrees C for ten minutes and appears to be susceptible to papain digestion. Precipitins specific for this antigen remained in sera from which all detectable Brucella agglutinating antibody had been removed by adsorption with live or heat killed B. abortus. The antigen has been extracted from smooth and rough strains of B abortus. Precipitins specific for this antigen have been detected in antisera produced against Brucella canis. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:405088

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.

Indirect Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Kohl, Thomas O; Ascoli, Carl A

2017-07-05

The indirect competitive ELISA (indirect cELISA) pits plate-immobilized antigen against antigens in solution for binding to antigen-specific antibody. The antigens in solution are in the test sample and are first incubated with antigen-specific antibody. These antibody-antigen complexes are then added to microtiter plates whose wells have been coated with purified antigen. The wells are washed to remove unbound antigen-antibody complexes and free antigen. A reporter-labeled secondary antibody is then added followed by the addition of substrate. Substrate hydrolysis yields a signal that is inversely proportional to antigen concentration within the sample. This is because when antigen concentration is high in the test sample, most of the antibody is bound before adding the solution to the plate. Most of the antibody remains in solution (as complexes) and is thus washed away before the addition of the reporter-labeled secondary antibody and substrate. Thus, the higher the antigen concentration in the test sample, the weaker the resultant signal in the detection step. The indirect cELISA is often used for competitive detection and quantification of antibodies against viral diseases in biological samples. © 2017 Cold Spring Harbor Laboratory Press.

Platinum porous nanoparticles hybrid with metal ions as probes for simultaneous detection of multiplex cancer biomarkers.

PubMed

Wang, Zifeng; Liu, Na; Ma, Zhanfang

2014-03-15

In this work, platinum porous nanoparticles (PtPNPs) absorbed metal ions as electrochemical signals were fabricated. Clean-surface PtPNPs were prepared by a surfactant-free method and decorated with amino groups via 2-aminoethanethiol. Amino capped PtPNPs complexation with Cd(2+) and Cu(2+) to form PtPNPs-Cd(2+) and PtPNPs-Cu(2+) hybrids, respectively. Anti-CEA and Anti-AFP separately labeled with PtPNPs-Cd(2+) and PtPNPs-Cu(2+) were used as distinguishable signal tags for capturing antigens. The metal ions were detected in a single run through differential pulse voltammetry (DPV) without acid dissolution, electric potentials and peak heights of which reflected the identity and concentrations of the corresponding antigen. Ionic liquid reduced graphene oxide (IL-rGO) modified glassy carbon electrode (GCE) was used as a substrate, which was rich in amino groups to immobilize antibodies by glutaraldehyde through cross-link between aldehyde groups and amino groups. Using the proposed probes and platform, a novel sandwich-type electrochemical immunosensor for simultaneous detecting carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) was successfully developed. This immunoassay possessed good linearity from 0.05 ng mL(-1) to 200 ng mL(-1) for both CEA and AFP. The detection limit of CEA was 0.002 ng mL(-1) and that of AFP was 0.05 ng mL(-1) (S/N=3). Furthermore, analysis of clinical serum samples using this immunosensor was well consistent with the data determined by the enzyme-linked immunosorbent assay (ELISA). It suggested that the proposed electrochemical immunoassay provided a potential application of clinical screening for early-stage cancers. © 2013 Published by Elsevier B.V.

Blood characterization using UV/vis spectroscopy

NASA Astrophysics Data System (ADS)

Mattley, Yvette D.; Mitrani-Gold, F.; Orton, S.; Bacon, Christina P.; Leparc, German F.; Bayona, M.; Potter, Robert L.; Garcia-Rubio, Luis H.

1995-05-01

The current methods used for typing blood involve an agglutination reaction which results from the association of specific antibodies with antigens present on the erythrocyte cell surface. While this method is effective, it requires involved laboratory procedures to detect the cell surface antigens. As an alternative technique, uv/vis spectroscopy has been investigated as a novel way to characterize and differentiate the blood types. Typing with this technique is based on spectral differences which appear throughout portions of both the ultraviolet and visible range. The origin of these spectral differences is unknown and presently under investigation. They may be due to intrinsic absorption differences at the molecular level, and/or they may be due to scattering differences brought about by either subtle variation in cell surface characteristics, cell shape or state of aggregation. As the background optical density in these samples is identified and accounted for, the spectral differences become more defined. This work and the continuation of this project will be included in a general database encompassing a wide range of blood samples. In addition, long term goals involve the investigation of diseased blood with the potential of providing a more rapid diagnosis for blood borne pathogens.

Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

PubMed

Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

1989-04-01

Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

Evaluation of mucosal adjuvants and immunization routes for the induction of systemic and mucosal humoral immune responses in macaques.

PubMed

Veazey, Ronald S; Siddiqui, Asna; Klein, Katja; Buffa, Viviana; Fischetti, Lucia; Doyle-Meyers, Lara; King, Deborah F; Tregoning, John S; Shattock, Robin J

2015-01-01

Delivering vaccine antigens to mucosal surfaces is potentially very attractive, especially as protection from mucosal infections may be mediated by local immune responses. However, to date mucosal immunization has had limited successes, with issues of both safety and poor immunogenicity. One approach to improve immunogenicity is to develop adjuvants that are effective and safe at mucosal surfaces. Differences in immune responses between mice and men have overstated the value of some experimental adjuvants which have subsequently performed poorly in the clinic. Due to their closer similarity, non-human primates can provide a more accurate picture of adjuvant performance. In this study we immunised rhesus macaques (Macaca mulatta) using a unique matrix experimental design that maximised the number of adjuvants screened while reducing the animal usage. Macaques were immunised by the intranasal, sublingual and intrarectal routes with the model protein antigens keyhole limpet haemocyanin (KLH), β-galactosidase (β-Gal) and ovalbumin (OVA) in combination with the experimental adjuvants Poly(I:C), Pam3CSK4, chitosan, Thymic Stromal Lymphopoietin (TSLP), MPLA and R848 (Resiquimod). Of the routes used, only intranasal immunization with KLH and R848 induced a detectable antibody response. When compared to intramuscular immunization, intranasal administration gave slightly lower levels of antigen specific antibody in the plasma, but enhanced local responses. Following intranasal delivery of R848, we observed a mildly inflammatory response, but no difference to the control. From this we conclude that R848 is able to boost antibody responses to mucosally delivered antigen, without causing excess local inflammation.

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.

Real-time quantification of antibody-short interfering RNA conjugate in serum by antigen capture reverse transcription-polymerase chain reaction.

PubMed

Tan, Martha; Vernes, Jean-Michel; Chan, Joyce; Cuellar, Trinna L; Asundi, Aarati; Nelson, Christopher; Yip, Victor; Shen, Ben; Vandlen, Richard; Siebel, Christian; Meng, Y Gloria

2012-11-15

Short interfering RNA (siRNA) has therapeutic potential. However, efficient delivery is a formidable task. To facilitate delivery of siRNA into cells, we covalently conjugated siRNA to antibodies that bind to cell surface proteins and internalize. Understanding how these antibody-siRNA conjugates function in vivo requires pharmacokinetic analysis. Thus, we developed a simple real-time antigen capture reverse transcription-polymerase chain reaction (RT-PCR) assay to detect intact antibody-siRNA conjugates. Biotinylated antigen bound to streptavidin-coated PCR tubes was used to capture antibody-siRNA conjugate. The captured antibody-siRNA conjugate was then reverse-transcribed in the same tube, avoiding a sample transfer step. This reproducible assay had a wide standard curve range of 0.029 to 480ng/ml and could detect as low as 0.58ng/ml antibody-siRNA conjugates in mouse serum. The presence of unconjugated antibody that could be generated from siRNA degradation in vivo did not affect the assay as long as the total antibody concentration in the antigen capture step did not exceed 480ng/ml. Using this assay, we observed a more rapid decrease in serum antibody-siRNA conjugate concentrations than the total antibody concentrations in mice dosed with antibody-siRNA conjugates, suggesting loss of siRNA from the antibody. This assay is useful for optimizing antibody-siRNA and likely aptamer-siRNA conjugates to improve pharmacokinetics and aid siRNA delivery. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular interferometric imaging study of molecular interactions

NASA Astrophysics Data System (ADS)

Zhao, Ming; Wang, Xuefeng; Nolte, David

2008-02-01

Molecular Interferometric Imaging (MI2) is a sensitive detection platform for direct optical detection of immobilized biomolecules. It is based on inline common-path interferometry combined with far-field optical imaging. The substrate is a simple thermal oxide on a silicon surface with a thickness at or near the quadrature condition that produces a π/2 phase shift between the normal-incident wave reflected from the top oxide surface and the bottom silicon surface. The presence of immobilized or bound biomolecules on the surface produces a relative phase shift that is converted to a far-field intensity shift and is imaged by a reflective microscope onto a CCD camera. Shearing interferometry is used to remove the spatial 1/f noise from the illumination to achieve shot-noise-limited detection of surface dipole density profiles. The lateral resolution of this technique is diffraction limited at 0.4 micron, and the best longitudinal resolution is 10 picometers. The minimum detectable mass at the metrology limit is 2 attogram, which is 8 antibody molecules of size 150 kDa. The corresponding scaling mass sensitivity is 5 fg/mm compared with 1 pg/mm for typical SPR sensitivity. We have applied MI2 to immunoassay applications, and real-time binding kinetics has been measured for antibody-antigen reactions. The simplicity of the substrate and optical read-out make MI2 a promising analytical assay tool for high-throughput screening and diagnostics.

Ochratoxin A Detection on Antibody- Immobilized on BSA-Functionalized Gold Electrodes.

PubMed

Badea, Mihaela; Floroian, Laura; Restani, Patrizia; Cobzac, Simona Codruta Aurora; Moga, Marius

2016-01-01

Ochratoxin A (OTA)-a toxin produced by Aspergillus carbonarius, Aspergillus ochraceus, and Penicillium verrucosum-is one of the most-abundant food-contaminating mycotoxins. To avoid the risk of OTA consumption for humans and animals, the rapid detection and quantitation of OTA level in different commodities are of great importance. In this work, an impedimetric immunosensor for ochratoxin A (OTA) detection, a common toxic botanical contaminant, was developed via the immobilization of anti-OTA antibody on bovine serum albumin modified gold electrodes. A four-step reaction protocol was tested to modify the gold electrode and obtain the sensing substrate. All the steps of the immunosensor elaboration and also the immunochemical reaction between surface-bound antibody and ochratoxin A were analyzed using cyclic voltammetry and electrochemical impedance spectroscopy. Modification of the impedance due to the specific antigen-antibody reaction at immunosensor surface, was used in order to detect ochratoxin A. Linear proportionality of the charge transfer resistance to the concentration of OTA allows ochratoxin A detection in the range of 2.5-100 ng/mL.

T-Cell Surface Antigens and sCD30 as Biomarkers of the Risk of Rejection in Solid Organ Transplantation.

PubMed

Wieland, Eberhard; Shipkova, Maria

2016-04-01

T-cell activation is a characteristic of organ rejection. T cells, located in the draining lymph nodes of the transplant recipient, are faced with non-self-molecules presented by antigen presenting cells and become activated. Activated T cells are characterized by up-regulated surface antigens, such as costimulatory molecules, adhesion molecules, chemokine receptors, and major histocompatibility complex class II molecules. Surface antigen expression can be followed by flow cytometry using monoclonal antibodies in either cell function assays using donor-specific or nonspecific stimulation of isolated cells or whole blood and without stimulation on circulating lymphocytes. Molecules such as CD30 can be proteolytically cleaved off the surface of activated cells in vivo, and the determination of the soluble protein (sCD30) in serum or plasma is performed by immunoassays. As promising biomarkers for rejection and long-term transplant outcome, CD28 (costimulatory receptor for CD80 and CD86), CD154 (CD40 ligand), and sCD30 (tumor necrosis factor receptor superfamily, member 8) have been identified. Whereas cell function assays are time-consuming laboratory-developed tests which are difficult to standardize, commercial assays are frequently available for soluble proteins. Therefore, more data from clinical trials have been published for sCD30 compared with the surface antigens on activated T cells. This short review summarizes the association between selected surface antigens and immunosuppression, and rejection in solid organ transplantation.

Rational Design of Peptide-Functionalized Surface Plasmon Resonance Sensor for Specific Detection of TNT Explosive.

PubMed

Wang, Jin; Muto, Masaki; Yatabe, Rui; Onodera, Takeshi; Tanaka, Masayoshi; Okochi, Mina; Toko, Kiyoshi

2017-09-30

In this study, a rationally-designed 2,4,6-trinitrotoluene (TNT) binding peptide derived from an amino acid sequence of the complementarity-determining region (CDR) of an anti-TNT monoclonal antibody was used for TNT detection based on a maleimide-functionalized surface plasmon resonance (SPR) sensor. By antigen-docking simulation and screening, the TNT binding candidate peptides were obtained as TNTHCDR1 derived from the heavy chain of CDR1, TNTHCDR2 derived from CDR2, and TNTHCDR3 from CDR3 of an anti-TNT antibody. The binding events between candidate peptides and TNT were evaluated using the SPR sensor by direct determination based on the 3-aminopropyltriethoxysilane (APTES) surface. The TNT binding peptide was directly immobilized on the maleimide-functionalized sensor chip surface from N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). The results demonstrated that peptide TNTHCDR3 was identified and selected as a TNT binding peptide among the other two candidate peptides. Five kinds of TNT analogues were also investigated to testify the selectivity of TNT binding peptide TNTHCDR3. Furthermore, the results indicated that the APTES-GMBS-based SPR sensor chip procedure featured a great potential application for the direct detection of TNT.

Rational Design of Peptide-Functionalized Surface Plasmon Resonance Sensor for Specific Detection of TNT Explosive

PubMed Central

Wang, Jin; Muto, Masaki; Yatabe, Rui; Onodera, Takeshi; Okochi, Mina; Toko, Kiyoshi

2017-01-01

In this study, a rationally-designed 2,4,6-trinitrotoluene (TNT) binding peptide derived from an amino acid sequence of the complementarity-determining region (CDR) of an anti-TNT monoclonal antibody was used for TNT detection based on a maleimide-functionalized surface plasmon resonance (SPR) sensor. By antigen-docking simulation and screening, the TNT binding candidate peptides were obtained as TNTHCDR1 derived from the heavy chain of CDR1, TNTHCDR2 derived from CDR2, and TNTHCDR3 from CDR3 of an anti-TNT antibody. The binding events between candidate peptides and TNT were evaluated using the SPR sensor by direct determination based on the 3-aminopropyltriethoxysilane (APTES) surface. The TNT binding peptide was directly immobilized on the maleimide-functionalized sensor chip surface from N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). The results demonstrated that peptide TNTHCDR3 was identified and selected as a TNT binding peptide among the other two candidate peptides. Five kinds of TNT analogues were also investigated to testify the selectivity of TNT binding peptide TNTHCDR3. Furthermore, the results indicated that the APTES-GMBS-based SPR sensor chip procedure featured a great potential application for the direct detection of TNT. PMID:28973962

Protein-diazonium adduct direct electrografting onto SPRi-biochip.

PubMed

Corgier, Benjamin P; Bellon, Sophie; Anger-Leroy, Marielle; Blum, Loïc J; Marquette, Christophe A

2009-08-18

A direct protein immobilization method for surface plasmon resonance imaging (SPRi) gold chip arraying is exposed. The biomolecule electroaddressing strategy, previously demonstrated by our team on carbon surfaces, is here valuably involved and adapted to create a straightforward and efficient protein immobilization process onto SPRi-biochips. The proteins, modified with an aryl-diazonium adduct, are addressed to the SPRi chip surface through the electroreduction of the aryl-diazonium. The biomolecule deposition was followed through SPRi live measurements during the electrografting process. A specially designed setup enabled us to directly observe the mass increasing at the sensor surface while the proteins were electrografted. A pin electrospotting method, allowing the achievement of distinct sensing layers on gold SPRi-biochips, was used to generate microarray biochips. The integrity of the immobilized proteins and the specificity of the detection, based on antigen/antibody interactions, were demonstrated for the detection of specific antibodies and ovalbumin. The SPRi detection limit of ovalbumin using the electroaddressing of anti-ovalbumin IgG was compared with two other immobilization procedures, cystamine-glutaraldehyde self-assembled monolayer and pyrrole, and was found to be a decade lower than these ones (100 ng/mL, i.e., 2 nM).

Solid surface fluorescence immunosensor for ultrasensitive detection of hepatitis B virus surface antigen using PAMAM/CdTe@CdS QDs nanoclusters.

PubMed

Babamiri, Bahareh; Hallaj, Rahman; Salimi, Abdollah

2018-06-20

In the present study, we constructed an ultrasensitive solid surface fluorescence-immunosensor based on highly luminescent CdTe@CdS-PAMAM structures as nanoprobe for determination of HBsAg by monitoring fluorescence intensity. This strategy was achieved by using PAMAM as a signal amplifier; the PAMAM dendrimer with the many functional amine groups can amplify the fluorescence signal of QDs by covalent attachment of CdTe@CdS on PAMAM and hence, improve the sensitivity of the proposed method significantly. A sandwich type immunosensor was formed after the addition of HBsAg and the PAMAM-QD-Ab 2 , respectively. Under optimal conditions, the designed immunosensor demonstrates a good analytical performance for the HBsAg detection in an excellent linear range from 5 fg ml -1 to 0.15 ng ml -1 with the detection limit (LOD) of 0.6 fg ml -1 at a S/N ratio of 3. In addition, the analysis of human serum samples shows that the fluorescent immunoassay has the great potential for early diagnosis of hepatitis B and can be used for the detection of other tumor markers in clinical applications.

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

Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

PubMed

Liao, Ting-Yu Angela; Lau, Alice; Joseph, Sunil; Hytönen, Vesa; Hmama, Zakaria

2015-01-01

Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

Distinct galactofuranose antigens in the cell wall and culture supernatants as a means to differentiate Fusarium from Aspergillus species.

PubMed

Wiedemann, Annegret; Kakoschke, Tamara Katharina; Speth, Cornelia; Rambach, Günter; Ensinger, Christian; Jensen, Henrik Elvang; Ebel, Frank

2016-09-01

Detection of carbohydrate antigens is an important means for diagnosis of invasive fungal infections. For diagnosis of systemic Aspergillus infections, galactomannan is commonly used, the core antigenic structure of which consists of chains of several galactofuranose moieties. In this study, we provide evidence that Fusarium produces at least two distinct galactofuranose antigens: Smaller amounts of galactomannan and larger quantities of a novel antigen recognized by the monoclonal antibody AB135-8. In A. fumigatus, only minor amounts of the AB135-8 antigen are found in supernatants and in the apical regions of hyphae. A galactofuranose-deficient A. fumigatus mutant lacks the AB135-8 antigen, which strongly suggests that galactofuranose is an essential constituent of this antigen. Using a combination of AB135-8 and a galactomannan-specific antibody, we were able to unambiguously differentiate A. fumigatus and Fusarium hyphae in immunohistology. Moreover, since Fusarium releases the AB135-8 antigen, it appears to be a promising target antigen for a serological detection of Fusarium infections. Copyright © 2016 Elsevier GmbH. All rights reserved.

Molecular analysis of human papillomavirus virus-like particle activated Langerhans cells in vitro.

PubMed

Woodham, Andrew W; Raff, Adam B; Da Silva, Diane M; Kast, W Martin

2015-01-01

Langerhans cells (LC) are the resident antigen-presenting cells in human epithelium, and are therefore responsible for initiating immune responses against human papillomaviruses (HPV) entering the epithelial and mucosal layers in vivo. Upon proper pathogenic stimulation, LC become activated causing an internal signaling cascade that results in the up-regulation of co-stimulatory molecules and the release of inflammatory cytokines. Activated LC then migrate to lymph nodes where they interact with antigen-specific T cells and initiate an adaptive T-cell response. However, HPV manipulates LC in a suppressive manner that alters these normal maturation responses. Here, in vitro LC activation assays for the detection of phosphorylated signaling intermediates, the up-regulation of activation-associated surface markers, and the release of inflammatory cytokines in response to HPV particles are described.

Detection of hepatitis B virus core antigen by phage display mediated TaqMan real-time immuno-PCR.

PubMed

Monjezi, Razieh; Tan, Sheau Wei; Tey, Beng Ti; Sieo, Chin Chin; Tan, Wen Siang

2013-01-01

The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10ng of HBcAg with 10(8)pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples. Copyright © 2012 Elsevier B.V. All rights reserved.

A comparative Study of Aptasensor Vs Immunosensor for Label-Free PSA Cancer Detection on GQDs-AuNRs Modified Screen-Printed Electrodes.

PubMed

Srivastava, Monika; Nirala, Narsingh R; Srivastava, S K; Prakash, Rajiv

2018-01-31

Label-free and sensitive detection of PSA (Prostate Specific Antigen) is still a big challenge in the arena of prostate cancer diagnosis in males. We present a comparative study for label-free PSA aptasensor and PSA immunosensor for the PSA-specific monoclonal antibody, based on graphene quantum dots-gold nanorods (GQDs-AuNRs) modified screen-printed electrodes. GQDs-AuNRs composite has been synthesized and used as an electro-active material, which shows fast electron transfer and catalytic property. Aptamer or anti-PSA has immobilized onto the surface of modified screen printed electrodes. Three techniques are used simultaneously, viz. cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedence spectroscopy (EIS) to investigate the analytical performance of both PSA aptasensor and PSA immunosensor with its corresponding PSA antigen. Under optimum conditions, both sensors show comparable results with an almost same limit of detection (LOD) of 0.14 ng mL -1 . The results developed with aptasensor and anti-PSA is also checked through the detection of PSA in real samples with acceptable results. Our study suggests some advantages of aptasensor in terms of better stability, simplicity and cost effectiveness. Further our present work shows enormous potential of our developed sensors for real application using voltammetric and EIS techniques simultaneous to get reliable detection of the disease.

Prediction of conformational changes by single mutation in the hepatitis B virus surface antigen (HBsAg) identified in HBsAg-negative blood donors.

PubMed

Ie, Susan I; Thedja, Meta D; Roni, Martono; Muljono, David H

2010-11-18

Selection of hepatitis B virus (HBV) by host immunity has been suggested to give rise to variants with amino acid substitutions at or around the 'a' determinant of the surface antigen (HBsAg), the main target of antibody neutralization and diagnostic assays. However, there have never been successful attempts to provide evidence for this hypothesis, partly because the 3 D structure of HBsAg molecules has not been determined. Tertiary structure prediction of HBsAg solely from its primary amino acid sequence may reveal the molecular energetic of the mutated proteins. We carried out this preliminary study to analyze the predicted HBsAg conformation changes of HBV variants isolated from Indonesian blood donors undetectable by HBsAg assays and its significance, compared to other previously-reported variants that were associated with diagnostic failure. Three HBV variants (T123A, M133L and T143M) and a wild type sequence were analyzed together with frequently emerged variants T123N, M133I, M133T, M133V, and T143L. Based on the Jameson-Wolf algorithm for calculating antigenic index, the first two amino acid substitutions resulted in slight changes in the antigenicity of the 'a' determinant, while all four of the comparative variants showed relatively more significant changes. In the pattern T143M, changes in antigenic index were more significant, both in its coverage and magnitude, even when compared to variant T143L. These data were also partially supported by the tertiary structure prediction, in which the pattern T143M showed larger shift in the HBsAg second loop structure compared to the others. Single amino acid substitutions within or near the 'a' determinant of HBsAg may alter antigenicity properties of variant HBsAg, which can be shown by both its antigenic index and predicted 3 D conformation. Findings in this study emphasize the significance of variant T143M, the prevalent isolate with highest degree of antigenicity changes found in Indonesian blood donors. This highlights the importance of evaluating the effects of protein structure alterations on the sensitivity of screening methods being used in detection of ongoing HBV infection, as well as the use of vaccines and immunoglobulin therapy in contributing to the selection of HBV variants.

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.

Characterisation of surface antigens of Strongylus vulgaris of potential immunodiagnostic importance.

PubMed

Nichol, C; Masterson, W J

1987-08-01

When antigens prepared by detergent washes of Strongylus vulgaris and Parascaris equorum were probed in an enzyme-linked immunosorbent assay test with horse sera from single species infections of S. vulgaris and P. equorum, a high degree of cross-reaction between the species was demonstrated. Western blot analysis of four common horse nematode species showed a large number of common antigens when probed with horse infection sera. Antisera raised in rabbits against the four species, including S. vulgaris, were also found to cross-react considerably. Rabbit anti-S. vulgaris sera were affinity adsorbed over a series of affinity chromatography columns, bound with cross-reactive surface antigens, to obtain S. vulgaris-specific antisera and thereby identify S. vulgaris-specific antigens by Western blotting. These studies revealed potentially specific antigens of apparent molecular weights of 100,000, 52,000, and 36,000. Of these bands, only the 52 kDa and 36 kDa appeared to be found on the surface as judged by 125I-labelling of intact worms by the Iodogen method, although neither protein was immunoprecipitated by horse infection sera. Finally, immunoprecipitation of in vitro translated proteins derived from larval S. vulgaris RNA suggests that two proteins may be parasite-derived. These findings are discussed both with respect to the surface of S. vulgaris and to the use of these species-specific antigens in immunodiagnosis.

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.

Surface antigens of Plasmodium falciparum gametocytes--a new class of transmission-blocking vaccine targets?

PubMed

Sutherland, Colin J

2009-08-01

The re-establishment of elimination and eradication on the malaria control agenda has led to calls for renewed effort in the development of parasite transmission-blocking interventions. Vaccines are ideally suited to this task, but progress towards an anti-gamete transmission-blocking vaccine, designed to act on parasites in blood-fed mosquitoes, has been slow. Recent work has confirmed that the surface of the gametocyte-infected erythrocyte presents antigens to the host immune system, and elicits specific humoral immune responses to these antigens, termed gametocyte surface antigens (GSAs). Likely candidate molecules, including antigens encoded by sub-telomeric multi-gene families, are discussed, and a hypothetical group of parasite molecules involved in spatial and temporal signal transduction in the human host is proposed, the tropins and circadins. The next steps for development of anti-gametocyte transmission-blocking vaccines for P. falciparum and the other human malaria species are considered.

Development of a Strategy Based on the Surface Plasmon Resonance Technology for Platelet Compatibility Testing.

PubMed

Wu, Chang-Lin; He, Jian-An; Gu, Da-Yong; Shao, Chao-Peng; Zhu, Yi; Dang, Xin-Tang

2018-01-01

This study was aimed to establish a novel strategy based on the surface plasmon resonance (SPR) technology for platelet compatibility testing. A novel surface matrix was prepared based on poly (OEGMA-co-HEMA) via surface-initiated polymerization as a biosensor surface platform. Type O universal platelets and donor platelets were immobilized on these novel matrices via amine-coupling reaction and worked as a capturing ligand for binding the platelet antibody. Antibodies binding to platelets were monitored in real time by injecting the samples into a microfluidic channel. Clinical serum samples (n = 186) with multiple platelet transfusions were assayed for platelet antibodies using the SPR technology and monoclonal antibody-immobilized platelet antigen (MAIPA) assay. The novel biosensor surface achieved nonfouling background and high immobilization capacity and showed good repeatability and stability after regeneration. The limit of detection of the SPR biosensor for platelet antibody was estimated to be 50 ng/mL. The sensitivity and specificity were 92% and 98.7%. It could detect the platelet antibody directly in serum samples, and the results were similar to MAIPA assay. A novel strategy to facilitate the sensitive and reliable detection of platelet compatibility for developing an SPR-based biosensor was established in this study. The SPR-based biosensor combined with novel surface chemistry is a promising method for platelet compatibility testing.

Frequency of hepatitis B surface antigen variants (HBsAg) in hepatitis B virus genotype B and C infected East- and Southeast Asian patients: Detection by the Elecsys® HBsAg II assay.

PubMed

Kim, Hyon Suk; Chen, Xinyue; Xu, Min; Yan, Cunling; Liu, Yali; Deng, Haohui; Hoang, Bui Huu; Thuy, Pham Thi Thu; Wang, Terry; Yan, Yiwen; Zeng, Zhen; Gencay, Mikael; Westergaard, Gaston; Pabinger, Stephan; Kriegner, Albert; Nauck, Markus; Seffner, Anja; Gohl, Peter; Hübner, Kirsten; Kaminski, Wolfgang E

2018-06-01

To avoid false negative results, hepatitis B surface antigen (HBsAg) assays need to detect samples with mutations in the immunodominant 'a' determinant region, which vary by ethnographic region. We evaluated the prevalence and type of HBsAg mutations in a hepatitis B virus (HBV)-infected East- and Southeast Asian population, and the diagnostic performance of the Elecsys ® HBsAg II Qualitative assay. We analyzed 898 samples from patients with HBV infection from four sites (China [Beijing and Guangzhou], Korea and Vietnam). HBsAg mutations were detected and sequenced using highly sensitive ultra-deep sequencing and compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the 'a' determinant region using the Elecsys ® HBsAg II Qualitative assay. Overall, 237 distinct amino acid mutations in the major hydrophilic region were identified; mutations were present in 660 of 898 HBV-infected patient samples (73.5%). Within the pool of 237 distinct mutations, the majority of the amino acid mutations were found in HBV genotype C (64.8%). We identified 25 previously unknown distinct mutations, mostly prevalent in genotype C-infected Korean patients (n = 18) followed by Chinese (n = 12) patients. All 898 samples were correctly identified by the Elecsys ® HBsAg II Qualitative assay. We observed 237 distinct (including 25 novel) mutations, demonstrating the complexity of HBsAg variants in HBV-infected East- and Southeast Asian patients. The Elecsys ® HBsAg II Qualitative assay can reliably detect HBV-positive samples and is suitable for routine diagnostic use in East and Southeast Asia. Copyright © 2018 Roche Diagnostics International Ltd. Published by Elsevier B.V. All rights reserved.

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms.

PubMed

Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

2017-04-17

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation. Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments' ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

Feasibility of asymmetrical flow field-flow fractionation as a method for detecting protective antigen by direct recognition of size-increased target-captured nanoprobes.

PubMed

Shin, Kayeong; Choi, Jaeyeong; Cho, Jun-Haeng; Yoon, Moon-Young; Lee, Seungho; Chung, Hoeil

2015-11-27

Asymmetrical flow field-flow fractionation (AF4) was evaluated as a potential analytical method for detection of a protective antigen (PA), an Anthrax biomarker. The scheme was based on the recognition of altered AF4 retention through the generation of the size-increased Au nanoparticle probes as a result of PA binding, in which a PA-selective peptide was conjugated on the probe surface. In the visible absorption-based AF4 fractograms, the band position shifted to a longer retention time as the PA concentration increased due to the presence of probe bound with PAs. The shift was insignificant when the concentration was relatively low at 84.3pM. To improve sensitivity, two separate probes conjugated with two different peptides able to bind on different PA epitopes were used together. The band shift then became distinguishable even at 84.3pM of PA sample. The formation of larger PA-probe inter-connected species using the dual-probe system was responsible for the enhanced band shift. In parallel, the feasibility of surface-enhanced Raman scattering (SERS) as a potential AF4 detection method was also evaluated. In the off-line SERS fractogram constructed using fractions collected during AF4 separation, a band shift was also observed for the 84.3pM PA sample, and the band intensity was higher when using the dual-probe system. The combination of AF4 and SERS is promising for the detection of PA and will become a potential tool if the reproducibility of SERS measurement is improved. Copyright © 2015 Elsevier B.V. All rights reserved.

Rules of co-occurring mutations characterize the antigenic evolution of human influenza A/H3N2, A/H1N1 and B viruses.

PubMed

Chen, Haifen; Zhou, Xinrui; Zheng, Jie; Kwoh, Chee-Keong

2016-12-05

The human influenza viruses undergo rapid evolution (especially in hemagglutinin (HA), a glycoprotein on the surface of the virus), which enables the virus population to constantly evade the human immune system. Therefore, the vaccine has to be updated every year to stay effective. There is a need to characterize the evolution of influenza viruses for better selection of vaccine candidates and the prediction of pandemic strains. Studies have shown that the influenza hemagglutinin evolution is driven by the simultaneous mutations at antigenic sites. Here, we analyze simultaneous or co-occurring mutations in the HA protein of human influenza A/H3N2, A/H1N1 and B viruses to predict potential mutations, characterizing the antigenic evolution. We obtain the rules of mutation co-occurrence using association rule mining after extracting HA1 sequences and detect co-mutation sites under strong selective pressure. Then we predict the potential drifts with specific mutations of the viruses based on the rules and compare the results with the "observed" mutations in different years. The sites under frequent mutations are in antigenic regions (epitopes) or receptor binding sites. Our study demonstrates the co-occurring site mutations obtained by rule mining can capture the evolution of influenza viruses, and confirms that cooperative interactions among sites of HA1 protein drive the influenza antigenic evolution.

Evaluation of a Cryptococcal antigen Lateral Flow Assay in serum and cerebrospinal fluid for rapid diagnosis of cryptococcosis in Colombia.

PubMed

Cáceres, Diego H; Zuluaga, Alejandra; Tabares, Ángela M; Chiller, Tom; González, Ángel; Gómez, Beatriz L

2017-12-21

A Lateral Flow Assay to detect cryptococcal antigen (CrAg® LFA) in serum and cerebrospinal fluid for the rapid diagnosis of cryptococcosis was evaluated. A retrospective validation was performed. Sensitivity and specificity of the CrAg® LFA was 100%. High concordance (kappa index=1.0) between Cryptococcal Antigen Latex Agglutination System (CALAS®) and CrAg® LFA was observed. CrAg® LFA showed higher analytical sensitivity for detecting low concentrations of cryptococcal antigen.

Evaluation of a Cryptococcal antigen Lateral Flow Assay in serum and cerebrospinal fluid for rapid diagnosis of cryptococcosis in Colombia

PubMed Central

Cáceres, Diego H.; Zuluaga, Alejandra; Tabares, Ángela M.; Chiller, Tom; González, Ángel; Gómez, Beatriz L.

2017-01-01

ABSTRACT A Lateral Flow Assay to detect cryptococcal antigen (CrAg® LFA) in serum and cerebrospinal fluid for the rapid diagnosis of cryptococcosis was evaluated. A retrospective validation was performed. Sensitivity and specificity of the CrAg® LFA was 100%. High concordance (kappa index=1.0) between Cryptococcal Antigen Latex Agglutination System (CALAS®) and CrAg® LFA was observed. CrAg® LFA showed higher analytical sensitivity for detecting low concentrations of cryptococcal antigen. PMID:29267584

1-D grating based SPR biosensor for the detection of lung cancer biomarkers using Vroman effect

NASA Astrophysics Data System (ADS)

Teotia, Pradeep Kumar; Kaler, R. S.

2018-01-01

Grating based surface plasmon resonance waveguide biosensor have been reported for the detection of lung cancer biomarkers using Vroman effect. The proposed grating based multilayered biosensor is designed with high detection accuracy for Epidermal growth factor receptor (EGFR) and also analysed to show high detection accuracy with acceptable sensitivity for both cancer biomarkers. The introduction of periodic grating with multilayer metals generates a good resonance that make it possible for early detection of cancerous cells. Using finite difference time domain method, it is observed wavelength of biosensor get red-shifted on variations of the refractive index due to the presence of both the cancerous bio-markers. The reported detection accuracy and sensitivity of proposed biosensor is quite acceptable for both lung cancer biomarkers i.e. Carcinoembryonic antigen (CEA) and Epidermal growth factor receptor (EGFR) which further offer us label free early detection of lung cancer using these biomarkers.

Protamine-based nanoparticles as new antigen delivery systems.

PubMed

González-Aramundiz, José Vicente; Peleteiro Olmedo, Mercedes; González-Fernández, África; Alonso Fernández, María José; Csaba, Noemi Stefánia

2015-11-01

The use of biodegradable nanoparticles as antigen delivery vehicles is an attractive approach to overcome the problems associated with the use of Alum-based classical adjuvants. Herein we report, the design and development of protamine-based nanoparticles as novel antigen delivery systems, using recombinant hepatitis B surface antigen as a model viral antigen. The nanoparticles, composed of protamine and a polysaccharide (hyaluronic acid or alginate), were obtained using a mild ionic cross-linking technique. The size and surface charge of the nanoparticles could be modulated by adjusting the ratio of the components. Prototypes with optimal physicochemical characteristics and satisfactory colloidal stability were selected for the assessment of their antigen loading capacity, antigen stability during storage and in vitro and in vivo proof-of-concept studies. In vitro studies showed that antigen-loaded nanoparticles induced the secretion of cytokines by macrophages more efficiently than the antigen in solution, thus indicating a potential adjuvant effect of the nanoparticles. Finally, in vivo studies showed the capacity of these systems to trigger efficient immune responses against the hepatitis B antigen following intramuscular administration, suggesting the potential interest of protamine-polysaccharide nanoparticles as antigen delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Dry eye syndrome: developments and lifitegrast in perspective

PubMed Central

Lollett, Ivonne V; Galor, Anat

2018-01-01

Dry eye (DE) is a chronic ocular condition with high prevalence and morbidity. It has a complex pathophysiology and is multifactorial in nature. Chronic ocular surface inflammation has emerged as a key component of DE that is capable of perpetuating ocular surface damage and leading to symptoms of ocular pain, discomfort, and visual phenomena. It begins with stress to the ocular surface leading to the production of proinflammatory mediators that induce maturation of resident antigen-presenting cells which then migrate to the lymph nodes to activate CD4 T cells. The specific antigen(s) targeted by these pathogenic CD4+ T cells remains unknown. Two emerging theories include self-antigens by autoreactive CD4 T cells or harmless exogenous antigens in the setting of mucosal immunotolerance loss. These CD4 T cells migrate to the ocular surface causing additional inflammation and damage. Lifitegrast is the second topical anti-inflammatory agent to be approved by the US Food and Drug Administration for the treatment of DE and the first to show improvement in DE symptoms. Lifitegrast works by blocking the interaction between intercellular adhesion molecule-1 and lymphocyte functional associated antigen-1, which has been shown to be critical for the migration of antigen-presenting cells to the lymph nodes as well as CD4+ T cell activation and migration to the ocular surface. In four large multicenter, randomized controlled trials, lifitegrast has proven to be effective in controlling both the signs and symptoms of DE with minimal side effects. Further research should include comparative and combination studies with other anti-inflammatory therapies used for DE. PMID:29391773

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.

Characteristics of escape mutations from occult hepatitis B virus infected patients with hematological malignancies in South Egypt.

PubMed

Elkady, Abeer; Iijima, Sayuki; Aboulfotuh, Sahar; Mostafa Ali, Elsayed; Sayed, Douaa; Abdel-Aziz, Nashwa M; Ali, Amany M; Murakami, Shuko; Isogawa, Masanori; Tanaka, Yasuhito

2017-03-28

To investigate the prevalence and virological characteristics of occult hepatitis B virus (HBV) infections in patients with hematological malignancies in South Egypt. Serum samples were collected from 165 patients with hematological malignancies to monitor titers of HBV DNA, hepatitis B surface antigen (HBsAg), and antibodies to HBV core (anti-HBc) and surface antigens. Serum samples negative for HBsAg and positive for anti-HBc were subjected to nucleic acid extraction and HBV DNA detection by real-time polymerase chain reaction. DNA sequences spanning the S region were analyzed in cases with occult HBV infection. In vitro comparative study of constructed 1.24-fold wild type and S protein mutant HBV genotype D clones was further performed. HBV DNA was detected in 23 (42.6%) of 54 patients with hematological malignancies who were HBsAg negative, but anti-HBc positive, suggesting the presence of occult HBV infection. The complete HBV genome was retrieved from 6 occult HBV patients, and P120T and S143L were detected in 3 and 2 cases, respectively. Site directed mutagenesis was done to produce 1.24-fold genotype D clones with amino acid mutations T120 and L143. The in vitro analyses revealed that a lower level of extracellular HBsAg was detected by chemiluminescence enzyme immunoassay (CLEIA) with the clone containing T120 mutation, compared with the wild type or the clone with S143L mutation despite the similar levels of extracellular and intracellular HBsAg detected by Western blot. Southern blot experiments showed that the levels of intracellular HBV DNA were not different between these clones. Occult HBV infection is common in patients with hematological malignancies and associated with P120T and S143L mutations. 120T mutation impairs the detection of HBsAg by CLEIA.

Characteristics of escape mutations from occult hepatitis B virus infected patients with hematological malignancies in South Egypt

PubMed Central

Elkady, Abeer; Iijima, Sayuki; Aboulfotuh, Sahar; Mostafa Ali, Elsayed; Sayed, Douaa; Abdel-Aziz, Nashwa M; Ali, Amany M; Murakami, Shuko; Isogawa, Masanori; Tanaka, Yasuhito

2017-01-01

AIM To investigate the prevalence and virological characteristics of occult hepatitis B virus (HBV) infections in patients with hematological malignancies in South Egypt. METHODS Serum samples were collected from 165 patients with hematological malignancies to monitor titers of HBV DNA, hepatitis B surface antigen (HBsAg), and antibodies to HBV core (anti-HBc) and surface antigens. Serum samples negative for HBsAg and positive for anti-HBc were subjected to nucleic acid extraction and HBV DNA detection by real-time polymerase chain reaction. DNA sequences spanning the S region were analyzed in cases with occult HBV infection. In vitro comparative study of constructed 1.24-fold wild type and S protein mutant HBV genotype D clones was further performed. RESULTS HBV DNA was detected in 23 (42.6%) of 54 patients with hematological malignancies who were HBsAg negative, but anti-HBc positive, suggesting the presence of occult HBV infection. The complete HBV genome was retrieved from 6 occult HBV patients, and P120T and S143L were detected in 3 and 2 cases, respectively. Site directed mutagenesis was done to produce 1.24-fold genotype D clones with amino acid mutations T120 and L143. The in vitro analyses revealed that a lower level of extracellular HBsAg was detected by chemiluminescence enzyme immunoassay (CLEIA) with the clone containing T120 mutation, compared with the wild type or the clone with S143L mutation despite the similar levels of extracellular and intracellular HBsAg detected by Western blot. Southern blot experiments showed that the levels of intracellular HBV DNA were not different between these clones. CONCLUSION Occult HBV infection is common in patients with hematological malignancies and associated with P120T and S143L mutations. 120T mutation impairs the detection of HBsAg by CLEIA. PMID:28396718

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

PubMed

Davey, Sue; Navarrete, Cristina; Brown, Colin

2017-06-01

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

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

Nano-LISA for in vitro diagnostic applications

NASA Astrophysics Data System (ADS)

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

2011-03-01

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

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.

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.

Galactofuranose antigens, a target for diagnosis of fungal infections in humans

PubMed Central

Marino, Carla; Rinflerch, Adriana; de Lederkremer, Rosa M

2017-01-01

The use of biomarkers for the detection of fungal infections is of interest to complement histopathological and culture methods. Since the production of antibodies in immunocompromised patients is scarce, detection of a specific antigen could be effective for early diagnosis. D-Galactofuranose (Galf) is the antigenic epitope in glycoconjugates of several pathogenic fungi. Since Galf is not biosynthesized by mammals, it is an attractive candidate for diagnosis of infection. A monoclonal antibody that recognizes Galf is commercialized for detection of aspergillosis. The linkage of Galf in the natural glycans and the chemical structures of the synthesized Galf-containing oligosaccharides are described in this paper. The oligosaccharides could be used for the synthesis of artificial carbohydrate-based antigens, not enough exploited for diagnosis. PMID:28883999

Label-free biosensing with functionalized nanopipette probes.

PubMed

Umehara, Senkei; Karhanek, Miloslav; Davis, Ronald W; Pourmand, Nader

2009-03-24

Nanopipette technology can uniquely identify biomolecules such as proteins based on differences in size, shape, and electrical charge. These differences are determined by the detection of changes in ionic current as the proteins interact with the nanopipette tip coated with probe molecules. Here we show that electrostatic, biotin-streptavidin, and antibody-antigen interactions on the nanopipette tip surface affect ionic current flowing through a 50-nm pore. Highly charged polymers interacting with the glass surface modulated the rectification property of the nanopipette electrode. Affinity-based binding between the probes tethered to the surface and their target proteins caused a change in the ionic current due to a partial blockade or an altered surface charge. These findings suggest that nanopipettes functionalized with appropriate molecular recognition elements can be used as nanosensors in biomedical and biological research.

A monoclonal antibody inhibits calcium accumulation and skeleton formation in cultured embryonic cells of the sea urchin.

PubMed

Carson, D D; Farach, M C; Earles, D S; Decker, G L; Lennarz, W J

1985-06-01

The assembly of the spicules (primitive skeleton) of the sea urchin embryo is being studied in primary mesenchyme cells cultured in vitro. A monoclonal antibody (1223) has been prepared that inhibits the deposition of CaCO3 into the spicules. This antibody reacts with a 130,000 Mr cell-surface protein that is concentrated on the surface of approximately 5% of the cells of dissociated gastrula stage embryos. When primary mesenchyme cells in the embryo or cells cultured in vitro are examined, the 1223 antigen is detected on the surface of the cells and on the extracellular material associated with the spicule. We conclude that the 1223 antibody recognizes a cell-surface protein that plays an essential role in spicule formation.

A force-based protein biochip

NASA Astrophysics Data System (ADS)

Blank, K.; Mai, T.; Gilbert, I.; Schiffmann, S.; Rankl, J.; Zivin, R.; Tackney, C.; Nicolaus, T.; Spinnler, K.; Oesterhelt, F.; Benoit, M.; Clausen-Schaumann, H.; Gaub, H. E.

2003-09-01

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein-protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

DISTINCT ANTIBODY SPECIES: STRUCTURAL DIFFERENCES CREATING THERAPEUTIC OPPORTUNITIES

PubMed Central

Muyldermans, Serge; Smider, Vaughn V.

2016-01-01

Antibodies have been a remarkably successful class of molecules for binding a large number of antigens in therapeutic, diagnostic, and research applications. Typical antibodies derived from mouse or human sources use the surface formed by complementarity determining regions (CDRs) on the variable regions of the heavy chain/light chain heterodimer, which typically forms a relatively flat binding surface. Alternative species, particularly camelids and bovines, provide a unique paradigm for antigen recognition through novel domains which form the antigen binding paratope. For camelids, heavy chain antibodies bind antigen with only a single heavy chain variable region, in the absence of light chains. In bovines, ultralong CDR-H3 regions form an independently folding minidomain, which protrudes from the surface of the antibody and is diverse in both its sequence and disulfide patterns. The atypical paratopes of camelids and bovines potentially provide the ability to interact with different epitopes, particularly recessed or concave surfaces, compared to traditional antibodies. PMID:26922135

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

PubMed

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

2008-10-01

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

Development and Evaluation of Single Domain Antibodies for Vaccinia and the L1 Antigen

PubMed Central

Walper, Scott A.; Liu, Jinny L.; Zabetakis, Daniel; Anderson, George P.; Goldman, Ellen R.

2014-01-01

There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10−9 M to 7.0×10−10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×105 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies. PMID:25211488

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.

Surface enhanced Raman spectroscopy based nanoparticle assays for rapid, point-of-care diagnostics

NASA Astrophysics Data System (ADS)

Driscoll, Ashley J.

Nucleotide and immunoassays are important tools for disease diagnostics. Many of the current laboratory-based analytical diagnostic techniques require multiple assay steps and long incubation times before results are acquired. In the development of bioassays designed for detecting the emergence and spread of diseases in point-of-care (POC) and remote settings, more rapid and portable analytical methods are necessary. Nanoparticles provide simple and reproducible synthetic methods for the preparation of substrates that can be applied in colloidal assays, providing gains in kinetics due to miniaturization and plasmonic substrates for surface enhanced spectroscopies. Specifically, surface enhanced Raman spectroscopy (SERS) is finding broad application as a signal transduction method in immunological and nucleotide assays due to the production of narrow spectral peaks from the scattering molecules and the potential for simultaneous multiple analyte detection. The application of SERS to a no-wash, magnetic capture assay for the detection of West Nile Virus Envelope and Rift Valley Fever Virus N antigens is described. The platform utilizes colloid based capture of the target antigen in solution, magnetic collection of the immunocomplexes and acquisition of SERS spectra by a handheld Raman spectrometer. The reagents for a core-shell nanoparticle, SERS based assay designed for the capture of target microRNA implicated in acute myocardial infarction are also characterized. Several new, small molecule Raman scatterers are introduced and used to analyze the enhancing properties of the synthesized gold coated-magnetic nanoparticles. Nucleotide and immunoassay platforms have shown improvements in speed and analyte capture through the miniaturization of the capture surface and particle-based capture systems can provide a route to further surface miniaturization. A reaction-diffusion model of the colloidal assay platform is presented to understand the interplay of system parameters such as particle diameter, initial analyte concentration and dissociation constants. The projected sensitivities over a broad range of assay conditions are examined and the governing regime of particle systems reported. The results provide metrics in the design of more robust analytics that are of particular interest for POC diagnostics.

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

PubMed Central

Jones, Kenneth W.; Kaufman, Leo

1978-01-01

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

Comparative efficacy of antigen and antibody detection tests for human trichinellosis

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

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

1989-02-01

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

Detection of cancer biomarkers in serum using a hybrid mechanical and optoplasmonic nanosensor

NASA Astrophysics Data System (ADS)

Kosaka, P. M.; Pini, V.; Ruz, J. J.; da Silva, R. A.; González, M. U.; Ramos, D.; Calleja, M.; Tamayo, J.

2014-12-01

Blood contains a range of protein biomarkers that could be used in the early detection of disease. To achieve this, however, requires sensors capable of detecting (with high reproducibility) biomarkers at concentrations one million times lower than the concentration of the other blood proteins. Here, we show that a sandwich assay that combines mechanical and optoplasmonic transduction can detect cancer biomarkers in serum at ultralow concentrations. A biomarker is first recognized by a surface-anchored antibody and then by an antibody in solution that identifies a free region of the captured biomarker. This second antibody is tethered to a gold nanoparticle that acts as a mass and plasmonic label; the two signatures are detected by means of a silicon cantilever that serves as a mechanical resonator for ‘weighing’ the mass of the captured nanoparticles and as an optical cavity that boosts the plasmonic signal from the nanoparticles. The capabilities of the approach are illustrated with two cancer biomarkers: the carcinoembryonic antigen and the prostate specific antigen, which are currently in clinical use for the diagnosis, monitoring and prognosis of colon and prostate cancer, respectively. A detection limit of 1 × 10-16 g ml-1 in serum is achieved with both biomarkers, which is at least seven orders of magnitude lower than that achieved in routine clinical practice. Moreover, the rate of false positives and false negatives at this concentration is extremely low, ˜10-4.

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

PubMed

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

2018-03-07

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

SNSAG5 IS AN ALTERNATIVE SURFACE ANTIGEN OF SARCOCYSTIS NEURONA STRAINS THAT IS MUTUALLY EXCLUSIVE TO SNSAG1

USDA-ARS?s Scientific Manuscript database

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore can...