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Sample records for accurate antibody assays

  1. Fluorometric assay for red blood cell antibodies

    SciTech Connect

    Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

    1981-03-01

    A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

  2. Standardization of anti-DNA antibody assays.

    PubMed

    Pisetsky, David S

    2013-07-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA-anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.

  3. A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

    PubMed

    Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

    2013-11-13

    A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

  4. Assays of thyroid-stimulating antibody

    SciTech Connect

    McKenzie, J.M.; Zakarija, M.

    1985-01-01

    A comparison is presented of the two major assay methods of thyroid-stimulating antibody (TSAb) of Graves' disease. The basic procedures involve: (1) some index of thyroid stimulation, usually in vitro, using TSAb to indicate its activity; and (2) indirect recognition by assessment of the inhibition of binding of radioiodinated thyrotropin (TSH) to a preparation of its receptor, i.e., TSH-binding inhibition or TBI. There is potential for misinterpretation of data acquired by testing patients' sera by one or the other basic procedure.

  5. Production and assay of forskolin antibodies

    SciTech Connect

    Ho, L.T.; Ho, R.J.

    1986-05-01

    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.

  6. Solid-phase antibody capture hemadsorption assay for detection of hepatitis A virus immunoglobulin M antibodies.

    PubMed Central

    Summers, P L; Dubois, D R; Cohen, W H; Macarthy, P O; Binn, L N; Sjogren, M H; Snitbhan, R; Innis, B L; Eckels, K H

    1993-01-01

    A solid-phase antibody capture hemadsorption (SPACH) assay was developed to detect hepatitis A virus (HAV)-specific immunoglobulin M (IgM) antibodies in sera from humans recently infected with hepatitis. The assay is performed with microtiter plates coated with anti-human IgM antibodies to capture IgM antibodies from the test sera. HAV-specific IgM antibody is detected by the addition of HAV hemagglutinating antigen and goose erythrocytes. Hemadsorption of erythrocytes to antigen-antibody complexes attached to the solid phase indicate the presence of IgM antibodies. The SPACH assay was compared to a commercial radioimmunoassay and was found to be equally or more sensitive and specific for the detection of HAV IgM antibodies. The SPACH assay is an alternative, rapid assay that doesn't require hazardous substrates or radioactivity for the detection of HAV-specific antibodies. PMID:8388890

  7. Acquisition of accurate data from intramolecular quenched fluorescence protease assays.

    PubMed

    Arachea, Buenafe T; Wiener, Michael C

    2017-04-01

    The Intramolecular Quenched Fluorescence (IQF) protease assay utilizes peptide substrates containing donor-quencher pairs that flank the scissile bond. Following protease cleavage, the dequenched donor emission of the product is subsequently measured. Inspection of the IQF literature indicates that rigorous treatment of systematic errors in observed fluorescence arising from inner-filter absorbance (IF) and non-specific intermolecular quenching (NSQ) is incompletely performed. As substrate and product concentrations vary during the time-course of enzyme activity, iterative solution of the kinetic rate equations is, generally, required to obtain the proper time-dependent correction to the initial velocity fluorescence data. Here, we demonstrate that, if the IQF assay is performed under conditions where IF and NSQ are approximately constant during the measurement of initial velocity for a given initial substrate concentration, then a simple correction as a function of initial substrate concentration can be derived and utilized to obtain accurate initial velocity data for analysis.

  8. A rapid, simple, and accurate plaque assay for human respiratory syncytial virus (HRSV).

    PubMed

    Kim, Kyung Sook; Kim, Ah Ra; Piao, Ying; Lee, Ju-Hie; Quan, Fu-Shi

    2017-03-31

    Plaque assays of human respiratory syncytial virus (HRSV) are time-consuming, requiring 4 to 7 days for plaque formation and several hours for dye staining. Here, we describe a simple method by which RSV plaques can be visualized and counted with the naked eye only 2 days after infection of HEp-2 cells. In this assay, the infected cells are stained with monoclonal antibodies and the plaques are developed using diaminobenzidine (DAB). We tested the accuracy of this new plaque assay by comparing the results obtained on days 1, 2, 3, 4, 5, and 6 post-infection. The whole procedure is significantly simpler than the traditional method, with an immunostaining process of around 1.5h. Our method is rapid, accurate, and simple; thus, it has the potential to significantly contribute to studies related to RSV disease.

  9. Monoclonal antibody technologies and rapid detection assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

  10. Clinically relevant interpretation of solid phase assays for HLA antibody

    PubMed Central

    Bettinotti, Maria P.; Zachary, Andrea A.; Leffell, Mary S.

    2016-01-01

    Purpose of review Accurate and timely detection and characterization of human leukocyte antigen (HLA) antibodies are critical for pre-transplant and post-transplant immunological risk assessment. Solid phase immunoassays have provided increased sensitivity and specificity, but test interpretation is not always straightforward. This review will discuss the result interpretation considering technical limitations; assessment of relative antibody strength; and the integration of data for risk stratification from complementary testing and the patient's immunological history. Recent findings Laboratory and clinical studies have provided insight into causes of test failures – false positive reactions because of antibodies to denatured HLA antigens and false negative reactions resulting from test interference and/or loss of native epitopes. Test modifications permit detection of complement-binding antibodies and determination of the IgG subclasses. The high degree of specificity of single antigen solid phase immunoassays has revealed the complexity and clinical relevance of antibodies to HLA-C, HLA-DQ, and HLA-DP antigens. Determination of antibody specificity for HLA epitopes enables identification of incompatible antigens not included in test kits. Summary Detection and characterization of HLA antibodies with solid phase immunoassays has led to increased understanding of the role of those antibodies in graft rejection, improved treatment of antibody-mediated rejection, and increased opportunities for transplantation. However, realization of these benefits requires careful and accurate interpretation of test results. PMID:27200498

  11. Cooperative Immunoassays: Ultrasensitive Assays with Mixed Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ehrlich, Paul H.; Moyle, William R.

    1983-07-01

    Mixtures of certain monoclonal antibodies appear to bind human chorionic gonadotropin in a ``cooperative'' fashion because they form circular complexes with the hormone. Experiments illustrate how this property might be exploited to develop very sensitive immunoassays for human chorionic gonadotropin or any other antigen. Since the assays are not based on competitive inhibition between radiolabeled and unlabeled antigen, they are much more sensitive than a traditional radioimmunoassay in which either one of the same antibodies is used alone.

  12. Multicentre comparison of a diagnostic assay: aquaporin-4 antibodies in neuromyelitis optica

    PubMed Central

    Waters, Patrick; Reindl, Markus; Saiz, Albert; Schanda, Kathrin; Tuller, Friederike; Kral, Vlastimil; Nytrova, Petra; Sobek, Ondrej; Nielsen, Helle Hvilsted; Barington, Torben; Lillevang, Søren T; Illes, Zsolt; Rentzsch, Kristin; Berthele, Achim; Berki, Tímea; Granieri, Letizia; Bertolotto, Antonio; Giometto, Bruno; Zuliani, Luigi; Hamann, Dörte; van Pelt, E Daniëlle; Hintzen, Rogier; Höftberger, Romana; Costa, Carme; Comabella, Manuel; Montalban, Xavier; Tintoré, Mar; Siva, Aksel; Altintas, Ayse; Deniz, Günnur; Woodhall, Mark; Palace, Jacqueline; Paul, Friedemann; Hartung, Hans-Peter; Aktas, Orhan; Jarius, Sven; Wildemann, Brigitte; Vedeler, Christian; Ruiz, Anne; Leite, M Isabel; Trillenberg, Peter; Probst, Monika; Saschenbrecker, Sandra; Vincent, Angela; Marignier, Romain

    2016-01-01

    Objective Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). Methods Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). Results Results of tests on 92 controls identified 12assays as highly specific (0–1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5–100%) of all 21 assays. The specificities (85.8–100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. Conclusions The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology. PMID:27113605

  13. Inter-assay variation in antiphospholipid antibody testing.

    PubMed

    Robert, Judith M; Macara, Lena M; Chalmers, Elizabeth A; Smith, Gordon C S

    2002-03-01

    We evaluated inter-assay variation in anticardiolipin antibody status, comparing three centres, and using different assays among 36 women with recurrent miscarriage and 26 controls. There was no more agreement between the laboratories than would be predicted on the basis of chance for IgM and only fair agreement among the laboratories for IgG. None of the tests were significantly more likely to be positive in the cases compared with the controls.

  14. A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients.

    PubMed

    Shan, Chao; Xie, Xuping; Ren, Ping; Loeffelholz, Michael J; Yang, Yujiao; Furuya, Andrea; Dupuis, Alan P; Kramer, Laura D; Wong, Susan J; Shi, Pei-Yong

    2017-03-01

    The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV) infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT) that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV) diagnosis that can attain the "gold standard" of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

  15. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    PubMed

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

  16. Solid-Phase Antibody Capture Hemadsorption Assay for Detection of Hepatitis A Virus Immunoglobulin M Antibodies

    DTIC Science & Technology

    1993-02-25

    our tested for HAV IgM antibodies by SPACH and for human ELISAs was negative in our SPACH assay for HAV IgM IgM and IgG with radial immunodiffusion ...radioimmunoassays (RIAs) and ELISAs to de- RIA kits supplied by Abbott Laboratories, North Chicago, tect IgM antibodies to a variety of infectious... RID ) plates antibody despite having a HAV HAI titer of 5,120. (Calbiochem Behring, La Jolla, Calif.). These results suggest there is very little if any

  17. Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

    PubMed

    Di Cristofaro, Julie; Frassati, Coralie; Montagnie, Rolande; Basire, Agnes; Merieux, Yves; Picard, Christophe

    2015-01-01

    Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future.

  18. Rapid assessment of antibody-induced ricin neutralization by employing a novel functional cell-based assay.

    PubMed

    Gal, Yoav; Alcalay, Ron; Sabo, Tamar; Noy-Porat, Tal; Epstein, Eyal; Kronman, Chanoch; Mazor, Ohad

    2015-09-01

    Ricin is one of the most potent and lethal toxins known against which there is no available antidote. Currently, the most promising countermeasures against the toxin are based on neutralizing antibodies elicited by active vaccination or administered passively. A cell-based assay is widely applied for the primary screening and evaluation of anti-ricin antibodies, yet such assays are usually time-consuming (18-72 h). Here, we report of a novel assay to monitor ricin activity, based on HeLa cells that stably express the rapidly-degraded ubiquitin-luciferase (Ub-FL, half-life of 2 min). Ricin-induced arrest of protein synthesis could be quantified within 3 to 6h post intoxication (IC90 of 300 and 100 ng/ml, respectively). Furthermore, by stabilizing the intracellular levels of Ub-FL in the last hour of the assay, a 3-fold increase in the assay sensitivity was attained. We applied this assay to monitor the efficacy of a ricin holotoxin-based vaccine by measuring the formation of neutralizing antibodies throughout the immunization course. The potency of anti-ricin monoclonal antibodies (directed to either subunit of the toxin) could also be easily and accurately measured in this assay format. Owing to its simplicity, this assay may be implemented for high-throughput screening of ricin-neutralizing antibodies and for identification of small-molecule inhibitors of the toxin, as well as other ribosome-inactivating toxins.

  19. A comparison of the Genie and western blot assays in confirmatory testing for HIV-1 antibody.

    PubMed

    Chan, E L; Sidaway, F; Horsman, G B

    1996-03-01

    The Genie HIV-1/2 kit (Sanofi Diagnostics Pasteur, Montreal, Quebec), a synthetic-peptide solid-phase enzyme immunoassay, was evaluated as a confirmatory assay for HIV-1 antibodies in comparison with Western blot (BioRad, Hercules, CA, USA) on 50 stored HIV-1 antibody-positive sera and the 137 sera yielding repeated positive results in the conventional EIA screen out of 13405 fresh patient sera from Saskatchewan in 1993. The stored HIV-1-positive sera were uniformly positive in the Genie test. Of the 137 EIA screen-positive sera, 33 were uniformly positive and 64 were uniformly negative in Genie and Western blot; 36 were Genie-negative and indeterminate by Western blot; and four were Genie indeterminate, of which one was negative and three were indeterminate by Western blot. All HIV-1 Western blot-indeterminate and Genie-interdeterminate sera were negative in radio-immunoprecipitation assay (RIPA) and Western blot for HIV-1 and HIV-2 antibodies performed by a reference laboratory. Genie gave an accurate definitive result for 97% of EIA positive sera compared with 71% for Western blot. There was excellent correlation between Genie, Western blot and RIPA results. However, the Genie assay was faster, less costly and yielded fewer indeterminate results than Western blot in confirmatory testing for HIV-1 antibodies.

  20. Toward Sensitive and Accurate Analysis of Antibody Biotherapeutics by Liquid Chromatography Coupled with Mass Spectrometry

    PubMed Central

    An, Bo; Zhang, Ming

    2014-01-01

    Remarkable methodological advances in the past decade have expanded the application of liquid chromatography coupled with mass spectrometry (LC/MS) analysis of biotherapeutics. Currently, LC/MS represents a promising alternative or supplement to the traditional ligand binding assay (LBA) in the pharmacokinetic, pharmacodynamic, and toxicokinetic studies of protein drugs, owing to the rapid and cost-effective method development, high specificity and reproducibility, low sample consumption, the capacity of analyzing multiple targets in one analysis, and the fact that a validated method can be readily adapted across various matrices and species. While promising, technical challenges associated with sensitivity, sample preparation, method development, and quantitative accuracy need to be addressed to enable full utilization of LC/MS. This article introduces the rationale and technical challenges of LC/MS techniques in biotherapeutics analysis and summarizes recently developed strategies to alleviate these challenges. Applications of LC/MS techniques on quantification and characterization of antibody biotherapeutics are also discussed. We speculate that despite the highly attractive features of LC/MS, it will not fully replace traditional assays such as LBA in the foreseeable future; instead, the forthcoming trend is likely the conjunction of biochemical techniques with versatile LC/MS approaches to achieve accurate, sensitive, and unbiased characterization of biotherapeutics in highly complex pharmaceutical/biologic matrices. Such combinations will constitute powerful tools to tackle the challenges posed by the rapidly growing needs for biotherapeutics development. PMID:25185260

  1. [Assay of circulating anti-insulin antibodies by radioimmunoprecipitation].

    PubMed

    Cotisson, A; Hartmann, D J; Ville, G

    1984-01-01

    We report a radio-immunoprecipitation method for the measurement of circulating insulin antibodies using porcine, bovine or human radiolabeled insulin and precipitation of the bound fraction by a combined polyethylene glycol--second antibody method. The coefficient of variation for intra- and inter-assay is below 8,5 p. cent and 17 p. cent respectively. 100 insulin treated patients sera were tested in the three systems: the results showed a great scattering; for the same serum, the values obtained with porcine and human insulins are below that with bovine insulin. The data correlated statistically significantly with those found with a commercial kit (r = 0,97, n = 170, t = -4,91, p greater than 0,001).

  2. Measuring immunoglobulin g antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin with single-antigen enzyme-linked immunosorbent assays and a bead-based multiplex assay.

    PubMed

    Reder, Sabine; Riffelmann, Marion; Becker, Christian; Wirsing von König, Carl Heinz

    2008-05-01

    Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assay was compared to a standardized in-house IgG- anti-PT enzyme-linked immunosorbent assay (ELISA) of the German reference laboratory for bordetellae, as well as to various commercially available ELISAs for anti-PT IgG, anti-tetanus IgG, and anti-diphtheria IgG. The results of the multiplex assay regarding the antibodies against diphtheria toxin compared favorably with a regression coefficient of 0.938 for values obtained with an ELISA from the same manufacturer used as a reference. Similarly, antibodies to tetanus toxin showed a correlation of 0.910 between the reference ELISA and the multianalyte assay. A correlation coefficient of 0.905 was found when an "in-house" IgG anti-PT and the multiplex assay were compared. Compared to single ELISA systems from two other manufacturers, the multiplex assay performed similarly well or better. The multianalyte assay system was a robust system with fast and accurate results, analyzing three parameters simultaneously in one sample. The system was well suited to quantitatively determine relevant vaccine induced antibodies compared to in-house and commercially available single-antigen ELISA systems.

  3. ABAP: antibody-based assay for peptidylarginine deiminase activity.

    PubMed

    Zendman, Albert J W; Raijmakers, Reinout; Nijenhuis, Suzanne; Vossenaar, Erik R; Tillaart, Marloes van den; Chirivi, Renato G S; Raats, Jos M H; van Venrooij, Walther J; Drijfhout, Jan W; Pruijn, Ger J M

    2007-10-15

    Members of the family of peptidylarginine deiminases (PADs, EC 3.5.3.15) catalyze the posttranslational modification of peptidylarginine into peptidylcitrulline. Citrulline-containing epitopes have been shown to be major and specific targets of autoantibodies produced by rheumatoid arthritis patients. Recently, the citrullination of histone proteins by PAD enzyme was reported to influence gene expression levels. These findings greatly increase the interest in the PAD enzymes and their activities. A few procedures to monitor PAD activity in biological samples have been described previously. However, these assays either have low sensitivity or are rather laborious. Here we describe a reliable and reproducible method for the determination of PAD activity in both purified and crude samples. The method is based on the quantification of PAD-dependent citrullination of peptides, immobilized in microtiter plates, using antibodies that are exclusively reactive with the reaction product(s). Our results demonstrate that this antibody-based assay for PAD activity, called ABAP, is very sensitive and can be applied to monitor PAD activity in biological samples.

  4. Misleading FT4 measurement: Assay-dependent antibody interference

    PubMed Central

    Revet, Ingrid; Boesten, Lianne SM; Linthorst, Jan; Yildiz, Elif; Janssen, Johannes W; de Rijke, Yolanda B; Albersen, Arjan

    2016-01-01

    Introduction Commonly used free thyroxine (FT4) immunoassays can be falsely elevated due to interference causing misinterpreted thyroid function. We present two cases with high FT4 concentrations due to antibody interference. This study’s aim was to investigate the source of the FT4 immunoassay interference and possibility of its removal by two different techniques in order to correct the discrepancy between obtained FT4 values and the patient’s clinical status. Materials and methods Two patients presented at their general practitioners’ with elevated FT4 concentrations in combination with a normal and increased thyroid stimulating hormone (TSH) concentrations. Clinical symptoms differed between patients but did not correspond with the hyperthyroid status suggested by the laboratory results. FT4 concentrations from both patients were measured on four common commercial immunoassays and the dialysis method before and after treatment with heterophilic blocking tubes and protein A/G. Results Removal of interfering antibodies using protein A/G resulted in normal FT4 concentrations. Conclusion This report illustrates falsely elevated FT4 concentrations due to assay interference on the Immulite immunoassay analyser caused by heterophilic antibodies, which were eliminated by protein A/G treatment. We point out the importance of a close collaboration between doctors and the laboratory to avoid unnecessary clinical intervention.

  5. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

    NASA Astrophysics Data System (ADS)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason

    2016-09-01

    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  6. Monoclonal antibody binding-site diversity assessment with a cell-based clustering assay.

    PubMed

    Liao-Chan, Sindy; Zachwieja, Joseph; Gomez, Steven; Duey, Dana; Lippincott, John; Theunissen, Jan-Willem

    2014-03-01

    The diversity of a panel of antibodies that target a specific antigen can be established in various assay formats. In conventional epitope binning assays purified antibodies are tested in a pairwise manner: two antibodies that compete with each other for binding to an antigen are grouped into the same cluster or bin, while they are assigned to two different clusters when they do not compete. Here we present a high through put assay that enables grouping of crude hybridoma supernatants without a need for antibody purification. In addition, the assay does not require recombinant protein, because it is conducted on cells that express the antigen of interest. Hence, one can use the antibody-clustering assay for cell surface proteins that are not amenable to purification. Heavy chain variable region (VH) sequencing shows that VH composition within clusters is conserved. Finally, the assay is in good agreement with a conventional epitope binning assay with purified antigen.

  7. Development of an Anti-Elicitin Antibody-Based Immunohistochemical Assay for Diagnosis of Pythiosis

    PubMed Central

    Inkomlue, Ruchuros; Larbcharoensub, Noppadol; Karnsombut, Patcharee; Lerksuthirat, Tassanee; Aroonroch, Rangsima; Lohnoo, Tassanee; Yingyong, Wanta; Santanirand, Pitak; Sansopha, Lalana

    2015-01-01

    Pythiosis is an emerging and life-threatening infectious disease of humans and animals living in tropical and subtropical countries and is caused by the fungus-like organism Pythium insidiosum. Antifungals are ineffective against this pathogen. Most patients undergo surgical removal of the infected organ, and many die from advanced infections. Early and accurate diagnosis leads to prompt management and promotes better prognosis for affected patients. Immunohistochemical assays (IHCs) have been developed using rabbit antibodies raised against P. insidiosum crude extract, i.e., culture filtrate antigen (CFA), for the histodiagnosis of pythiosis, but cross-reactivity with pathogenic fungi compromises the diagnostic performance of the IHC. Therefore, there is a need to improve detection specificity. Recently, the elicitin protein, ELI025, was identified in P. insidiosum, but it was not identified in other human pathogens, including true fungi. The ELI025-encoding gene was successfully cloned and expressed as a recombinant protein in Escherichia coli. This study aims to develop a new IHC using the rabbit anti-ELI025 antibody (anti-ELI) and to compare its performance with the previously reported anti-CFA-based IHC. Thirty-eight P. insidiosum histological sections stained positive by anti-ELI-based and anti-CFA-based IHCs indicating 100% detection sensitivity for the two assays. The anti-ELI antibody stained negative for all 49 negative-control sections indicating 100% detection specificity. In contrast, the anti-CFA antibody stained positive for one of the 49 negative controls (a slide prepared from Fusarium-infected tissue) indicating 98% detection specificity. In conclusion, the anti-ELI based IHC is sensitive and specific for the histodiagnosis of pythiosis and is an improvement over the anti-CFA-based assay. PMID:26719582

  8. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies.

    PubMed

    Partridge, Michael A; Purushothama, Shobha; Elango, Chinnasamy; Lu, Yanmei

    2016-01-01

    Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline.

  9. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies

    PubMed Central

    Elango, Chinnasamy

    2016-01-01

    Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline. PMID:27556048

  10. Antibody-Based Assays for Phenotyping of Extracellular Vesicles

    PubMed Central

    Pugholm, Lotte Hatting; Revenfeld, Anne Louise Schacht; Søndergaard, Evo Kristina Lindersson; Jørgensen, Malene Møller

    2015-01-01

    Extracellular vesicles (EVs) are a heterogeneous population of membrane-enclosed vesicles. EVs are recognized as important players in cell-to-cell communication and are described to be involved in numerous biological and pathological processes. The fact that EVs are involved in the development and progression of several diseases has formed the basis for the use of EV analysis in a clinical setting. As the interest in EVs has increased immensely, multiple techniques have been developed aiming at characterizing these vesicles. These techniques characterize different features of EVs, like the size distribution, enumeration, protein composition, and the intravesicular cargo (e.g., RNA). This review focuses on techniques that exploit the specificity and sensitivity associated with antibody-based assays to characterize the protein phenotype of EVs. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. Thus, protein profiling of EVs holds great diagnostic and prognostic potential. PMID:26770974

  11. Development and optimization of a novel assay to measure neutralizing antibodies against Clostridium difficile toxins.

    PubMed

    Xie, Jinfu; Zorman, Julie; Indrawati, Lani; Horton, Melanie; Soring, Keri; Antonello, Joseph M; Zhang, Yuhua; Secore, Susan; Miezeiewski, Matthew; Wang, Su; Kanavage, Anthony D; Skinner, Julie M; Rogers, Irene; Bodmer, Jean-Luc; Heinrichs, Jon H

    2013-04-01

    Clostridium difficile produces two major virulence toxins, toxin A (TcdA) and toxin B (TcdB). Antitoxin antibodies, especially neutralizing antibodies, have been shown to be associated with a lower incidence of C. difficile infection (CDI) recurrence, and antibody levels are predictive of asymptomatic colonization. The development of an assay to detect the presence of neutralizing antibodies in animal and human sera for the evaluation of vaccine efficacy is highly desired. We have developed such an assay, which allows for the quantification of the effect of toxins on eukaryotic cells in an automated manner. We describe here the optimization of this assay to measure toxin potency as well as neutralizing antibody (NAb) activity against C. difficile toxins using a design-of-experiment (DOE) methodology. Toxin concentration and source, cell seeding density, and serum-toxin preincubation time were optimized in the assay using Vero cells. The assay was shown to be robust and to produce linear results across a range of antibody concentrations. It can be used to quantify neutralizing antibodies in sera of monkeys and hamsters immunized with C. difficile toxoid vaccines. This assay was shown to correlate strongly with traditional assays which rely on labor-intensive methods of determining neutralizing antibody titers by visual microscopic inspection of intoxicated-cell monolayers. This assay has utility for the selection and optimization of C. difficile vaccine candidates.

  12. Characterization of antibody binding to cell surface antigens using a plasma membrane-bound plate assay.

    PubMed

    Vater, C A; Reid, K; Bartle, L M; Goldmacher, V S

    1995-01-01

    A procedure has been developed for measuring antibody binding to cell surface antigens using an immobilized plasma membrane fraction. In this method, isolated plasma membranes are dried onto wells of a 96-well microtiter plate and incubated with antibodies that recognize a cell surface protein. Bound antibody is detected indirectly using an enzyme-linked or fluorescently tagged second antibody. Alternatively, the primary antibody itself can be labeled and its binding can be detected directly. The assay is simple and fast and provides several advantages over whole cell binding assays currently in widespread use.

  13. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

    PubMed

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng

    2016-09-01

    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.

  14. The line blot assay: problems with titrating first and second antibodies for Western blot and immunohistochemistry assays?

    PubMed

    Rojas-Espinosa, O; Silva-Miranda, M; Wek-Rodriguez, K; Arce-Paredes, P

    2006-01-01

    We describe a technique designed to assess the optimal dilution of primary and secondary antibodies, to be used in Western blot, dot blot, the multi-antigen print immunoassay (MAPIA) and immunohistochemistry assays. The method that we call "line blot" is not an alternative but a practical, complementary tool for the above techniques that assures definitive results are obtained from single assays, so there is no need to repeat the assay. As with most immunoenzymatic assays, the line blot assay is very sensitive, allowing the detection of absolute amounts of antigen as low as 2.5 ng in the 0.5 cm-long segment line (see Results), depending on the strength of the secondary, enzyme-labelled antibody.

  15. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples

    PubMed Central

    Ackerman, Margaret E.; Moldt, Brian; Wyatt, Richard T; Dugast, Anne-Sophie; McAndrew, Elizabeth; Tsoukas, Stephen; Jost, Stephanie; Berger, Christoph T.; Sciaranghella, Gaia; Liu, Qingquan; Irvine, Darrell J; Burton, Dennis R.; Alter, Galit

    2011-01-01

    Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptors—allowing complex phenotypes to be studied. We demonstrate the adaptability of this high-throughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function. PMID:21192942

  16. A Rapid, Self-confirming Assay for HIV: Simultaneous Detection of Anti-HIV Antibodies and Viral RNA

    PubMed Central

    Chen, Zongyuan; Zhu, Hui; Malamud, Daniel; Barber, Cheryl; Ongagna, Yhombi Yvon Serge; Yasmin, Rubina; Modak, Sayli; Janal, Malvin N.; Abrams, William R.; Montagna, Richard A.

    2016-01-01

    Objective We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest. Methods We first developed and optimized two separate manual assays for the detection of host anti-HIV antibodies and viral RNA and then converted them to the microfluidic system. We optimized a commercially available serologic assay to run within the microfluidic device while we incorporated the isothermal LAMP assay to detect the presence of viral RNA. The microfluidic device and instrumentation were developed to simultaneously perform both assays without any user intervention. Results The finalized system consists of a disposable injection molded and film-laminated microfluidic CARD disposable device and a portable, software controlled instrument, which together can automatically perform all steps of both assays without any user intervention after the initial loading of samples and reagents. The microfluidic CARD cartridge has multiple microchannels, valves, pumps and reservoirs, which perform the immunoassay, isolates viral RNA for detection by magnetic bead based purification, and Reverse Transcriptase loop-mediated isothermal amplification (RT-LAMP). The microfluidic system was able to detect host anti-HIV antibodies and viral RNA in either a blood or saliva sample. Conclusion The ability to detect antibodies and simultaneously confirm a seropositive HIV-RNA result provides healthcare workers with a complete and accurate appraisal of a patient's infection status in the earliest stages of the disease and represents an important tool for

  17. New monoclonal-antibody two-site solid-phase immunoradiometric assay for human thyrotropin evaluated

    SciTech Connect

    Pekary, A.E.; Hershman, J.M.

    1984-07-01

    The authors compared results with a commercial solid-phase two-site immunoradiometric assay kit for human thyrotropin in which monoclonal antibodies are used with those by our radioimmunoassay, which is optimized for measurement of low concentrations of thyrotropin. In the immunoradiometric assay a specific antibody to the beta subunit of human thyrotropin is immobilized on a polystyrene bead, and a radiolabeled monoclonal antibody directed against the alpha subunit provides a measure of bead-immobilized hormone. The mean thyrotropin concentrations in 70 euthyroid serum samples were similar in the two assays. Values for hypothyroid patients were clearly higher in both assays than values for euthyroid individuals. In commercial assays the major source of error in measurement of thyrotropin response to thyroliberin in terms of the increment over the basal concentration of thyrotropin has been systematic errors in the measurement of those basal concentrations. With the present assay, however, basal values are obtained with good precision and accuracy.

  18. Monoclonal antibody capture enzyme-linked immunosorbent assay for detection of bovine enteric coronavirus.

    PubMed Central

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

    1984-01-01

    Monoclonal antibodies reactive with three different viral polypeptides were evaluated singly and in combination as the capture antibody(s) in an enzyme-linked immunosorbent assay system for the detection of bovine enteric coronavirus. Similar levels of sensitivity were found for all combinations tested. A sensitive, highly specific, and reproducible assay for the detection of bovine enteric coronavirus was developed, using a mixture of two of these monoclonal antibodies reactive with antigenic components either external or internal to the virion. These monoclonal antibodies were bound indirectly to 96-well plates via rabbit anti-mouse immunoglobulin. After sample application and incubation, virus was detected by using rabbit anti-coronavirus peroxidase conjugate followed by enzyme substrate and chromagen. Fecal samples from a single herd of cows were screened for the presence of coronavirus by this assay. Five percent of clinically normal cows were found to be shedding coronavirus. Images PMID:6325490

  19. Generic anti-drug antibody assay with drug tolerance in serum samples from mice exposed to human antibodies.

    PubMed

    Stubenrauch, Kay; Mackeben, Klaus; Vogel, Rudolf; Heinrich, Julia

    2012-11-15

    Knowledge of the anti-drug antibody (ADA) status is necessary in early research studies. Because specific assay materials are sparse and time is pressing, a generic assay format with drug tolerance for detection of ADAs in serum samples from mice exposed to immunoglobulin G (IgG) or antigen-binding fragments (Fabs) is highly desirable. This article describes a generic immune complex assay in the sandwich enzyme-linked immunosorbent assay (ELISA) format based on (i) transformation of free ADAs to immune complexes by preincubation with excess drug, (ii) the use of a murine anti-human Fab constant domain Fab as capture reagent, (iii) detection of the immune complexes by a peroxidase-labeled rabbit anti-murine Fc antibody, and (iv) ADA-positive control conjugates consisting of human Fab and murine IgG. Results of the experiments suggest that the generic immune complex assay for mouse serum samples was at least equivalent to specific ADA immune assays and even superior regarding drug tolerance. The generic immune complex assay confers versatility as it detects ADAs in complex with full-length IgG as well as with Fabs independent of the target specificity in mouse serum samples. These features help to save the sparse amounts of specific antibodies available in early research and development and speed up drug candidate selection.

  20. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

    PubMed

    Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta

    2016-04-01

    Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

  1. Antibody Development and Immunoligand Assay Testing of MS2 Bacteriophage on the Light Addressable Potentiometric Sensor

    DTIC Science & Technology

    1994-04-01

    fluoresceinated immunocomplexes are immobilized followed by reaction with an anti-fluorescein urease - conjugated antibody. Analyte quantitation is...monoclonal 5A4-1 detector antibody ..... 11 Flgurm 7. ELISA sandwich assay using LAPS assay format with the ani-fluorescem urease antbody...accomplished by placing the urease -labeled immunocomplex into a reader containing a 100-mM solution of urea. Hydrolysis of the urea produces a

  2. Accurate Quantification of Disease Markers in Human Serum Using Iron Oxide Nanoparticle-linked Immunosorbent Assay

    PubMed Central

    Zhang, Linlin; Tong, Sheng; Zhou, Jun; Bao, Gang

    2016-01-01

    Accurate and reliable quantification of biomarkers in the blood is essential in disease screening and diagnosis. Here we describe an iron oxide nanoparticle (IONP)-linked immunosorbent assay (ILISA) for detecting biomolecules in human serum. Sandwich ILISA was optimized for the detection of four important serological markers, IgA, IgG, IgM, and C-reactive protein (CRP), and assessed with normal sera, simulated disease-state sera and the serum samples from patients infected with West Nile virus (WNV) or human herpes virus (HHV). Our study shows that using the detection assay formulated with 18.8 nm wüstite nanocrystals, ILISA can achieve sub-picomolar detection sensitivity, and all four markers can be accurately quantified over a large dynamic range. In addition, ILISA is not susceptible to variations in operating procedures and shows better linearity and higher stability compared with ELISA, which facilitates its integration into detection methods suitable for point of care. Our results demonstrate that ILISA is a simple and versatile nanoplatform for highly sensitive and reliable detection of serological biomarkers in biomedical research and clinical applications. PMID:27375784

  3. The criticality of high-resolution N-linked carbohydrate assays and detailed characterization of antibody effector function in the context of biosimilar development.

    PubMed

    Brady, Lowell J; Velayudhan, Jyoti; Visone, Devi B; Daugherty, Ken C; Bartron, Jeff L; Coon, Michael; Cornwall, Cabot; Hinckley, Peter J; Connell-Crowley, Lisa

    2015-01-01

    Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development.

  4. A versatile assay for the accurate, time-resolved determination of cellular viability.

    PubMed

    Amano, Toyoki; Hirasawa, Ken ichi; O'Donohue, Michael J; Pernolle, Jean Claude; Shioi, Yuzo

    2003-03-01

    A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.

  5. Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus.

    PubMed

    Kusano-Arai, Osamu; Fukuda, Rie; Kamiya, Wakana; Iwanari, Hiroko; Hamakubo, Takao

    2016-07-01

    The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface.

  6. A sensitive assay for anti-phosphatidylethanol-amine antibody in patients with recurrent fetal loss.

    PubMed

    Hayashi, Y; Aoki, K; Kunimatsu, M; Sasaki, M; Yagami, Y

    1990-11-01

    There is strong evidence that anti-phospholipid antibodies is implicated in thrombosis and recurrent fetal death. In recent years, it has been suggested that anti-phosphatidylethanolamine (PE) antibody is an important antibody of this type. In the present study, we established a sensitive enzyme linked immunosorbent assay (ELISA) to detect anti-PE antibodies and examined the relationship between the anti-PE antibody level in serum and recurrent abortion. The improvement of the assay was made by treating PE with 1.0% acetic acid in methanol solution prior to its application to the microplates. This treatment markedly increased the antigenicity of PE. Using this modified ELISA, anti-PE antibodies in 10 patients with a history of recurrent fetal loss were measured before and after therapy during the last period of their pregnancies. IgG anti-PE antibodies were detected in all 10 patients. Four patients exhibiting high titers of IgG anti-PE antibody experienced subsequent intrauterine fetal death (IUFD), while the other 6 patients, whose titers of IgG anti-PE antibody had decreased with therapy, had live births. These results suggest that this modified ELISA for estimating IgG anti-PE antibody is a valuable tool for the diagnosis and prognosis of patients with recurrent fetal loss.

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  8. Immune Infertility Should Be Positively Diagnosed Using an Accurate Method by Monitoring the Level of Anti-ACTL7a Antibody

    PubMed Central

    Fu, Jun; Yao, Rongyan; Luo, Yanyun; Yang, Dantong; Cao, Yang; Qiu, Yi; Song, Wei; Miao, Shiying; Gu, Yiqun; Wang, Linfang

    2016-01-01

    Infertility is currently a major public health problem. Anti-sperm antibodies (ASAs) markedly reduce sperm quality, which can subsequently lead to male and/or female infertility. The accurate detection of ASAs derived from specific spermatozoa is, therefore, clinically useful. We have focused on the spermatozoa-specific expression protein ACTL7a for many years and have developed an enzyme-linked immunosorbent assay (ELISA) to detect the concentration of anti-ACTL7a antibodies in fertile sera (n = 267) and infertile sera (n = 193). Infertile sera were collected from the positive sera of tray agglutination tests (TAT), which is a routine ASA screening methodology. We found that the concentration of anti-ACTL7a antibodies was significantly higher in the infertile sera (than in the fertile sera, P < 0.0001) and much higher in the TAT ≥ 16 infertile sera. The ELISA was much better for male sera detection (AUC = 0.9899). If we set the standard at a strongly positive value (calculated by ROC curve), the positive predictive value of the antibody detection reached 100 percent, with a false positive rate of zero. The developed ELISA method for anti-ACTL7a antibody detection is therefore sensitive, accurate, and easy to perform, making it an excellent potential tool for future clinical use. PMID:26957350

  9. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    EPA Science Inventory

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

  10. Development of and assay methodology for antibodies to benzo(a) pyrene (BP)

    SciTech Connect

    Griffin, G.D.; Thomason, R.; Murchison, C.; St. Wecker, P.; Kurka, K.; Ambrose, K.R.

    1986-05-01

    Rabbits, rats and mice have been immunized with BP-bovine serum albumin (BSA) conjugates, administered subcutaneously in Freund's adjuvant. Activity and specificity of antisera preparations from immunized animals was determined by: Double immunodiffusion, passive hemagglutination, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). All techniques could be successfully used to demonstrate the presence of BP antibodies in immune sera. The fist three assays were used only with BP antigen coupled to a protein. If BSA was used, cross-reactivity against BSA was observed, but differences in extent of reactivity toward BP-BSA and BSA were readily apparent. Antisera from highly immune animals showed anti-BP reactivity at 1-10 x 10/sup 6/ dilutions in the ELISA assay, but did not produce positive reactions in the passive hemagglutination assay at >1:256 dilutions. The RIA assay was used with either /sup 14/C-or /sup 3/H-BP, and relied upon an activated charcoal separation step to effectively remove (>98% efficiency) free BP from antibody-bound BP. Using this RIA assay, estimates of amounts of BP antibodies in antisera (0.1-1% of the immunoglobulin fraction) and antibody specificity for BP structural determinants could be made. These antibodies may form the basis for a novel detection system for BP and/or other polycyclic compounds.

  11. Normalized enzyme-linked immunosorbent assay for determining immunoglobulin G antibodies to cytomegalovirus.

    PubMed Central

    Kiefer, D J; Phelps, D A; Halbert, S P

    1983-01-01

    A normalized enzyme-linked immunosorbent assay for the determination of immunoglobulin G antibodies to cytomegalovirus is described. The rapid assay involves three 30-min incubations and permits the quantitation of antibody levels with a single-specimen dilution in conjunction with a reference antibody preparation. The results obtained with the normalized enzyme-linked immunosorbent assay correlated closely with the results of complement fixation titrations and another commercially available enzyme-linked immunosorbent assay. The specificity of the procedure was further demonstrated by viral absorption, using cytomegalovirus from two different sources and other viral antigen preparations, including rubella and influenza. The reproducibility of the normalized test results is good and allows for greater uniformity of reporting on a day-to-day basis, as well as between laboratories. PMID:6309900

  12. Assay dependence of Brucella antibody prevalence in a declining Alaskan harbor seal (Phoca vitulina) population

    PubMed Central

    2013-01-01

    Background Brucella is a group of bacteria that causes brucellosis, which can affect population health and reproductive success in many marine mammals. We investigated the serological prevalence of antibodies against Brucella bacteria in a declining harbor seal population in Glacier Bay National Park, Alaska. Results Prevalence ranged from 16 to 74 percent for those tests detecting antibodies, indicating that harbor seals in Glacier Bay have been exposed to Brucella bacteria. However, the actual level of serological prevalence could not be determined because results were strongly assay-dependent. Conclusions This study reinforces the need to carefully consider assay choice when comparing different studies on the prevalence of anti–Brucella antibodies in pinnipeds and further highlights the need for species- or taxon-specific assay validation for both pathogen and host species. PMID:23324565

  13. Two-Component Direct Fluorescent-Antibody Assay for Rapid Identification of Bacillus Anthracis

    DTIC Science & Technology

    2002-10-01

    Bacillus spp. (n=56) Five closely related Bacillus species—B. cereus (n=23), B. megaterium (n=11), B. subtilis (n=9), B. thuringiensis (n=12), and B...Rapid Identification of Bacillus anthracis Barun K. De,* Sandra L. Bragg,* Gary N. Sanden,* Kathy E. Wilson,* Lois A. Diem,* Chung K. Marston...antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA

  14. Henipavirus microsphere immuno-assays for detection of antibodies against Hendra virus.

    PubMed

    McNabb, Leanne; Barr, J; Crameri, G; Juzva, S; Riddell, S; Colling, A; Boyd, V; Broder, C; Wang, L-F; Lunt, R

    2014-05-01

    Hendra and Nipah viruses (HeV and NiV) are closely related zoonotic pathogens of the Paramyxoviridae family. Both viruses belong to the Henipavirus genus and cause fatal disease in animals and humans, though only HeV is endemic in Australia. In general and due to the acute nature of the disease, agent detection by PCR and virus isolation are the primary tools for diagnostic investigations. Assays for the detection of antibodies against HeV are fit more readily for the purpose of surveillance testing in disease epidemiology and to meet certification requirements in the international movement of horses. The first generation indirect ELISA has been affected by non-specific reactions which must be resolved using virus neutralisation serology conducted at laboratory bio-safety level 4 containment (PC4). Recent developments have enabled improvements in the available serology assays. The production of an expressed recombinant truncated HeV G protein has been utilised in ELISA and in Luminex-based multiplexed microsphere assays. In the latter format, two Luminex assays have been developed for use in henipavirus serology: a binding assay (designed for antibody detection and differentiation) and a blocking assay (designed as a surrogate for virus neutralisation). Equine and canine field sera were used to evaluate the two Luminex assays relative to ELISA and virus neutralisation serology. Results showed that Luminex assays can be effective as rapid, sensitive and specific tests for the detection of HeV antibody in horse and dog sera. The tests do not require PC4 containment and are appropriate for high throughput applications as might be required for disease investigations and other epidemiological surveillance. Also, the results show that the Luminex assays detect effectively HeV vaccine-induced antibodies.

  15. Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria.

    PubMed

    Llewellyn, David; Miura, Kazutoyo; Fay, Michael P; Williams, Andrew R; Murungi, Linda M; Shi, Jianguo; Hodgson, Susanne H; Douglas, Alexander D; Osier, Faith H; Fairhurst, Rick M; Diakite, Mahamadou; Pleass, Richard J; Long, Carole A; Draper, Simon J

    2015-09-16

    The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field.

  16. Detection of antinuclear antibodies by indirect immunofluorescence and by solid phase assay.

    PubMed

    Op De Beeck, Katrijn; Vermeersch, Pieter; Verschueren, Patrick; Westhovens, René; Mariën, Godelieve; Blockmans, Daniel; Bossuyt, Xavier

    2011-10-01

    Testing for antinuclear antibodies is useful for the diagnosis of systemic rheumatic diseases. Solid phase assays are increasingly replacing indirect immunofluorescence for detection of antinuclear antibodies. In the most recent generation of solid phase assays, manufacturers attempt to improve the performance of the assays by adding extra antigens. Solid phase assay (EliA CTD Screen, Phadia, in which antibodies to 17 antigens are detected) was compared to indirect immunofluorescence for the detection of antinuclear antibodies in diagnostic samples of 236 patients with autoimmune connective tissue diseases, in 149 healthy blood donors, 139 patients with chronic fatigue syndrome, and 134 diseased controls. The sensitivity of EliA CTD Screen for systemic lupus erythematosus, systemic sclerosis, primary Sjögren's syndrome, mixed connective tissue disease, and inflammatory myopathy was 74%, 72%, 89%, 100%, and 39%, respectively. The reactivity in blood donors, in patients with chronic fatigue syndrome, and in diseased controls was <4%. Likelihood ratios increased with increasing antibody concentrations. Generally, a positive test result by EliA CTD Screen had a higher likelihood ratio for systemic rheumatic disease than a positive test result by indirect immunofluorescence. A negative test result by indirect immunofluorescence, however, had a lower likelihood ratio than a negative test result by EliA CTD Screen, indicating that the negative predictive value was higher for indirect immunofluorescence than for EliA CTD screen.

  17. Development a monoclonal antibody-based enzyme-linked immunosorbent assay for screening carotenoids in eggs.

    PubMed

    Peng, Dapeng; Liao, Feng; Pan, Yuanhu; Chen, Dongmei; Liu, Zhenli; Wang, Yulian; Yuan, Zonghui

    2016-07-01

    In this study, a monoclonal antibody (mAb) with broad-specificity against several carotenoid analogs with equal or similar efficacy was prepared. The obtained mAb C11, with the IgG1 isotype, showed cross-reactivity (CR) with canthaxanthin (100%), β-ionone acid (140.4%), β-carotene (92.9%), capsanthin (90.1%), β-apo-8'-carotenal (92.7%), and xanthophyll (95.8%). Using the mAb C11, a highly sensitive and inexpensive indirect competitive enzyme linked immunosorbent assay (ic-ELISA) was developed with a simple sample preparation procedure for the simultaneous detection of these carotenoid compounds in eggs. The limit of detection of the various carotenoids ranged from 1.31mgkg(-1) to 1.48mgkg(-1). Recoveries from egg yolks spiked with the above carotenoids ranged from 91.8% to 113.3%, with coefficients of variation (CVs) of less than 14.8%. These results suggest that the developed ic-ELISA is a sensitive, specific, accurate, and inexpensive method that is suitable for the screening of carotenoid residues in routine monitoring.

  18. Antibody binding in altered gravity: implications for immunosorbent assay during space flight

    NASA Technical Reports Server (NTRS)

    Maule, Jake; Fogel, Marilyn; Steele, Andrew; Wainwright, Norman; Pierson, Duane L.; McKay, David S.

    2003-01-01

    A single antibody-incubation step of an indirect, enzyme-linked immunosorbent assay (ELISA) was performed during microgravity, Martian gravity (0.38 G) and hypergravity (1.8 G) phases of parabolic flight, onboard the NASA KC-135 aircraft. Antibody-antigen binding occurred within 15 seconds; the level of binding did not differ between microgravity, Martian gravity and 1 G (Earth's gravity) conditions. During hypergravity and 1 G, antibody binding was directly proportional to the fluid volume (per microtiter well) used for incubation; this pattern was not observed during microgravity. These effects in microgravity may be due to "fluid spread" within the chamber (observed during microgravity with digital photography), leading to greater fluid-surface contact and subsequently antibody-antigen contact. In summary, these results demonstrate that: i) ELISA antibody-incubation and washing steps can be successfully performed by human operators during microgravity, Martian gravity and hypergravity; ii) there is no significant difference in antibody binding between microgravity, Martian gravity and 1 G conditions; and iii) a smaller fluid volume/well (and therefore less antibody) was required for a given level of binding during microgravity. These conclusions indicate that reduced gravity would not present a barrier to successful operation of immunosorbent assays during spaceflight.

  19. Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.

    PubMed

    Liu, Ang; Kozhich, Alexander; Passmore, David; Gu, Huidong; Wong, Richard; Zambito, Frank; Rangan, Vangipuram S; Myler, Heather; Aubry, Anne-Françoise; Arnold, Mark E; Wang, Jian

    2015-10-01

    Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys.

  20. The use of antibodies labelled with dyes for fast and simple assay of some human proteins by immunofiltration technique.

    PubMed

    Szewczuk, A; Kuropatwa, M; Rapak, A

    1992-01-01

    Immunofiltration technique with polyclonal and monoclonal antibodies for semi-quantitative assays of human albumin, chorionic gonadotropin, immunoglobulin G and transferrin was elaborated. An amount of antibody was immobolized in the form of 6 radially located small bars on a dry test filter made of glass microfibre sheet. The other amount of antibody, used in solution, was labelled with some dyes like commercial disperse dyes, colloidal elements, formazans and polypyrrole. Number of colour bars appearing on the test filter showed ranged of analyte concentration. Good results were obtained using antibodies labelled with colloidal gold, Disperse Red 11 and formazan from MTT. Assays with monoclonal antibodies were more sensitive than with polyclonal antibodies.

  1. An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies

    PubMed Central

    Estep, Patricia; Caffry, Isabelle; Yu, Yao; Sun, Tingwan; Cao, Yuan; Lynaugh, Heather; Jain, Tushar; Vásquez, Maximiliano; Tessier, Peter M; Xu, Yingda

    2015-01-01

    The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties. PMID:25790175

  2. Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation.

    PubMed

    Prince, J A; Feuk, L; Howell, W M; Jobs, M; Emahazion, T; Blennow, K; Brookes, A J

    2001-01-01

    We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by approximately 30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.

  3. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

  4. Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

    USGS Publications Warehouse

    Alcorn, S.W.; Pascho, R.J.

    2000-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibaclerium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

  5. International reference standards: antibody standards for the indirect enzyme-linked immunosorbent assay.

    PubMed

    Wright, P F; Tounkara, K; Lelenta, M; Jeggo, M H

    1997-12-01

    Reference standards are used to calibrate similar assay systems against an international reference protocol and to provide a template for the preparation of secondary and/or working standards. Three reference standards are recommended for the indirect enzyme-linked immunosorbent assay: a strong positive standard, a weak positive standard and a negative serum standard. The negative standard should be derived from a single serum or from a serum pool which exhibits typical background activity in the reference protocol. The strong and weak positive standards should be derived from a single serum or from a serum pool which typifies the humoral response (antibody) to natural infection. Suitable candidates for the positive reference standards should exhibit dose/response curves in the mid-range of antibody activity. The strong and weak positive standards should each be prepared from a one-time dilution in the negative standard, to yield antibody activities which are defined by specific points on the linear portion of the dose/response curve. The strong positive standard should represent an antibody activity (absorbance value) midway between the upper and central points and the weak positive standard should represent an antibody activity midway between the central and lower points of the linear portion of the curve. Owing to inherent differences among assay systems, antibody activities should be expressed in relative rather than in absolute terms. It is recommended that the antibody activity of the strong positive standard should denote 100% positivity. The activities of the weak positive and negative standards should then be expressed as relative percentages. Every set of international reference standards should be accompanied by an information sheet which includes, among other things, a plot of the dose/response curve and an indication of the dilutions used to prepare the standards.

  6. Effect of treatment on serum antibody to Hymenolepis nana detected by enzyme-linked immunosorbent assay.

    PubMed Central

    Castillo, R M; Grados, P; Carcamo, C; Miranda, E; Montenegro, T; Guevara, A; Gilman, R H

    1991-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum immunoglobulin G antibodies in 65 patients infected with Hymenolepis nana and 30 noninfected patients. Antibody was detected in 51 of 65 (sensitivity, 79%) and 5 of 30 H. nana-negative patients (specificity, 83%). Nine patients infected with H. nana were treated with praziquantel (20 to 25 mg/kg of body weight). Antibody disappeared from the sera at 90 days in six patients, five of whom had eliminated H. nana. Antibody persisted in three patients in whom H. nana infection did not clear after treatment. The H. nana ELISA had a high rate of cross-reactions with sera from patients with cysticercosis (8 of 29 [28%]) and hydatidosis (8 of 23 [35%]). The ELISA for H. nana may be useful for defining the epidemiology of H. nana infections, especially in areas free from cysticercosis and hydatidosis. PMID:2007652

  7. Effect of treatment on serum antibody to Hymenolepis nana detected by enzyme-linked immunosorbent assay.

    PubMed

    Castillo, R M; Grados, P; Carcamo, C; Miranda, E; Montenegro, T; Guevara, A; Gilman, R H

    1991-02-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum immunoglobulin G antibodies in 65 patients infected with Hymenolepis nana and 30 noninfected patients. Antibody was detected in 51 of 65 (sensitivity, 79%) and 5 of 30 H. nana-negative patients (specificity, 83%). Nine patients infected with H. nana were treated with praziquantel (20 to 25 mg/kg of body weight). Antibody disappeared from the sera at 90 days in six patients, five of whom had eliminated H. nana. Antibody persisted in three patients in whom H. nana infection did not clear after treatment. The H. nana ELISA had a high rate of cross-reactions with sera from patients with cysticercosis (8 of 29 [28%]) and hydatidosis (8 of 23 [35%]). The ELISA for H. nana may be useful for defining the epidemiology of H. nana infections, especially in areas free from cysticercosis and hydatidosis.

  8. Anti-DNA antibodies--overview of assays and clinical correlations.

    PubMed

    Rahman, A; Hiepe, F

    2002-01-01

    Many authors have studied the links between levels of anti-dsDNA antibodies and disease activity in patients with SLE. Interpretation of these studies must take into account the facts that there are a range of possible assays for anti-dsDNA and a number of indices available for assessing disease activity. A recent study compared levels of various autoantibodies with organ specific disease activity assessed during the British Isles Lupus Assessment Group (BILAG) index. Anti-dsDNA and anti-heparan sulphate levels were more likely to be raised in patients with renal than non-renal disease. Some anti-DNA antibodies are actually anti-nucleosome antibodies, which lose DNA reactivity when purified under dissociating conditions. Patients with SLE have significantly increased levels of nucleosomes in their sera compared with healthy controls. In patients with SLE, reduced clearance of nucleosomes released from apoptotic cells may induce the formation of anti-nucleosome antibodies.

  9. Comparison of measles virus-specific antibody titres as measured by enzyme-linked immunosorbent assay and virus neutralisation assay.

    PubMed

    van den Hof, Susan; van Gageldonk-Lafeber, Arianne B; van Binnendijk, Robert S; van Gageldonk, Pieter G M; Berbers, Guy A M

    2003-10-01

    We assessed whether measles virus-specific antibody levels in the Dutch population as estimated by an enzyme-linked immunosorbent assay (ELISA) were comparable with estimates by virus neutralisation assay (NT), prompted by a relatively low ELISA seroprevalence in the 10-21-year-old group. We tested 791 sera from individuals aged 2-49 years both in ELISA and NT. Seroprevalence in the 10-21-year-old group was 93.4% (95% confidence interval (CI) 89.5-97.2%) in ELISA versus 97.2% (CI 94.7-99.6%) in NT. There was good agreement between NT and ELISA seroprevalences in the vaccinated 2-9-year-olds and the unvaccinated 22-49-year-olds.

  10. Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine.

    PubMed

    Goodell, C K; Prickett, J; Kittawornrat, A; Johnson, J; Zhang, J; Wang, C; Zimmerman, J J

    2016-02-01

    Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti-IAV antibodies using homologous and heterologous haemagglutination-inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)-blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut-off of S/N ≤ 0.60, the sensitivity and specificity of the NP-blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post-inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost-effective approach for the detection and surveillance of IAV infections in swine populations.

  11. Commercially available antibodies can be applied in quantitative multiplexed peptide immunoaffinity enrichment targeted mass spectrometry assays

    PubMed Central

    Schoenherr, Regine M.; Zhao, Lei; Ivey, Richard G.; Voytovich, Uliana J.; Kennedy, Jacob; Yan, Ping; Lin, Chenwei; Whiteaker, Jeffrey R.; Paulovich, Amanda G.

    2016-01-01

    Immunoaffinity enrichment of peptides coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM) enables highly specific, sensitive, and precise quantification of peptides and post-translational modifications. Major obstacles to developing a large number of immuno-MRM assays are the poor availability of monoclonal antibodies (mAbs) validated for immunoaffinity enrichment of peptides and the cost and lead time of developing the antibodies de novo. Although many thousands of mAbs are commercially offered, few have been tested for application to immunoaffinity enrichment of peptides. In this study we tested the success rate of using commercially available mAbs for peptide immuno-MRM assays. We selected 105 commercial mAbs (76 targeting non-modified “pan” epitopes, 29 targeting phosphorylation) to proteins associated with the DNA damage response network. We found that 8 of the 76 pan (11%) and 5 of the 29 phospho-specific mAbs (17%) captured tryptic peptides (detected by LC-MS/MS) of their protein targets from human cell lysates. Seven of these mAbs were successfully used to configure and analytically characterize immuno-MRM assays. By applying selection criteria upfront, the results indicate that a screening success rate of up to 24% is possible, establishing the feasibility of screening a large number of catalog antibodies to provide readily-available assay reagents. PMID:27094115

  12. Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics

    PubMed Central

    Joubert, Marisa K.; Deshpande, Meghana; Yang, Jane; Reynolds, Helen; Bryson, Christine; Fogg, Mark; Baker, Matthew P.; Herskovitz, Jonathan; Goletz, Theresa J.; Zhou, Lei; Moxness, Michael; Flynn, Gregory C.; Narhi, Linda O.; Jawa, Vibha

    2016-01-01

    An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed. PMID:27494246

  13. [Evaluation of a Computer-Aided Microscope System and Its Anti-Nuclear Antibody Test Kit for Indirect Immunofluorescence Assay].

    PubMed

    Hayashi, Nobuhide; Saegusa, Jun; Uto, Kenichi; Oyabu, Chinami; Saito, Toshiharu; Sato, Itsuko; Kawano, Seiji; Kumagai, Shunichi

    2016-02-01

    Antinuclear antibody (ANA) testing is indispensable for diagnosing and understanding clinical conditions of autoimmune diseases. The indirect immunofluorescence assay (IFA) is the gold standard for ANA screening, and it can detect more than 100 different antibodies, such as anti-PCNA as well as anti-cytoplasmic antibodies. However, complicated procedures of conventional IFA and visual interpretation require highly skilled laboratory staff. This study evaluates the capability, characteristics, and applicability of the recently developed ANA detection system (EUROPattern Cosmic IFA System, EPA) using HEp20-10 cells and the automated pattern recognition microscope. Findings using EPA and conventional methods were compared in 282 sera obtained from connective tissue disease patients and 250 sera from healthy individuals. The concordance of the positivity rate, antibody titer (within +/- 1 tube difference), and the accurate recognition rate of ANA patterns between the automated EPA method and the microscopic judgement of the EPA image by eye was 98.9, 97.4, and 55.3%, respectively. The EPA method showed concordance of the positivity rate as high as 93.3% and concordance of the antibody titer as high as 94.0% (within +/- 1 titer) compared with the conventional method. Regarding the four typical patterns of ANA (homogeneous, speckled, nucleolar, and centromere), large differences between the EPA and conventional methods were not observed, and the rate of concordance between the final EPA result and the conventional method was from 94.1 to 100%. The positivity rate of ANA using the EPA and conventional methods showed marked agreement among the six connective tissue diseases (SLE, MCTD, SSc, PM/DM, and SS) and healthy individuals. Although the EPA system is not considered a complete system and laboratory staff should verify the results, it is a useful system for routine ANA analysis because it contributes to ANA standardization and an efficient workflow.

  14. High Concentrations of Measles Neutralizing Antibodies and High-Avidity Measles IgG Accurately Identify Measles Reinfection Cases

    PubMed Central

    Rota, Jennifer S.; Hickman, Carole J.; Mercader, Sara; Redd, Susan; McNall, Rebecca J.; Williams, Nobia; McGrew, Marcia; Walls, M. Laura; Rota, Paul A.; Bellini, William J.

    2016-01-01

    In the United States, approximately 9% of the measles cases reported from 2012 to 2014 occurred in vaccinated individuals. Laboratory confirmation of measles in vaccinated individuals is challenging since IgM assays can give inconclusive results. Although a positive reverse transcription (RT)-PCR assay result from an appropriately timed specimen can provide confirmation, negative results may not rule out a highly suspicious case. Detection of high-avidity measles IgG in serum samples provides laboratory evidence of a past immunologic response to measles from natural infection or immunization. High concentrations of measles neutralizing antibody have been observed by plaque reduction neutralization (PRN) assays among confirmed measles cases with high-avidity IgG, referred to here as reinfection cases (RICs). In this study, we evaluated the utility of measuring levels of measles neutralizing antibody to distinguish RICs from noncases by receiver operating characteristic curve analysis. Single and paired serum samples with high-avidity measles IgG from suspected measles cases submitted to the CDC for routine surveillance were used for the analysis. The RICs were confirmed by a 4-fold rise in PRN titer or by RT-quantitative PCR (RT-qPCR) assay, while the noncases were negative by both assays. Discrimination accuracy was high with serum samples collected ≥3 days after rash onset (area under the curve, 0.953; 95% confidence interval [CI], 0.854 to 0.993). Measles neutralizing antibody concentrations of ≥40,000 mIU/ml identified RICs with 90% sensitivity (95% CI, 74 to 98%) and 100% specificity (95% CI, 82 to 100%). Therefore, when serological or RT-qPCR results are unavailable or inconclusive, suspected measles cases with high-avidity measles IgG can be confirmed as RICs by measles neutralizing antibody concentrations of ≥40,000 mIU/ml. PMID:27335386

  15. Prevalence of antibodies to type A influenza virus in wild avian species using two serologic assays

    USGS Publications Warehouse

    Brown, Justin D.; Luttrell, M. Page; Berghaus, Roy D.; Kistler, Whitney; Keeler, Shamus P.; Howey, Andrea; Wilcox, Benjamin; Hall, Jeffrey; Niles, Larry; Dey, Amanda; Knutsen, Gregory; Fritz, Kristen; Stallknecht, David E.

    2010-01-01

    Serologic testing to detect antibodies to avian influenza (AI) virus has been an underused tool for the study of these viruses in wild bird populations, which traditionally has relied on virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR). In a preliminary study, a recently developed commercial blocking enzyme-linked immunosorbent assay (bELISA) had sensitivity and specificity estimates of 82% and 100%, respectively, for detection of antibodies to AI virus in multiple wild bird species after experimental infection. To further evaluate the efficacy of this commercial bELISA and the agar gel immunodiffusion (AGID) test for AI virus antibody detection in wild birds, we tested 2,249 serum samples collected from 62 wild bird species, representing 10 taxonomic orders. Overall, the bELISA detected 25.4% positive samples, whereas the AGID test detected 14.8%. At the species level, the bELISA detected as many or more positive serum samples than the AGID in all 62 avian species. The majority of positive samples, detected by both assays, were from species that use aquatic habitats, with the highest prevalence from species in the orders Anseriformes and Charadriiformes. Conversely, antibodies to AI virus were rarely detected in the terrestrial species. The serologic data yielded by both assays are consistent with the known epidemiology of AI virus in wild birds and published reports of host range based on virus isolation and RT-PCR. The results of this research are also consistent with the aforementioned study, which evaluated the performance of the bELISA and AGID test on experimental samples. Collectively, the data from these two studies indicate that the bELISA is a more sensitive serologic assay than the AGID test for detecting prior exposure to AI virus in wild birds. Based on these results, the bELISA is a reliable species-independent assay with potentially valuable applications for wild bird AI surveillance.

  16. Development of an enzyme-linked immunosorbent assay (ELISA) using highly-specific monoclonal antibodies against plumbagin.

    PubMed

    Sakamoto, Seiichi; Putalun, Waraporn; Tsuchihashi, Ryota; Morimoto, Satoshi; Kinjo, Junei; Tanaka, Hiroyuki

    2008-01-21

    Plumbagin (PL; 5-hydroxy-2-methyl-1,4-naphthoquinone) is a natural compound mainly isolated from Plumbago zeylanica. This plant is distributed in Southeast Asia, and well known as Ayurvedic medicine in India for its medicinal properties. PL has been shown to have various pharmacological activities. We have successfully prepared monoclonal antibodies against PL, and developed an enzyme-linked immunosorbent assay (ELISA) system for determination of PL. 3-(5-Hydroxy-2-methyl-1,4-naphthoquinone-3-yl) propanoic acid was synthesized and purified to prepare PL-bovine serum albumin conjugate (PL-BSA), which was used as an immunogen. PL-BSA conjugate was administered into BALB/c male mice for production of monoclonal antibodies against PL. The monoclonal antibody against PL which is secreted from established hybridoma cell line 3A3 (MAb 3A3) has been proven to have highly-specific to PL resulting from cross-reactivities test. The range for calibration of PL by ELISA was 0.2-25 microg mL(-1). Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of PL in plant.

  17. A rapid assay for Hendra virus IgG antibody detection and its titre estimation using magnetic nanoparticles and phycoerythrin.

    PubMed

    Gao, Yuan; Pallister, Jackie; Lapierre, Florian; Crameri, Gary; Wang, Lin-Fa; Zhu, Yonggang

    2015-09-15

    Detection of Hendra viral IgG antibody in animal sera is useful for surveillance following a virus outbreak. The commonly used enzyme-linked immunosorbent assay and fluorescence-based Luminex assay typically consist of three steps and take at least several hours to complete. We have simplified the procedure to two steps in an effort to develop a rapid procedure for IgG antibody, but not IgM antibody, detection. This is achieved by conjugating the fluorescence label R-phycoerythrin directly onto the IgG binding protein Protein G. The use of magnetic nanoparticles, due to their large specific surface area, has helped reduce each of the binding steps to 20 min. As a result, the whole assay can be completed in 60 min. We also demonstrate a method to quickly estimate IgG antibody titres by assaying the sera at only two dilutions (i.e. 1:20 and 1:1000) and using the fluorescence ratio at these dilutions as an indicator of antibody titre. The results of this approach correlated well with the well-regarded serum neutralization test in virus antibody assays. This protocol reported here can be adopted in Luminex assays, fluorescence-linked immunosorbent assays and assays on microfluidics platforms for rapid antibody surveillance of Hendra and other viruses.

  18. Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to Pisum and Avena phytochrome

    SciTech Connect

    Cordonnier, M.M.; Greppin, H.; Pratt, L.H.

    1984-01-01

    Nine monoclonal antibodies to pea (Pisum sativum L.) and 16 to oat (Avena sativa L.) phytochrome are characterized by enzyme-linked immunosorbent assay against phytochrome from six different sources: pea, zucchini (Cucurbita pepo L.), lettuce (Lactuca sativa L.), oat, rye (Secale cereale L.), and barley (Hordeum vulgare L.). All antibodies were raised against phytochrome with a monomer size near 120,000 daltons. Nevertheless, none of them discriminated qualitatively between 118/114-kilodalton oat phytochrome and a photoreversible, 60-kilodalton proteolytic degradation product derived from it. In addition, none of the 23 antibodies tested discriminated substantially between phytochrome - red-absorbing form and phytochrome - far red-absorbing form. Two antibodies to pea and six to oat phytochrome also bound strongly to phytochrome from the other species, even though these two plants are evolutionarily widely divergent. Of these eight antibodies, two bound significantly to all of the six phytochrome preparations tested, indicating that these two may recognize highly conserved regions of the chromoprotein. Since the molecular function of phytochrome is unknown, these two antibodies may serve as unique probes for regions of this pigment that are important to its mode of action. 27 references, 3 figures, 1 table.

  19. Enzyme-linked immunosorbent assay for immunoglobulin M specific antibody for the diagnosis of melioidosis.

    PubMed Central

    Kunakorn, M; Boonma, P; Khupulsup, K; Petchclai, B

    1990-01-01

    Indirect hemagglutination (IHA) is commonly used for serodiagnosis of melioidosis. However, in endemic areas, high background titers in normal populations and occasional low titers in patients with septicemic melioidosis prompted a search for a more sensitive and more specific method of serodiagnosis. An indirect fluorescent-antibody test for immunoglobulin M (IgM) specific antibody to Pseudomonas pseudomallei was more sensitive and more specific, but fluorescence microscopes are rarely available in the endemic areas. An enzyme-linked immunosorbent assay (ELISA) for IgM antibody is an attractive alternative. An indirect ELISA for IgM antibody (IgM ELISA) and an IgM antibody capture ELISA for melioidosis were developed. Both tests, together with IHA, were evaluated for 153 serum specimens from blood donors and 16 serum specimens from 16 melioidosis patients. It was found that IHA, the IgM ELISA, and the IgM antibody capture ELISA had sensitivities of 88, 88, and 75%, respectively, with specificities of 97.4, 92.2, and 91.5%, respectively. When IHA was combined with IgM ELISA, a sensitivity of 100% and a specificity of 95.4% were obtained. The IgM ELISA and IHA should be used in combination for serodiagnosis of melioidosis. PMID:2199494

  20. A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies

    PubMed Central

    Stengl, Andreas; Hörl, David; Leonhardt, Heinrich; Helma, Jonas

    2016-01-01

    Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2′-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell–based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2′-deoxyuridine (EdU) and 4′,6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs. PMID:27909235

  1. Monoclonal antibody-based competitive enzyme-linked immunosorbent assay for detection of morbillivirus antibody in marine mammal sera.

    PubMed

    Saliki, J T; Lehenbauer, T W

    2001-05-01

    A competitive enzyme-linked immunosorbent assay (cELISA), using two monoclonal antibodies (MAbs), was developed and compared with the standard virus neutralization test (VNT) for detecting antibodies against canine distemper virus (CDV) and phocine distemper virus (PDV) in sera from dogs and various species of marine mammals. The test depends on the blocking of MAb binding to solid-phase antigen in the presence of positive serum. Test conditions were optimized by using control VNT-negative and -positive sera specific for CDV and PDV. A positive cutoff value of 30% inhibition, which represents the mean cutoff of a VNT-negative population (n = 623) plus 2 standard deviations, was adopted for the test. A total of 736 serum samples were tested by the new cELISA and by the VNT as the "gold standard." An unexpected but useful finding was the ability of this CDV- and PDV-specific cELISA to also detect antibodies against the related pair dolphin morbillivirus and porpoise morbillivirus. Based on a subpopulation of 625 sera used in statistical analyses, the overall sensitivity and specificity of cELISA relative to those of the VNT were 94.9 and 97.7%, respectively. Because the cELISA proved to be nearly as sensitive and specific as the VNT while being simpler and more rapid, it would be an adequate screening test for suspect CDV or PDV cases and would also be useful for epidemiological surveillance of morbilliviral infections in marine mammal populations.

  2. Highly Accurate Antibody Assays for Early and Rapid Detection of Tuberculosis in African and Asian Elephants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current methods for screening and diagnosis rely on trunk wash culture, which has serious limitations due to low test sensitivity, slow turn-around time, and variable sample quality. Inn...

  3. Specific immunoradiometric assay of insulin-like growth factor I with use of monoclonal antibodies.

    PubMed

    Scott, M G; Cuca, G C; Petersen, J R; Lyle, L R; Burleigh, B D; Daughaday, W H

    1987-11-01

    We identified two monoclonal antibodies that bind spatially distinct epitopes on insulin-like growth factor I (IGF-I). Using these two antibodies, we developed a simultaneous, two-site immunoradiometric assay (IRMA) specific for IGF-I. This IRMA has no detectable cross reactivity with insulin, proinsulin, prolactin, or somatotropin, and less than 2% crossreactivity with IGF-II. The assay response varies linearly with IGF-I concentrations of 0-800 micrograms/L in serum; the detection limit is about 10 micrograms/L. A comparison of 26 IGF-I serum values from the IRMA and from a previously reported IGF-I specific RIA gave a correlation coefficient of 0.96 with no substantial bias (slope = 1.10). IGF-I values for serum, as an aid in assessing growth abnormalities, are easily (only three pipetting steps) obtained in less than 4 h.

  4. Application of a Rapid Assay for Detection of Antibodies to Human Immunodeficiency Virus in Urine

    DTIC Science & Technology

    1994-01-01

    epidemiologic surveys, and in devel- and no false-negative results when compared with the Western blot . oping countries. The authors evaluated the...in urine. AL of sample was required. By Western blot analysis, antibody profiles Test performance and applicability of the SUDS test were compared in...compromised when larger volumes were used. The au- immunosorbent assay (ELISA) and Western blot using 139 serum and thors concluaed that this rapid

  5. Detection of anti-infliximab antibodies is impacted by antibody titer, infliximab level and IgG4 antibodies: a systematic comparison of three different assays

    PubMed Central

    Afonso, Joana; Lopes, Susana; Gonçalves, Raquel; Caldeira, Paulo; Lago, Paula; Tavares de Sousa, Helena; Ramos, Jaime; Gonçalves, Ana Rita; Ministro, Paula; Rosa, Isadora; Vieira, Ana Isabel; Coelho, Rosa; Tavares, Patrícia; Soares, João; Sousa, Ana Lúcia; Carvalho, Diana; Sousa, Paula; da Silva, João Pereira; Meira, Tânia; Silva Ferreira, Filipa; Dias, Cláudia Camila; Chowers, Yehuda; Ben-Horin, Shomron; Magro, Fernando

    2016-01-01

    Background: There is scant information on the accuracy of different assays used to measure anti-infliximab antibodies (ADAs), especially in the presence of detectable infliximab (IFX). We thus aimed to evaluate and compare three different assays for the detection of IFX and ADAs and to clarify the impact of the presence of circulating IFX on the accuracy of the ADA assays. Methods: Blood samples from 79 ulcerative colitis (UC) patients treated with infliximab were assessed for IFX levels and ADAs using three different assays: an in-house assay and two commercial kits, Immundiagnostik and Theradiag. Sera samples with ADAs and undetectable levels of IFX were spiked with exogenous IFX and analyzed for ADAs. Results: The three assays showed 81–96% agreement for the measured IFX level. However, the in-house assay and Immundiagnostik assays detected ADAs in 34 out of 79 samples, whereas Theradiag only detected ADAs in 24 samples. Samples negative for ADAs with Theradiag, but ADA-positive in both the in-house and Immundiagnostik assays, were positive for IFX or IgG4 ADAs. In spiking experiments, a low concentration of exogenous IFX (5 µg/ml) hampered ADA detection with Theradiag in sera samples with ADA levels of between 3 and 10 µg/ml. In the Immundiagnostik assay detection interference was only observed at concentrations of exogenous IFX higher than 30 µg/ml. However, in samples with high levels of ADAs (>25 µg/ml) interference was only observed at IFX concentrations higher than 100 µg/ml in all three assays. Binary (IFX/ADA) stratification of the results showed that IFX+/ADA- and IFX-/ADAs+ were less influenced by the assay results than the double-positive (IFX+/ADAs+) and double-negative (IFX-/ADAs-) combination. Conclusions: All three methodologies are equally suitable for measuring IFX levels. However, erroneous therapeutic decisions may occur when patients show double-negative (IFX-/ADAs-) or double-positive (IFX+/ADAs+) status, since agreement between

  6. Rapid detection of infectious mononucleosis-associated heterophile antibodies by a novel immunochromatographic assay and a latex agglutination test.

    PubMed Central

    Farhat, S E; Finn, S; Chua, R; Smith, B; Simor, A E; George, P; Diena, B B; Diena, D; Skulnick, M

    1993-01-01

    A novel immunochromatographic assay, the CARDS O.S. MONO test (Pacific Biotech, San Diego, Calif.), and a latex agglutination test, the Infectious Mononucleosis Kit (Unipath Ltd., Hampshire, United Kingdom) were compared with the Paul-Bunnell-Davidsohn test. Of the 957 serum specimens studied, 78 were positive and 879 were negative by the Paul-Bunnell-Davidsohn test. After discrepancies were resolved by determining Epstein-Barr virus serology, the sensitivities of the CARDS O.S. MONO test and the Infectious Mononucleosis Kit were 91.0 and 96.2%, respectively, and both tests had a specificity and a positive predictive value of 100% and a negative predictive value and overall agreement of greater than 99%. The results show that both tests can accurately detect infectious mononucleosis-associated heterophile antibodies. PMID:8315001

  7. Thrombotic risk assessment in antiphospholipid syndrome: the role of new antibody specificities and thrombin generation assay.

    PubMed

    Sciascia, Savino; Baldovino, Simone; Schreiber, Karen; Solfietti, Laura; Radin, Massimo; Cuadrado, Maria J; Menegatti, Elisa; Erkan, Doruk; Roccatello, Dario

    2016-01-01

    Antiphospholipid syndrome (APS) is an autoimmune condition characterized by the presence of antiphospholipid antibodies (aPL) in subjects presenting with thrombosis and/or pregnancy loss. The currently used classification criteria were updated in the international consensus held in Sidney in 2005. Vascular events seem to result of local procoagulative alterations upon triggers influence (the so called "second-hit theory"), while placental thrombosis and complement activation seem to lead to pregnancy morbidity. The laboratory tests suggested by the current classification criteria include lupus anticoagulant, a functional coagulation assay, and anticardiolipin and anti-β2-glycoprotein-I antibodies, generally detected by solid phase enzyme-linked immunosorbent assay. The real challenge for treating physicians is understanding what is the actual weight of aPL in provoking clinical manifestations in each case. As thrombosis has a multi-factorial cause, each patient needs a risk-stratified approach. In this review we discuss the role of thrombotic risk assessment in primary and secondary prevention of venous and arterial thromboembolic disease in patients with APS, focusing on new antibody specificities, available risk scoring models and new coagulation assays.

  8. Domain based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein

    PubMed Central

    Meng, Q.; Li, M.; Silberg, M.A.; Conrad, F.; Bettencourt, J.; To, R.; Huang, C.; Ma, J.; Meyer, K.; Shimizu, R.; Cao, L.; Tomic, M.T.; Marks, J.D.

    2014-01-01

    Quantitation of individual mAbs within a combined antibody drug product is required for preclinical and clinical drug development including pharmacokinetics (PK), toxicology, stability and biochemical characterization studies of such drugs. We have developed an antitoxin (XOMA 3AB) consisting of three recombinant monoclonal antibodies (mAbs) that potently neutralizes the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind non-overlapping BoNT/A epitopes with high affinity. XOMA3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from E. coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. MAb specific domains were used to develop an ELISA for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay was also developed that is robust to interference from components in serum and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein and is superior to anti-idiotype approaches. PMID:22037290

  9. An improved Bathocuproine assay for accurate valence identification and quantification of copper bound by biomolecules.

    PubMed

    Chen, Dinglong; Darabedian, Narek; Li, Zhiqiang; Kai, Tianhan; Jiang, Dianlu; Zhou, Feimeng

    2016-03-15

    Copper is an essential metal in all organisms. Reliably quantifying and identifying the copper content and oxidation state is crucial, since the information is essential to understanding protein structure and function. Chromophoric ligands, such as Bathocuproine (BC) and its water-soluble analog, Bathocuproinedisulfonic acid (BCS), preferentially bind Cu(I) over Cu(II), and therefore have been widely used as optical probes to determine the oxidation state of copper bound by biomolecules. However, the BCS assay is commonly misused, leading to erroneous conclusions regarding the role of copper in biological processes. By measuring the redox potential of Cu(II)-BCS2 and conducting UV-vis absorption measurements in the presence of oxidizable amino acids, the thermodynamic origin of the potential artifacts becomes evident. The BCS assay was improved by introducing a strong Cu(II) chelator EDTA prior to the addition of BCS to prevent interference that might arise from Cu(II) present in the sample. The strong Cu(II) chelator rids of all the potential errors inherent in the conventional BCS assay. Applications of the improved assay to peptides and protein containing oxidizable amino acid residues confirm that free Cu(II) no longer leads to artifacts, thereby resolving issues related to this persistently misused colorimetric assay of Cu(I) in biological systems.

  10. Measurement of antibodies to pneumococcal, meningococcal and haemophilus polysaccharides, and tetanus and diphtheria toxoids using a 19-plexed assay.

    PubMed

    Whitelegg, Alison M E; Birtwistle, Jane; Richter, Alex; Campbell, John P; Turner, James E; Ahmed, Tarana M; Giles, Lynda J; Fellows, Mark; Plant, Tim; Ferraro, Alastair J; Cobbold, Mark; Drayson, Mark T; MacLennan, Calman A

    2012-03-30

    The measurement of antibody responses to vaccination is useful in the assessment of immune status in suspected immune deficiency. Previous reliance on enzyme-linked immunoabsorbent assays (ELISA) has been cumbersome, time-consuming and expensive. The availability of flow cytometry systems has led to the development of multiplexed assays enabling simultaneous measurement of antibodies to several antigens. We optimized a flow cytometric bead-based assay to measure IgG and IgM concentrations in serum to 19 antigens contained in groups of bacterial subunit vaccines: pneumococcal vaccines, meningococcal vaccines, Haemophilus influenzae b (Hib), and tetanus and diphtheria toxoid vaccines. 89-SF was employed as the standard serum. The assay was used to determine specific antibody levels in serum from 193 healthy adult donors. IgG and pneumococcal IgM antibody concentrations were measurable across 3 log10 ranges encompassing the threshold protective IgG antibody levels for each antigen. There was little interference between antibody measurements by the 19-plexed assay compared with monoplexed assays, and a lack of cross-reactive IgG antibody, but evidence for cross-reacting IgM antibody for 3/19 pneumococcal antigens. 90th centile values for 15/19 IgG concentrations and 12/12 IgM concentrations of the 193 adult sera were within these ranges and percentages of sera containing protective IgG antibody levels varied from 4% to 95% depending on antigen. This multiplexed assay can simultaneously measure antibody levels to 19 bacterial vaccine antigens. It is suitable for use in standard clinical practice to assess the in vivo immune response to test vaccinations and measure absolute antibody levels to these antigens.

  11. Comparison of Antibody Responses to Human Papillomavirus Vaccination as Measured by Three Assays

    PubMed Central

    Robbins, Hilary A.; Kemp, Troy J.; Porras, Carolina; Rodriguez, Ana Cecilia; Schiffman, Mark; Wacholder, Sholom; Gonzalez, Paula; Schiller, John; Lowy, Douglas; Poncelet, Sylviane; Esser, Mark; Matys, Katie; Hildesheim, Allan; Pinto, Ligia A.; Herrero, Rolando; Safaeian, Mahboobeh

    2014-01-01

    Background: Different assays, including the competitive Luminex immunoassay (cLIA), secreted alkaline phosphatase neutralization assay (SEAP-NA), and virus-like particle-based ELISA, are commonly used to measure antibody responses after human papillomavirus (HPV) vaccination. Direct assay comparisons aid interpretation of immunogenicity data evaluated by different assays. Methods: We compared cLIA to SEAP-NA and ELISA among 51 HPV16/18-vaccinated women enrolled in the Costa Rica Vaccine Trial. We tested replicate serum samples collected at months 0, 1, and 12 by HPV16/18 cLIA, SEAP-NA, and ELISA. For a subset (N = 10), we further tested month 6, 24 and 36 samples. We calculated seroprevalence estimates and Spearman rank correlation coefficients comparing cLIA to SEAP-NA and ELISA. Results: After one vaccine dose, seroprevalence by SEAP-NA and ELISA was 100% (both HPV16 and HPV18), and by cLIA was 96% (95% CI 87–100%) for HPV16 and 71% (95% CI 56–83%) for HPV18. Seroprevalence was 100% by all assays after three doses. Correlation between assays was high after one vaccine dose [cLIA/SEAP-NA ρ = 0.91 (HPV16) and ρ = 0.86 (HPV18); cLIA/ELISA ρ = 0.84 (HPV16) and ρ = 0.74 (HPV18); all p < 0.001] and remained high through month 36. Ratios of mean antibody levels to seropositivity cutoffs at month 36 were lower for cLIA than for SEAP-NA or ELISA, particularly for HPV18 (HPV18 ratio for cLIA 1.9, SEAP-NA 3.5, ELISA 3.4). Conclusion: Though correlation between cLIA and SEAP-NA/ELISA is high and stable after vaccination, the assays differ in scale and sensitivity, with notable differences after one vaccine dose and for HPV18. Our results demonstrate that comparisons of antibody responses to HPV vaccination measured by different assays are approximate, and must consider biological and technical differences between assays. PMID:24455487

  12. Improvement in the specificity of assays for detection of antibody to hepatitis B core antigen.

    PubMed Central

    Weare, J A; Robertson, E F; Madsen, G; Hu, R; Decker, R H

    1991-01-01

    Reducing agents dramatically alter the specificity of competitive assays for antibody to hepatitis B core antigen (anti-HBc). A specificity improvement was demonstrated with a new assay which utilizes microparticle membrane capture and chemiluminescence detection as well as a current radioimmunoassay procedure (Corab: Abbott Laboratories, Abbott Park, Ill.). The effect was most noticeable with elevated negative and weakly reactive samples. In both systems, reductants increased separation of a negative population (n = 160) from assay cutoffs. With a selected population (n = 307), inclusion of reductant eliminated apparent anti-HBc activity in 54 of 81 samples in the 30 to 70% inhibition range. Reductant-stable anti-HBc samples were strongly associated with the presence of antibody to hepatitis B surface antigen (21 of 27). The association persisted below the detection limits of current assays to 0.3 to 0.4 Paul Ehrlich Institute units per ml. Only 1 of 54 reduction-sensitive borderline samples was confirmed to be positive for antibody to hepatitis B surface antigen. The modified procedures had unchanged or slightly improved sensitivity for immunoglobulin G (IgG)-associated anti-HBc activity. Although IgM anti-HBc detection was reduced from four- to eightfold in the presence of reductants, sensitivities remained at least twofold greater than tha of an enzyme immunoassay (Corzyme M; Abbott) designed to detect acute-phase levels of IgM anti-HBc. The use of reducing agents should significantly improve the reliability of anti-HBc testing, especially near assay cutoffs. PMID:2037678

  13. How-to-do-it: Immunological Assays for the Classroom 1. Enzyme Linked Immunosorbent Assay (ELISA): A Laboratory Tool for Demonstration of Antibody-Antigen Interaction.

    ERIC Educational Resources Information Center

    Russo, A. J.; And Others

    1984-01-01

    Background information, list of required materials, and procedures are provided for an immunological assay which has been modified for use as a classroom/laboratory demonstration of antigen-antibody reaction. The assay is designed for a two and one-half hour laboratory period but may be modified for one hour laboratories. (JN)

  14. Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

    PubMed

    Riffelmann, M; Thiel, K; Schmetz, J; Wirsing von Koenig, C H

    2010-12-01

    Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of ELISA kits that were commercially available in Germany. Eleven measured IgG antibodies, and nine measured IgA antibodies. An in-house ELISA with purified antigens served as a reference method. Samples included two WHO reference preparations, the former Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research (CBER) reference preparations, serum samples from patients with clinically suspected pertussis, and serum samples from patients having received a combined tetanus, diphtheria, and pertussis (Tdap) vaccination. Kits using pertussis toxin (PT) as an antigen showed linearity compared to the WHO Reference preparation (r2 between 0.82 and 0.99), and these kits could quantify antibodies according to the reference preparation. ELISA kits using mixed antigens showed no linear correlation to the reference preparations. Patient results were compared to results of in-house ELISAs using a dual cutoff of either ≥100 IU/ml anti-PT IgG or ≥40 IU/ml anti-PT IgG together with ≥12 IU/ml anti-PT IgA. The sensitivities of kits measuring IgG antibodies ranged between 0.84 and 1.00. The specificities of kits using PT as an antigen were between 0.81 and 0.93. The specificities of kits using mixed antigens were between 0.51 and 0.59 and were thus not acceptable. The sensitivities of kits measuring IgA antibodies ranged between 0.53 and 0.73, and the specificities were between 0.67 and 0.94, indicating that IgA antibodies may be of limited diagnostic value. Our data suggest that ELISAs should use purified PT as an antigen and be standardized to the 1st International Reference preparation.

  15. An accurate DNA marker assay for stem rust resistance gene Sr2 in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The stem rust resistance gene Sr2 has provided broad-spectrum protection against stem rust (Puccinia graminis) since its wide spread deployment in wheat from the 1940s. Because Sr2 confers partial resistance which is difficult to select under field conditions, a DNA marker is desirable that accurate...

  16. Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis

    PubMed Central

    Smith, William L.; Chadwick, Sean G.; Toner, Geoffrey; Mordechai, Eli; Adelson, Martin E.; Aguin, Tina J.; Sobel, Jack D.

    2016-01-01

    Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease: Gardnerella vaginalis, Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the order Clostridiales), Megasphaera phylotype 1 or 2, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. We generated a logistic regression model that identified G. vaginalis, A. vaginae, and Megasphaera phylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion of Lactobacillus spp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV. PMID:26818677

  17. An extended set of yeast-based functional assays accurately identifies human disease mutations

    PubMed Central

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L.; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E.; Vidal, Marc; Andrews, Brenda J.; Boone, Charles; Dolinski, Kara; Roth, Frederick P.

    2016-01-01

    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods. PMID:26975778

  18. Evaluation of the antibody response in pigs vaccinated against Streptococcus suis capsular type 2 using a double-antibody sandwich enzyme-linked immunosorbent assay.

    PubMed Central

    Blouin, C; Higgins, R; Gottschalk, M; Simard, J

    1994-01-01

    A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was standardized for the detection of specific antibodies following vaccination with Streptococcus suis capsular type 2 bacterins. No statistically significant increase of antibody titers was detected in vaccinated piglets compared to the nonvaccinated control group, even if a minority of piglets demonstrated an important postvaccinal response. Three of four vaccinated sows showed a low antibody response to vaccine and specific immunity was detected in piglets of only one litter of these three sows. Passive protection studies showed that none of the sera from vaccinated piglets were protective for mice whereas serum obtained from hyperimmunized pigs gave protection. PMID:8143253

  19. An improved method for utilization of peptide substrates for antibody characterization and enzymatic assays.

    PubMed

    Ghosh, Inca; Sun, Luo; Evans, Thomas C; Xu, Ming-Qun

    2004-10-01

    Synthetic peptides have become an important tool in antibody production and enzyme characterization. The small size of peptides, however, has hindered their use in assays systems, such as Western blots, and as immunogens. Here, we present a facile method to improve the properties of peptides for multiple applications by ligating the peptides to intein-generated carrier proteins. The stoichiometric ligation of peptide and carrier achieved by intein-mediated protein ligation (IPL) results in the ligation product migrating as a single band on a SDS-PAGE gel. The carrier proteins, HhaI methylase (M.HhaI) and maltose-binding protein (MBP), were ligated to various peptides; the ligated carrier-peptide products gave sharp, reproducible bands when used as positive controls for antibodies raised against the same peptides during Western blot analysis. We further show that ligation of the peptide antigens to a different thioester-tagged carrier protein, paramyosin, produced immunogens for the production of antisera in rabbits or mice. Furthermore, we demonstrate the generation of a substrate for enzymatic assays by ligating a peptide containing the phosphorylation site for Abl protein tyrosine kinase to a carrier protein. This carrier-peptide protein was used as a kinase substrate that could easily be tested for phosphorylation using a phosphotyrosine antibody in Western blot analysis. These techniques do not require sophisticated equipment, reagents, or skills thereby providing a simple method for research and development.

  20. Enzyme-linked immunosorbent assay screening then indirect immunofluorescence confirmation of antinuclear antibodies: a statistical analysis.

    PubMed

    Copple, Susan S; Sawitzke, Allen D; Wilson, Andrew M; Tebo, Anne E; Hill, Harry R

    2011-05-01

    The purpose of this study was to analyze antinuclear antibody (ANA) screening by enzyme-linked immunosorbent assay (ELISA) followed by indirect fluorescent antibody (IFA) testing to confirm and characterize the pattern and titer of the antibody. We evaluated 4 ANA ELISAs and 1 HEp-2 IFA substrate in 224 clinically defined serum samples consisting of 30 from systemic lupus erythematosus (SLE) cases, 94 from rheumatoid arthritis cases, and 100 from healthy donors plus 495 serum samples submitted for routine ANA testing and 12 reference serum samples distributed by the Centers for Disease Control and Prevention. IFA tests were read independently by 2 certified medical technologists. ELISA sensitivities ranged from 90% to 97% compared with 80% by IFA in the SLE serum samples. The ELISAs had specificities of 36% to 94%, whereas the IFA had 99% specificity. Overall, ELISAs for ANA assays demonstrated better sensitivity and good specificity, suggesting ELISA is a more cost-effective alternative to IFA testing for initial ANA screening. Samples positive by ANA ELISA should be tested on HEp-2 to determine the titer and pattern.

  1. Development and evaluation of an immunochromatographic assay using a gp51 monoclonal antibody for the detection of antibodies against the bovine leukemia virus

    PubMed Central

    Kim, Eun-Ju; Cheong, Kwang-Myun; Joung, Ha-Kyung; Kim, Bo-Hye; Song, Jae-Young; Cho, In-Soo; Lee, Kyoung-Ki

    2016-01-01

    Infection of cattle with bovine leukemia virus (BLV) has been observed and reported worldwide, including in Korea. The onsite identification of infected cattle would help decreasing and eradicating BLV infections on farms. Here, we present a new immunochromatographic assay that employs monoclonal antibodies (MAbs) for the detection of antibodies against BLV in the field. BLV envelope glycoprotein (gp)51 was expressed in E. coli, and MAbs against recombinant BLV gp51 were generated for the development of an immunochromatographic assay to detect BLV antibodies in cattle. The sensitivity and specificity of the assay were determined by comparing these results with those obtained from a standard enzyme linked immunosorbent assay (ELISA). A total of 160 bovine sera were used to evaluate the new immunochromatographic assay. Using ELISA as a reference standard, the relative specificity and sensitivity of this assay were determined to be 94.7% and 98%, respectively. Because of its high sensitivity and specificity, this BLV antibody detection assay would be suitable for the onsite identification of BLV infection in the field. PMID:27030192

  2. Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

    PubMed

    Choudhary, Nandlal; Roy, Avijit; Govindarajulu, A; Nakhla, M K; Levy, L; Brlansky, R H

    2014-09-01

    Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing.

  3. Development of a high-throughput opsonophagocytic assay for the determination of functional antibody activity against Streptococcus pyogenes using bioluminescence.

    PubMed

    Lorenz, Natalie; Loh, Jacelyn M S; Moreland, Nicole J; Proft, Thomas

    2017-03-01

    The lack of standardised protocols for the assessment of functional antibodies has hindered Streptococcus pyogenes research and the development of vaccines. A robust, high throughput opsonophagocytic bactericidal assay to determine protective antibodies in human and rabbit serum has been developed that utilises bioluminescence as a rapid read out.

  4. A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination in Chinese agricultural smaples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC50 and IC10 of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries recovery rate...

  5. Optimization of a phage amplification assay to permit accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis cells.

    PubMed

    Foddai, Antonio; Elliott, Christopher T; Grant, Irene R

    2009-06-01

    A commercially available phage amplification assay, FASTPlaqueTB (Biotec Laboratories, Ipswich, United Kingdom), when used according to the manufacturer's instructions, does not permit accurate enumeration of Mycobacterium avium subsp. paratuberculosis. The aim of this study was to optimize the phage amplification assay conditions to permit accurate quantification of viable M. avium subsp. paratuberculosis cells. The burst time for M. avium subsp. paratuberculosis was initially determined to inform decisions about optimal incubation time before plating, and then other test parameters were altered to evaluate how the correlation between plaque and colony counts was affected. The D29 mycobacteriophage replicates more slowly in M. avium subsp. paratuberculosis than in Mycobacterium smegmatis (used to optimize the commercial test originally), and the mean burst time for four M. avium subsp. paratuberulosis strains was 210 +/- 36.8 min at 37 degrees C compared to 63 +/- 17.5 min for M. smegmatis mc(2) 155. To achieve 100% correlation between plaque and colony counts, the optimized phage assay includes the following: (i) resuspension of the samples to be tested in Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase and 2 mM calcium chloride, followed by overnight incubation at 37 degrees C before performance of the phage assay; (ii) a 2-h incubation of the sample with D29 mycobacteriophage before viricide treatment; and (iii) a further 90-min incubation after viricide treatment and neutralization up to the burst time (total incubation time, 210 min) before plating with M. smegmatis mc(2) 155 in 7H9 agar. The optimized phage amplification assay was able to detect 1 to 10 CFU/ml of M. avium subsp. paratuberculosis in spiked milk or broth within 48 h, as demonstrated by the results of several blind trials.

  6. Miniaturized Growth Inhibition Assay to Assess the Anti-blood Stage Activity of Antibodies.

    PubMed

    Duncan, Elizabeth H; Bergmann-Leitner, Elke S

    2015-01-01

    While no immune correlate for blood-stage specific immunity against Plasmodium falciparum malaria has been identified, there is strong evidence that antibodies directed to various malarial antigens play a crucial role. In an effort to evaluate the role of antibodies in inhibiting growth and/or invasion of erythrocytic stages of the malaria parasite it will be necessary to test large sample sets from Phase 2a/b trials as well as epidemiological studies. The major constraints for such analyses are (1) availability of sufficient sample quantities (especially from infants and small children) and (2) the throughput of standard growth inhibition assays. The method described here assesses growth- and invasion inhibition by measuring the metabolic activity and viability of the parasite (by using a parasite lactate dehydrogenase-specific substrate) in a 384-microtiter plate format. This culture method can be extended beyond the described detection system to accommodate other techniques commonly used for growth/invasion-inhibition.

  7. A study on the temperature dependency and time course of the cold capture antibody secretion assay.

    PubMed

    Pichler, Johannes; Hesse, Friedemann; Wieser, Matthias; Kunert, Renate; Galosy, Sybille S; Mott, John E; Borth, Nicole

    2009-04-20

    The cold capture assay as described by Brezinsky et al. [Brezinsky, S.C.G., Chiang, G.G., Szilvasi, A., Mohan, S., Shapiro, R.I., MacLean, A., Sisk, W., Thill, G., 2003. A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J. Immunol. Methods 277, 141-155] stands out as the most simple of single cell secretion assays which can be used to sort for high productivity in recombinant cell lines. At low temperatures the process of protein release from transport vesicles is assumed to be delayed as both vesicle fusion and product release is slowed, so that secreted proteins can be stained on the cell surface using a fluorescent antibody. Typically, the fluorescent signal obtained correlates to the cell specific production rate of the analysed cell. In the present study we compared staining of human antibody producing CHO cells performed at different temperatures and we observed the fluorescent signal over 24h. We found that the staining temperature did not influence signal intensity. The fluorescent signal was stable for 24h at 4 degrees C, decreased to 80% at room temperature (21 degrees C), while it decreased significantly already after 2h at 37 degrees C. Initially, the fluorescent signal was observed on the cell surface, however, at later stages it was found in compartments in the cytoplasm. Finally we compared differences in signal stability depending on whether the antibody used for staining bound to the light or heavy chain of the product and on whether the fluorescent label was a relatively stable protein (phycoerythrin) or a pH-dependent small molecule (FITC). Our results indicate that the secreted product is trapped by the staining antibody on the cell surface at all temperatures. Subsequently these aggregates are endocytosed by the cells, a process which is slowed down at low temperatures.

  8. Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay

    PubMed Central

    Tait, Brian D.

    2016-01-01

    This review outlines the development of human leukocyte antigen (HLA) antibody detection assays and their use in organ transplantation in both antibody screening and crossmatching. The development of sensitive solid phase assays such as the enzyme-linked immunosorbent assay technique, and in particular the bead-based technology has revolutionized this field over the last 10–15 years. This revolution however has created a new paradigm in clinical decision making with respect to the detection of low level pretransplant HLA sensitization and its clinical relevance. The relative sensitivities of the assays used are discussed and the relevance of conflicting inter-assay results. Each assay has its advantages and disadvantages and these are discussed. Over the last decade, the bead-based assay utilizing the Luminex® fluorocytometer instrument has become established as the “gold standard” for HLA antibody testing. However, there are still unresolved issues surrounding this technique, such as the presence of denatured HLA molecules on the beads which reveal cryptic epitopes and the issue of appropriate fluorescence cut off values for positivity. The assay has been modified to detect complement binding (CB) in addition to non-complement binding (NCB) HLA antibodies although the clinical relevance of the CB and NCB IgG isotypes is not fully resolved. The increase sensitivity of the Luminex® bead assay over the complement-dependent cytotoxicity crossmatch has permitted the concept of the “virtual crossmatch” whereby the crossmatch is predicted to a high degree of accuracy based on the HLA antibody specificities detected by the solid phase assay. Dialog between clinicians and laboratory staff on an individual patient basis is essential for correct clinical decision making based on HLA antibody results obtained by the various techniques. PMID:28018342

  9. Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in european wild boar

    PubMed Central

    Aurtenetxe, Olaia; Barral, Marta; Vicente, Joaquín; de la Fuente, José; Gortázar, Christian; Juste, Ramón A

    2008-01-01

    Background Bovine tuberculosis (bTB) remains a significant problem in some parts of Spain largely because of contacts between cattle and wildlife reservoirs in extensive grazing systems. European Wild boar (Sus scrofa) is one of the species involved in the transmission of the disease to other species. Fast and simple detection methods would be critical for assessing infection prevalence, study the mechanisms of pathogen transmission and monitoring the effects of TB control measures. Results An enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Mycobacterium bovis in wild boar serum was developed and validated on 185 sera from TB positive and negative wild boar. Based on antigen inoculation of captive animals as well as tuberculosis compatible lesions, culture results and molecular analysis of hunted individuals, animals were allocated into two groups: tuberculosis positive group and tuberculosis negative group. After optimization of the positive to negative ratio using different combinations of serum dilutions and conjugate concentrations, the test yielded a sensitivity of 72.60% and a specificity of 96.43% for the best cut-off. Conclusion Although some negative group animals showed an ELISA positive reaction (< 3%), this assay showed a high potential for accurate diagnosis of TB in wild boar, as its large dynamic range supported a good discriminatory power and a satisfactory balance between sensitivity and specificity. PMID:18976491

  10. Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b.

    PubMed

    Pickering, Jerry W; Martins, Thomas B; Schroder, M Carl; Hill, Harry R

    2002-07-01

    We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.

  11. Use of a Standardized MxA Protein Measurement-Based Assay for Validation of Assays for the Assessment of Neutralizing Antibodies Against Interferon-β

    PubMed Central

    Subramanyam, Meena; Goelz, Susan; Goyal, Jaya; Jethwa, Vijay; Jones, Wendy; Files, James G.; Kramer, Daniel; Bird, Chris; Dilger, Paula; Tovey, Michael; Lallemand, Christophe; Thorpe, Robin

    2013-01-01

    Effective monitoring of the development of neutralizing antibodies (NAbs) against IFN-β in multiple sclerosis (MS) patients on IFN-β therapy is important for clinical decision making and disease management. To date, antiviral assays have been the favored approach for NAb determination, but variations in assay conditions between laboratories and the increasing use of novel assays have contributed to the reporting of inconsistent antibody data between laboratories and between products. This study, undertaken at the request of the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA), is a joint effort by manufacturers of IFN-β products (approved in Europe) towards harmonization of a NAb assay that facilitates generation of comparable NAb data, which, in conjunction with clinical outcomes, should prove useful for clinicians treating MS patients with IFN-β products. This article describes the standardized cellular myxovirus resistance protein A (MxA) protein measurement-based assay for detection of IFN-β NAbs and its use for the validation of assays used for the quantitative determination of such antibodies. Although titers varied between laboratories and the products used, utilization of IFN-β1a rather than IFN-β1b as the challenge antigen produced more consistent results in the NAb assay. Adoption of the standardized assay improves comparability between laboratories circumventing problems that arise when different, nonstandardized assays are employed for immunogenicity assessment. Based on the data, the EMA recommended for standardization purposes, the use of IFN-β1a in NAb assays, independent of the therapeutic product used for therapy and validation of new NAb procedures against the standardized assay described. PMID:23848523

  12. Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis.

    PubMed

    Hilbert, David W; Smith, William L; Chadwick, Sean G; Toner, Geoffrey; Mordechai, Eli; Adelson, Martin E; Aguin, Tina J; Sobel, Jack D; Gygax, Scott E

    2016-04-01

    Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease:Gardnerella vaginalis,Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the orderClostridiales),Megasphaeraphylotype 1 or 2,Lactobacillus iners,Lactobacillus crispatus,Lactobacillus gasseri, andLactobacillus jensenii We generated a logistic regression model that identifiedG. vaginalis,A. vaginae, andMegasphaeraphylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion ofLactobacillusspp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV.

  13. Serum virus neutralization assay for detection and quantitation of serum neutralizing antibodies to influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The serum virus neutralization (SVN) assay is a serological test to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus. The SVN assay is a highly sensitive and specific test that may be applied to influenza A viruses (IAV) in swine to measure the ...

  14. International technology transfer of a GCLP-compliant HIV-1 neutralizing antibody assay for human clinical trials.

    PubMed

    Ozaki, Daniel A; Gao, Hongmei; Todd, Christopher A; Greene, Kelli M; Montefiori, David C; Sarzotti-Kelsoe, Marcella

    2012-01-01

    The Collaboration for AIDS Vaccine Discovery/Comprehensive Antibody-Vaccine Immune Monitoring Consortium (CAVD/CA-VIMC) assisted an international network of laboratories in transferring a validated assay used to judge HIV-1 vaccine immunogenicity in compliance with Good Clinical Laboratory Practice (GCLP) with the goal of adding quality to the conduct of endpoint assays for Human Immunodeficiency Virus I (HIV-1) vaccine human clinical trials. Eight Regional Laboratories in the international setting (Regional Laboratories), many located in regions where the HIV-1 epidemic is most prominent, were selected to implement the standardized, GCLP-compliant Neutralizing Antibody Assay for HIV-1 in TZM-bl Cells (TZM-bl NAb Assay). Each laboratory was required to undergo initial training and implementation of the immunologic assay on-site and then perform partial assay re-validation, competency testing, and undergo formal external audits for GCLP compliance. Furthermore, using a newly established external proficiency testing program for the TZM-bl NAb Assay has allowed the Regional Laboratories to assess the comparability of assay results at their site with the results of neutralizing antibody assays performed around the world. As a result, several of the CAVD/CA-VIMC Regional Laboratories are now in the process of conducting or planning to conduct the GCLP-compliant TZM-bl NAb Assay as an indicator of vaccine immunogenicity for ongoing human clinical trials.

  15. International Technology Transfer of a GCLP-Compliant HIV-1 Neutralizing Antibody Assay for Human Clinical Trials

    PubMed Central

    Todd, Christopher A.; Greene, Kelli M.; Montefiori, David C.; Sarzotti-Kelsoe, Marcella

    2012-01-01

    The Collaboration for AIDS Vaccine Discovery/Comprehensive Antibody – Vaccine Immune Monitoring Consortium (CAVD/CA-VIMC) assisted an international network of laboratories in transferring a validated assay used to judge HIV-1 vaccine immunogenicity in compliance with Good Clinical Laboratory Practice (GCLP) with the goal of adding quality to the conduct of endpoint assays for Human Immunodeficiency Virus I (HIV-1) vaccine human clinical trials. Eight Regional Laboratories in the international setting (Regional Laboratories), many located in regions where the HIV-1 epidemic is most prominent, were selected to implement the standardized, GCLP-compliant Neutralizing Antibody Assay for HIV-1 in TZM-bl Cells (TZM-bl NAb Assay). Each laboratory was required to undergo initial training and implementation of the immunologic assay on-site and then perform partial assay re-validation, competency testing, and undergo formal external audits for GCLP compliance. Furthermore, using a newly established external proficiency testing program for the TZM-bl NAb Assay has allowed the Regional Laboratories to assess the comparability of assay results at their site with the results of neutralizing antibody assays performed around the world. As a result, several of the CAVD/CA-VIMC Regional Laboratories are now in the process of conducting or planning to conduct the GCLP-compliant TZM-bl NAb Assay as an indicator of vaccine immunogenicity for ongoing human clinical trials. PMID:22303476

  16. An accurate DNA marker assay for stem rust resistance gene Sr2 in wheat.

    PubMed

    Mago, R; Simkova, H; Brown-Guedira, G; Dreisigacker, S; Breen, J; Jin, Y; Singh, R; Appels, R; Lagudah, E S; Ellis, J; Dolezel, J; Spielmeyer, W

    2011-03-01

    The stem rust resistance gene Sr2 has provided broad-spectrum protection against stem rust (Puccinia graminis Pers. f. sp. tritici) since its wide spread deployment in wheat from the 1940s. Because Sr2 confers partial resistance which is difficult to select under field conditions, a DNA marker is desirable that accurately predicts Sr2 in diverse wheat germplasm. Using DNA sequence derived from the vicinity of the Sr2 locus, we developed a cleaved amplified polymorphic sequence (CAPS) marker that is associated with the presence or absence of the gene in 115 of 122 (95%) diverse wheat lines. The marker genotype predicted the absence of the gene in 100% of lines which were considered to lack Sr2. Discrepancies were observed in lines that were predicted to carry Sr2 but failed to show the CAPS marker. Given the high level of accuracy observed, the marker provides breeders with a selection tool for one of the most important disease resistance genes of wheat.

  17. DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening

    PubMed Central

    Navrátil, Václav; Schimer, Jiří; Tykvart, Jan; Knedlík, Tomáš; Vik, Viktor; Majer, Pavel; Konvalinka, Jan; Šácha, Pavel

    2017-01-01

    Human diseases are often diagnosed by determining levels of relevant enzymes and treated by enzyme inhibitors. We describe an assay suitable for both ultrasensitive enzyme quantification and quantitative inhibitor screening with unpurified enzymes. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with a small-molecule inhibitor attached to a reporter DNA and detected by quantitative PCR. We validate the approach using the putative cancer markers prostate-specific membrane antigen and carbonic anhydrase IX. We show that DIANA has a linear range of up to six logs and it selectively detects zeptomoles of targets in complex biological samples. DIANA's wide dynamic range permits determination of target enzyme inhibition constants using a single inhibitor concentration. DIANA also enables quantitative screening of small-molecule enzyme inhibitors using microliters of human blood serum containing picograms of target enzyme. DIANA's performance characteristics make it a superior tool for disease detection and drug discovery. PMID:27679479

  18. Comparison of five commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

    PubMed

    Kösters, K; Riffelmann, M; Dohrn, B; von König, C H

    2000-05-01

    Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of five commercially available ELISA kits with the help of 65 serum specimens which were repetitively tested for evaluation of the kits. The specimens contained 20 paired serum samples from patients with clinical pertussis, 15 samples were from children vaccinated with a diphtheria-tetanus-acellular pertussis vaccine, seven specimens were taken from an interlaboratory comparison of ELISAs, and there were three reference preparations from the Food and Drug Administration's (FDA's) Laboratory of Pertussis and from our laboratory. Reference values were obtained from the FDA or from results obtained with an in-house ELISA. Commercial ELISAs were compared with respect to their reproducibility and variability, their ability to detect significant titer rises in paired serum samples, their ability to detect an immune response after vaccination, and the comparability of semiquantitative and quantitative results. Reproducibility was generally good (>89%), intra-assay variation ranged from 2.4 to 28.7%, and indeterminate results were recorded in up to 18.5% of all specimens. Most kits correctly identified the antibody response to an acellular pertussis vaccine. None of the commercial kits identified all cases of pertussis correctly, and the sensitivity ranged between 60 and 95%. All five commercial ELISAs showed great discrepancies when comparing semiquantitative results and contained obviously different antigen preparations. Our data suggest that the five commercial ELISAs tested here need further improvement and standardization.

  19. Evaluation of the LIAISON ANA screen assay for antinuclear antibody testing in autoimmune diseases.

    PubMed

    Ghillani, P; Rouquette, A M; Desgruelles, C; Hauguel, N; Le Pendeven, C; Piette, J C; Musset, L

    2007-08-01

    Antinuclear antibodies (ANA) are widely detected by immunofluorescence on HEp-2 cells in patients with connective tissue diseases and other pathological conditions. We evaluated the first-automated chemiluminescence immunoassay for the detection of ANA (LIAISON ANA screen, DiaSorin). This study was carried out simultaneously in two laboratories by testing 327 patient samples with clinically defined connective diseases, 273 routine samples for ANA screening, and 300 blood donors. A total of 268 out of 337 IIF-positive sera were positive with LIAISON ANA screen (79.5% of agreement) and 240 out of 263 IIF-negative sera were negative with LIAISON ANA screen (91.2% of agreement). After resolution of discrepant results, the concordance reached, respectively, 94.9% and 98.8%. The specificity was 99.3% and the sensitivity was 94%. Unlike results obtained by other ANA screening assays, we observed acceptable sensitivity and specificity. Despite the presence of HEp-2 cell extract, we failed to detect some antibodies as antinucleolar, antinuclear envelope, and antiproliferating cell nuclear antigen. This automated assay allows quick process to results and exhibits satisfactory sensitivity for the detection of the main ANA specificities of connective tissue diseases.

  20. 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.

  1. Comparing Assay Performance of ELISA and Chemiluminescence Immunoassay in Detecting Antibodies to Hepatitis B Surface Antigen

    PubMed Central

    Sagar, Siddharth; Vishwanath, Shashidhar; Banerjee, Barnini; Eshwara, Vandana Kalwaje; Chawla, Kiran

    2016-01-01

    Introduction Antibodies to Hepatitis B surface Antigen (Anti-HBs) levels are measured as markers for immune response to vaccination and in decision making for post-exposure prophylaxis against Hepatitis-B. Several immunoassay formats are used to measure Anti-HBs, thus carrying the possibility of variation in measured levels between different assays. This study compares the performance of Chemiluminescence Immunoassay (CLIA) against Enzyme-linked Immunosorbent Assay (ELISA) in measuring Anti-HBs titer by looking into concordance between the two test reports. Aim To compare the agreement between ELISA and CLIA in measurement of Anti–HBs antibody titers. Materials and Methods This prospective comparative study conducted at Kasturba Medical College, Manipal measured consecutive serum samples (69) sent for anti-HBs levels during May-June 2016 using both CLIA (Abbott Architect) and ELISA (Bio-Rad). Anti-HBs values of ≤10mIU/ml was considered as non-protective and >10mIU/ml as protective. The agreement between the tests in classifying the antibody titers as non-protective or protective was computed using Kappa coefficient, and the difference in individual titer values between the tests compared using Bland-Altman plot on SPSS (v.15). Results Out of the 69 samples analysed, 18 samples (26.1%) were of health-care personnel and remaining of patients. Agreement between ELISA and CLIA in identifying the antibody titers as protective and non-protective were 96.5% and 90.9% respectively, resulting in an agreement of 0.84. The coefficient-of-variation of ELISA and CLIA were 74.5% and 113.1%, respectively. Three value based discordant results were noted; two samples deemed protective by ELISA were reported as non-protective by CLIA. One non-protective titer by ELISA was reported as protective by CLIA. Conclusion Analytical agreement is good between the two immunoassays. However there are some discrepancies in quantitative measurement. This may have been due the variation in

  2. Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin.

    PubMed

    Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae; Kang, Hwan-Goo

    2015-01-01

    Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food.

  3. A Quantitative Flurometric Assay for the Measurement of Antibody to Pasteurella haemolytica in Cattle

    PubMed Central

    Confer, A.W.; Fox, J.C.; Newman, P.R.; Lawson, G.W.; Corstvet, R.E.

    1983-01-01

    A rapid, simple fluorometric method is described for measuring antibody to Pasteurella haemolytica in sera of cattle. Various antigen preparations were compared for the test including live, formalin-killed and phenol-killed P. haemolytica. A preparation composed of formalin-killed organisms from a 22 hour culture gave consistent results and was used in the studies. The test was reproduciable with percent coefficients of variation for fluorescent signal unit values on ten or more replicate samples ranging from 5.7 to 28.0. Sera from calves vaccinated by aerosol exposure to live P. haemolytica had up to a five-fold increase in antibody titer as measured by the flurometric method test during a 21 day period. Fluorometric method titers were comparable to those obtained by the indirect bacterial agglutination test. There was no seroconversion to P. haemolytica in calves vaccinated by aerosol exposure of P. multocida. The major advantages of the fluorometric method test over conventional methods are that the assay does not require serial dilutions of serum samples and thus limits time and effort to determine antibody titers. PMID:6339016

  4. Bacteria-modified amperometric immunosensor for a Brucella melitensis antibody assay.

    PubMed

    Li, Zhi-Zhang; Gong, Fu-Chun; Shen, Guo-Li; Yu, Ru-Qin

    2002-06-01

    A novel amperometric immunosensor setup is described which uses horseradish peroxidase (HRP) as a label in conjunction with a current-based Brucella sensor. The Bacteria modified immunosensor was constructed by using a biocomposite formed by dispersing graphite powder into a mixture of Brucella melitensis and silicate polymer gel. The enzyme-labeled antibody can readily diffuse toward the encapsulated antigen (Brucella melitensis), which retains its binding properties, and the association reaction is easily detected at the surface exposed to the solution. The use of an oaminophenol (o-AP) substrate and amperometric detection at -150 mV (vs. SCE) results in a relatively low detection limit of 3.5 ng/ml and a linear detection range of 3.5 ng/ml to 200 ng/ml. Based on an optimized parameter, the prepared sensor was used to detect the Brucella melitensis antibody in serum samples by using a competitive binding assay. The results demonstrate the feasibility of employing the proposed immunosensor for the detection for Brucella melitensis antibody in a clinical analysis.

  5. Measurement of anti-IgA antibodies by a two-site immunoradiometric assay

    SciTech Connect

    Homburger, H.A.; Smith, J.R.; Jacob, G.L.; Laschinger, C.; Naylor, D.H.; Pineda, A.A.

    1981-01-01

    To enable the detection of IgG class, anti-IgA antibodies and to investigate the possible occurrence of IgE class, anti-IgA antibodies, we developed a solid phase immunoradiometric assay, which uses purified IgA coupled covalently to microcrystalline cellulose as an immunosorbent. Radiolabeled, Fc specific anti-IgG and anti-IgE antibodies were used to detect specific aIgA after incubation of test sera or controls with the immunosorbent. IgG-aIgA were detected by the IRA in 100 and 67% of control sera with class specific and limited specificity aIgA. The IRA was sensitive to approximately two ng of class specific IgG-aIgA. IgG-aIgA also were detected by IRA in 7.9% of sera from patients with urticarial transfusion reactions and 73% of sera from patients with ataxia telangiectasia and IgA deficiency. Sera from 50 normal blood donors did not have detectable IgG-aIgA. Tests for IgE-aIgA were negative in all cases, including control sera with class specific IgG-aIgA. We conclude that the IRA is a sensitive and reproducible method for detection of class specific and limited specificity IgG-aIgA, and that IgE-aIgA do not mediate urticarial transfusion reactions.

  6. Diagnostic value of animal-side antibody assays for rapid detection of Mycobacterium bovis or Mycobacterium microti infection in South American camelids.

    PubMed

    Lyashchenko, Konstantin P; Greenwald, Rena; Esfandiari, Javan; Rhodes, Shelley; Dean, Gillian; de la Rua-Domenech, Ricardo; Meylan, Mireille; Vordermeier, H Martin; Zanolari, Patrik

    2011-12-01

    Tuberculosis (TB) in South American camelids (SAC) is caused by Mycobacterium bovis or Mycobacterium microti. Two serological methods, rapid testing (RT) and the dual-path platform (DPP) assay, were evaluated using naturally infected SAC. The study population included 156 alpacas and 175 llamas in Great Britain, Switzerland, and the United States. TB due to M. bovis (n = 44) or M. microti (n = 8) in 35 alpacas and 17 llamas was diagnosed by gross pathology examination and culture. Control animals were from herds with no TB history. The RT and the DPP assay showed sensitivities of 71% and 74%, respectively, for alpacas, while the sensitivity for llamas was 77% for both assays. The specificity of the DPP assay (98%) was higher than that of RT (94%) for llamas; the specificities of the two assays were identical (98%) for alpacas. When the two antibody tests were combined, the parallel-testing interpretation (applied when either assay produced a positive result) enhanced the sensitivities of antibody detection to 89% for alpacas and 88% for llamas but at the cost of lower specificities (97% and 93%, respectively), whereas the serial-testing interpretation (applied when both assays produced a positive result) maximized the specificity to 100% for both SAC species, although the sensitivities were 57% for alpacas and 65% for llamas. Over 95% of the animals with evidence of TB failed to produce skin test reactions, thus confirming concerns about the validity of this method for testing SAC. The findings suggest that serological assays may offer a more accurate and practical alternative for antemortem detection of camelid TB.

  7. Accurate and High-Coverage Immune Repertoire Sequencing Reveals Characteristics of Antibody Repertoire Diversification in Young Children with Malaria

    NASA Astrophysics Data System (ADS)

    Jiang, Ning

    Accurately measuring the immune repertoire sequence composition, diversity, and abundance is important in studying repertoire response in infections, vaccinations, and cancer immunology. Using molecular identifiers (MIDs) to tag mRNA molecules is an effective method in improving the accuracy of immune repertoire sequencing (IR-seq). However, it is still difficult to use IR-seq on small amount of clinical samples to achieve a high coverage of the repertoire diversities. This is especially challenging in studying infections and vaccinations where B cell subpopulations with fewer cells, such as memory B cells or plasmablasts, are often of great interest to study somatic mutation patterns and diversity changes. Here, we describe an approach of IR-seq based on the use of MIDs in combination with a clustering method that can reveal more than 80% of the antibody diversity in a sample and can be applied to as few as 1,000 B cells. We applied this to study the antibody repertoires of young children before and during an acute malaria infection. We discovered unexpectedly high levels of somatic hypermutation (SHM) in infants and revealed characteristics of antibody repertoire development in young children that would have a profound impact on immunization in children.

  8. Label free checkerboard assay to determine overlapping epitopes of Ebola virus VP-40 antibodies using surface plasmon resonance.

    PubMed

    Anderson, George P; Liu, Jinny L; Zabetakis, Dan; Legler, Patricia M; Goldman, Ellen R

    2017-03-01

    Immunoassay formats, in which antibodies provide sensitivity and specificity, are often utilized to provide rapid and simple diagnostic tests. Surface plasmon resonance is frequently used to evaluate the suitability of antibodies by determining binding kinetics to agents or surrogate antigens. We used SPR to evaluate a number of commercial monoclonal antibodies as well as single domain antibodies produced in-house. All the antibodies targeted the Ebola virus viral protein 40 (VP40). We determined the ability of each antibody to bind to immobilized VP40, and ensured they did not bind Ebola glycoprotein or the nucleoprotein. A subset of the monoclonal antibodies was immobilized to characterize antigen capture in solution. It can be advantageous to utilize antibodies that recognize distinct epitopes when choosing reagents for detection and diagnostic assays. We determined the uniqueness of the epitope recognized by the anti-VP40 antibodies using a checkerboard format that exploits the 6×6 array of interactions monitored by the Bio-Rad ProteOn XPR36 SPR instrument. The results demonstrate the utility of surface plasmon resonance to characterize monoclonal and recombinant antibodies. Additionally, the analysis presented here enabled the identification of pairs of anti-VP40 antibodies which could potentially be utilized in sandwich type immunoassays for the detection of Ebola virus.

  9. Detection of antibodies against Bordetella avium in turkeys by avidin-biotin enhancement of the enzyme-linked immunosorbent assay and the dot-immunobinding assay.

    PubMed

    Tsai, H J; Saif, Y M

    1991-01-01

    An avidin-biotin-enhanced enzyme-linked immunosorbent assay (AB-ELISA) and an avidin-biotin-enhanced dot-immunobinding (AB-DIB) assay for detecting antibody to Bordetella avium in turkey sera were developed and compared with the microagglutination (MA) test. Whole-cell antigen, biotin-labeled goat anti-turkey IgG conjugate, and horseradish-peroxidase-labeled streptavidin were used in the AB-ELISA and AB-DIB assay. The AB-ELISA and AB-DIB assay were sensitive, specific, and reproducible. These assays were superior to the MA test for measuring acquired and maternal antibodies against B. avium. All MA-positive sera were positive by two assays, but some sera negative by MA test had titers in the AB-ELISA and AB-DIB assay. AB-ELISA and AB-DIB titers showed a positive correlation (r = 0.866), and AB-ELISA was more sensitive than the AB-DIB assay.

  10. Accuracy of the Fluorescence-Activated Cell Sorting Assay for the Aquaporin-4 Antibody (AQP4-Ab): Comparison with the Commercial AQP4-Ab Assay Kit

    PubMed Central

    Kim, Yoo-Jin; Cheon, So Young; Kim, Boram; Jung, Kyeong Cheon; Park, Kyung Seok

    2016-01-01

    Background The aquaporin-4 antibody (AQP4-Ab) is a disease-specific autoantibody to neuromyelitis optica (NMO). We aimed to evaluate the accuracy of the FACS assay in detecting the AQP4-Ab compared with the commercial cell-based assay (C-CBA) kit. Methods Human embryonic kidney-293 cells were transfected with human aquaporin-4 (M23) cDNA. The optimal cut off values of FACS assay was tested using 1123 serum samples from patients with clinically definite NMO, those at high risk for NMO, patients with multiple sclerosis, patients with other idiopathic inflammatory demyelinating diseases, and negative controls. The accuracy of FACS assay and C-CBA were compared in consecutive 225 samples that were collected between January 2014 and June 2014. Results With a cut-off value of MFIi of 3.5 and MFIr of 2.0, the receiver operating characteristic curve for the FACS assay showed an area under the curve of 0.876. Among 225 consecutive sera, the FACS assay and C-CBA had a sensitivity of 77.3% and 69.7%, respectively, in differentiating the sera of definite NMO patients from sera of controls without IDD or of MS. Both assay had a good specificity of 100% in it. The overall positivity of the C-CBA among FACS-positive sera was 81.5%; moreover, its positivity was low as 50% among FACS-positive sera with relatively low MFIis. Conclusions Both the FACS assay and C-CBA are sensitive and highly specific assays in detecting AQP4-Ab. However, in some sera with relatively low antibody titer, FACS-assay can be a more sensitive assay option. In real practice, complementary use of FACS assay and C-CBA will benefit the diagnosis of NMO patients, because the former can be more sensitive among low titer sera and the latter are easier to use therefore can be widely used. PMID:27658059

  11. Comparison of radioimmunoassay and enzyme-linked immunosorbent assay in measurement of antibodies to Neisseria meningitidis group A capsular polysaccharide.

    PubMed Central

    Beuvery, E C; Kayhty, M H; Leussink, A B; Kanhai, V

    1984-01-01

    Antibodies to meningococcal group A polysaccharide were determined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) in serum samples from 16 adults vaccinated with bivalent meningococcal group A and C polysaccharide vaccine. The specific antibody levels in the serum samples were expressed as micrograms of antibody protein per milliliter of serum. For RIA the polysaccharide was radiolabeled extrinsically with 125I. Both native polysaccharide and polysaccharide labeled with 127I were used in ELISA. Because these antigens gave similar results, it can be concluded that the introduction of tyramine and iodine by the labeling procedure did not alter the antigenic activity of the polysaccharide. The reproducibility of RIA was clearly better than that of ELISA. The antibody levels detected by the methods were equal, which means that ELISA can be used satisfactorily to measure antibodies to meningococcal group A polysaccharide quantitatively. Some discrepant results were found due to an underestimation of immunoglobulin M antibodies in ELISA. This was shown by a correlation test in which a weakly significant negative correlation was found between the immunoglobulin M antibody level/immunoglobulin G antibody level ratio and the RIA antibody level/ELISA antibody level ratio. PMID:6436314

  12. Analysis of cross-reactive neutralizing antibodies in human HFMD serum with an EV71 pseudovirus-based assay.

    PubMed

    Zhang, Huafei; An, Dong; Liu, Wei; Mao, Qunying; Jin, Jun; Xu, Lin; Sun, Shiyang; Jiang, Liping; Li, Xiaojun; Shao, Jie; Ma, Hongxia; Huang, Xueyong; Guo, Shijie; Chen, Haiying; Cheng, Tong; Yang, Lisheng; Su, Weiheng; Kong, Wei; Liang, Zhenglun; Jiang, Chunlai

    2014-01-01

    Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies.

  13. Comparison of the indirect immunobead, radiolabeled, and immunofluorescence assays for immunoglobulin G serum antibodies to human sperm

    SciTech Connect

    Haas, G.G. Jr.; D'Cruz, O.J.; DeBault, L.E. )

    1991-02-01

    The relative sensitivities of the indirect immunobead test, the indirect flo cytometric immunofluorescence assay, and an indirect radiolabeled antiglobulin assay were compared. Eighteen immunobead test positive sera and 18 negative sera were used as the standard for the other two assays. Of the 18 positive sera, 14 (77%) and 5 (27%) were positive in the immunofluorescence assay and the radiolabeled antiglobulin assay, respectively. Four (22%) of the low titer immunobead test positive sera were negative by both the immunofluorescence assay and the radiolabeled antiglobulin assay. However, there was a significant positive correlation between the results of the immunofluorescence assay and the radiolabeled antiglobulin assay (r = 0.73) and between the results of the radiolabeled antiglobulin assay and the titer of the immunobead test (r = 0.82). The use of an unselected sperm population in the radiolabeled antiglobulin assay and the classical indirect immunofluorescence method using methanol-fixed sperm gave false-positive results in the radiolabeled antiglobulin assay and the immunofluorescence assay. These results suggested that immunoglobulin G antisperm antibody positive sera may be reactive both to sperm surface and internalized sperm antigens.

  14. Engineered domain based assays to identify individual antibodies in oligoclonal combinations targeting the same protein

    PubMed Central

    Meng, Q.; Garcia-Rodriguez, C.; Manzanarez, G.; Silberg, M.A.; Conrad, F.; Bettencourt, J.; Pan, X.; Breece, T.; To, R.; Li, M.; Lee, D.; Thorner, L.; Tomic, M.T.; Marks, J.D.

    2014-01-01

    Quantitation of individual mAbs within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins (XOMA 3B and XOMA 3E) each consisting of three monoclonal antibodies (mAbs) that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (HN). Epitope mapping data was used to design LC-HN domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or 3E. Mutant LC-HN domains were cloned, expressed, and purified from E. coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of ELISAs that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B, and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein. PMID:22922799

  15. Evaluation of the Antibody in Lymphocyte Supernatant Assay to Detect Active Tuberculosis

    PubMed Central

    Sariko, Margaretha; Anderson, Caitlin; Mujaga, Buliga S.; Gratz, Jean; Mpagama, Stellah G.; Heysell, Scott; Kibiki, Gibson; Mmbaga, Blandina; Houpt, Eric

    2017-01-01

    Background We aimed to evaluate the antibody in lymphocyte supernatant (ALS) assay as a biomarker to diagnose tuberculosis among adults from Tanzania with and without HIV. Methods Adults admitted with suspicion for tuberculosis had sputa obtained for GeneXpert MTB/RIF, acid-fast bacilli smear and mycobacterial culture; blood was obtained prior to treatment initiation and after 4 weeks. Adults hospitalized with non-infectious conditions served as controls. Peripheral blood mononuclear cells were cultured unstimulated for 72 hours. Anti-mycobacterial antibodies were measured from culture supernatants by ELISA, using BCG vaccine as the coating antigen. Median ALS responses were compared between cases and controls at baseline and between cases over time. Results Of 97 TB cases, 85 were microbiologically confirmed and 12 were clinically diagnosed. Median ALS responses from TB cases (0.366 OD from confirmed cases and 0.285 from clinical cases) were higher compared to controls (0.085, p<0.001). ALS responses did not differ based on HIV status, CD4 count or sputum smear status. Over time, the median ALS values declined significantly (0.357 at baseline; 0.198 after 4-weeks, p<0.001). Conclusions Robust ALS responses were mounted by patients with TB regardless of HIV status, CD4 count, or low sputum bacillary burden, potentially conferring a unique niche for this immunologic biomarker for TB. PMID:28085899

  16. Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

    NASA Astrophysics Data System (ADS)

    Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

    2012-06-01

    There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

  17. Evaluation of a pan-serotype point-of-care rapid diagnostic assay for accurate detection of acute dengue infection.

    PubMed

    Vivek, Rosario; Ahamed, Syed Fazil; Kotabagi, Shalini; Chandele, Anmol; Khanna, Ira; Khanna, Navin; Nayak, Kaustuv; Dias, Mary; Kaja, Murali-Krishna; Shet, Anita

    2017-03-01

    The catastrophic rise in dengue infections in India and globally has created a need for an accurate, validated low-cost rapid diagnostic test (RDT) for dengue. We prospectively evaluated the diagnostic performance of NS1/IgM RDT (dengue day 1) using 211 samples from a pediatric dengue cohort representing all 4 serotypes in southern India. The dengue-positive panel consisted of 179 dengue real-time polymerase chain reaction (RT-PCR) positive samples from symptomatic children. The dengue-negative panel consisted of 32 samples from dengue-negative febrile children and asymptomatic individuals that were negative for dengue RT-PCR/NS1 enzyme-linked immunosorbent assay/IgM/IgG. NS1/IgM RDT sensitivity was 89.4% and specificity was 93.8%. The NS1/IgM RDT showed high sensitivity throughout the acute phase of illness, in primary and secondary infections, in different severity groups, and detected all 4 dengue serotypes, including coinfections. This NS1/IgM RDT is a useful point-of-care assay for rapid and reliable diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue.

  18. Characterization of serum antibody responses to natural rotavirus infections in children by VP7-specific epitope-blocking assays.

    PubMed Central

    Matson, D O; O'Ryan, M L; Pickering, L K; Chiba, S; Nakata, S; Raj, P; Estes, M K

    1992-01-01

    Knowledge of the immune response to rotavirus is crucial for vaccine development. We compared an epitope-blocking assay (EBA) that uses VP7-specific monoclonal antibodies with neutralization assays (NAs) with polyclonal antisera for detecting serum antibody responses after natural rotavirus infection in children. Twenty-six serum pairs from children living in an orphanage with and without symptoms during two rotavirus outbreaks were evaluated for VP7 type 1-, 2-, 3-, and 4-specific antibody responses. In the first outbreak, which was caused by a VP7 type 3 strain, homotypic antibody responses were detected in 11 of 11 symptomatic children by NA and in 10 of 11 symptomatic children by EBA. Heterotypic antibody responses were detected more frequently (12 of 15 children) by NA than by EBA, and the heterotypic epitope-blocking antibody responses occurred in children older than 14 months of age. Antibody responses in asymptomatic children were more commonly detected by EBA than by NA. EBA results from the sera of children in the second outbreak indicated that it was caused by VP7 type 4, whereas NA results suggested it was caused by VP7 type 3. Our results confirm that EBA is a sensitive and specific method for determining VP7 type-specific immune responses after natural rotavirus infections. PMID:1374761

  19. Detection of wheat gliadin contamination of gluten-free foods by a monoclonal antibody dot immunobinding assay.

    PubMed

    Freedman, A R; Galfre, G; Gal, E; Ellis, H J; Ciclitira, P J

    1987-07-15

    Unfractionated wheat gliadin was used to produce murine monoclonal antibodies to gliadin. A dot immunobinding assay, using these antibodies, was developed to detect possible gliadin contamination of nominally gluten-free flour, using dilute ethanol extracts spotted onto nitrocellulose membranes. The sensitivity of the assay was less than 10 micrograms/ml of unfractionated gliadin which permitted the detection of trace amounts of gliadin present in certain wheat starch based 'gluten-free' products. The assay detected not only wheat gliadin, but also prolamine extracts of rye, barley and oats; maize, soya and potato extracts as well as the control proteins casein and ovalbumin, gave negative results. The assay is of value as a simple and rapid method of screening foods for their suitability for consumption by patients with coeliac disease.

  20. Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human Myeloperoxidase.

    PubMed

    Bian, Zhi-Ping; Li, Xiong-Zhi; Wu, Heng-Fang; Xu, Jin-Dan; Gu, Chun-Rong; Chen, Xiang-Jian; Yang, Di

    2016-04-01

    Myeloperoxidase (MPO), a leukocyte hemoprotein released from neutrophils, is thought to be a potential participant in plaque formation and plaque rupture. Therefore, MPO is regarded as an early marker predicting the risk for atherosclerosis, especially for coronary artery disease and acute coronary syndrome. We generated hybridoma clones 1E3 and 3E8 secreting monoclonal antibodies (mAbs) specific to human MPO. BALB/c mice were immunized with MPO protein purified from human neutrophils. Splenocytes from these mice were fused with the mouse myeloma cell line SP2/0. Based on isotyping of the mAbs, both clones 1E3 and 3E8 were referred to the IgG1 subclass. The specificities of 1E3 and 3E8 were assessed by enzyme-linked immunosorbent assay (ELISA), and only 3E8 was confirmed by western blot. We developed a simple MPO-immunosorbent assay (MPO-ISA) on microplate based on both the immune activity and peroxidase activity of MPO. The mAb secreted by clone 3E8 was chosen as coating antibody to capture the plasma MPO without interfering with the peroxidase activity of MPO. Then, tetramethylbenzidine substrate was added to the microwell directly, catalyzed by captured MPO, and a colored product was formed. The simple MPO-ISA test has a sensitivity of 3.68 ng/mL. The linear concentration of MPO-ISA for commercial MPO standard ranged to 250 ng/mL. The average recovery rate is 101.02%. The imprecision within-day was <10% at three different MPO levels. The imprecision between-day was <10% at low and middle MPO levels and varied to 14.61% at the high MPO level. We found that the established MPO-ISA can detect the plasma MPO from human and cavy, but not from mouse and rat. Compared with the commercial human MPO ELISA assay, the MPO-ISA can be used to detect the natural human MPO protein, but not recombinant MPO polypeptides. The generated mAbs and MPO-ISA test may be useful tools to assess risk for inflammation and cardiac events.

  1. Enzyme-linked immunosorbent assay for total isoflavonoids in Pueraria candollei using anti-puerarin and anti-daidzin polyclonal antibodies.

    PubMed

    Pongkitwitoon, Benyakan; Sakamoto, Seiichi; Tanaka, Hiroyuki; Tsuchihashi, Ryota; Kinjo, Junei; Morimoto, Satoshi; Putalun, Waraporn

    2010-05-01

    Pueraria candollei (White Kwao Khuer) is a medicinal plant containing puerarin, daidzin, genistin, daidzein, and genistein as major isoflavonoids used for its rejuvenating and estrogenic effects. In order to analyze these compounds, a single enzyme-linked immunosorbent assay (ELISA) for total isoflavonoids was developed using anti-puerarin and anti-daidzin polyclonal antibodies (PAbs). The range for calibration of isoflavonoids by ELISA was 0.05-6.25 microg/mL. Total isoflavonoid concentrations in P. candollei samples determined by the newly developed assay system showed good agreement with those analyzed by HPLC. Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of total isoflavonoids in P. candollei.

  2. Simultaneous determination of soy isoflavone glycosides, daidzin and genistin by monoclonal antibody-based highly sensitive indirect competitive enzyme-linked immunosorbent assay.

    PubMed

    Sakamoto, Seiichi; Yusakul, Gorawit; Pongkitwitoon, Benyakan; Paudel, Madan Kumar; Tanaka, Hiroyuki; Morimoto, Satoshi

    2015-02-15

    Soy isoflavones are known as major bioactive compounds in soybean (Glycine max), which is an indispensable food. Despite their utility, the consumption of isoflavones has recently been limited because they exhibit oestrogenic and topoisomerase II inhibitory effects. To assess their intake limitation, accurate, sensitive, and effective quantitative analyses are necessary. In this study, we produced the monoclonal antibody (MAb) against daidzin (DZ) and applied it to an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the simultaneous determination of DZ and genistin (GEN), which are known as two major soy isoflavone glycosides in soy products. Using the DZ-MAb, we developed a sensitive icELISA method, where the limit of detection for DZ and GEN was 1.95ng/ml. Several validation analyses revealed that the icELISA is sufficiently accurate and sensitive to be used to assess the overconsumption of soy isoflavones, which would lead to the safe dietary intake of soy products.

  3. Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus

    USGS Publications Warehouse

    Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas M.; Toohey-Kurth, Kathy

    2014-01-01

    Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

  4. A Combination of Serological Assays to Detect Human Antibodies to the Avian Influenza A H7N9 Virus

    PubMed Central

    Gao, Rongbao; Dong, Jie; Zhang, Ye; Guo, Junfeng; Zou, Shumei; Zhou, Jianfang; Zhu, Yun; Xin, Li; Li, Xiaodan; Xu, Cuiling; Wang, Dayan; Shu, Yuelong

    2014-01-01

    Human infection with avian influenza A H7N9 virus was first identified in March 2013 and represents an ongoing threat to public health. There is a need to optimize serological methods for this new influenza virus. Here, we compared the sensitivity and specificity of the hemagglutinin inhibition (HI), microneutralization (MN), and Western blot (WB) assays for the detection of human antibodies against avian influenza A (H7N9) virus. HI with horse erythrocytes (hRBCs) and a modified MN assay possessed greater sensitivity than turkey erythrocytes and the standard MN assay, respectively. Using these assays, 80% of tested sera from confirmed H7N9 cases developed detectable antibody to H7N9 after 21 days. To balance sensitivity and specificity, we found serum titers of ≥20 (MN) or 160 (HI) samples were most effective in determining seropositive to H7N9 virus. Single serum with HI titers of 20–80 or MN titer of 10 could be validated by each other or WB assay. Unlike serum collected from adult or elderly populations, the antibody response in children with mild disease was low or undetectable. These combinations of assays will be useful in case diagnosis and serologic investigation of human cases. PMID:24755627

  5. A new flow-cytometry-based opsonophagocytosis assay for the rapid measurement of functional antibody levels against Group B Streptococcus.

    PubMed

    Fabbrini, Monica; Sammicheli, Chiara; Margarit, Immaculada; Maione, Domenico; Grandi, Guido; Giuliani, Marzia Monica; Mori, Elena; Nuti, Sandra

    2012-04-30

    Opsonophagocytosis is the primary mechanism for the clearance of Group B Streptococcus (GBS) by the host, and levels of opsonic antibodies may correlate with protection in preclinical models. A killing-based opsonophagocytosis assay (OPA), can be used to determine the functional activity of vaccine-induced GBS-specific antibodies. The assay, which measures the number of bacterial colonies surviving phagocytic killing in the presence of specific antibodies and complement, is rather expensive, time-consuming and poorly standardized. Here we describe a rapid, sensitive and reproducible fluorescent OPA assay (fOPA) based on flow cytometry analysis (FACS), which allows internalized bacteria to be distinguished from those associated to the plasma membrane of phagocytic cells. Fixed GBS were labeled with pHrodo™, a fluorescent dye which dramatically increases the emitted fluorescence at the acidic conditions present in the phagocytic endosomal compartment. Labeled bacteria were incubated with HL-60 cells differentiated to phagocytes, antibodies and complement, and then analyzed by FACS. A further improvement to our method, allowing to reduce assay variability, consisted on a step of selection of effector cells among the HL-60 population. Analysis of sera from mice immunized with different GBS vaccines revealed comparable sensitivity and specificity with the traditional killing OPA assay (kOPA), and a good correlation between the fluorescent signal of bacteria internalized by HL-60 phagocytes and killing. Remarkably, the pHrodo-based approach reduced the variability observed with other fOPA assays. The obtained data indicate the proposed fOPA as a reliable and useful tool for functional antibody assessment.

  6. Development and evaluation of a flow cytometry microsphere assay to detect anti-histone antibody in dogs.

    PubMed

    Paul, Shoma; Wilkerson, Melinda J; Shuman, Wilma; Harkin, Kenneth R

    2005-09-15

    Anti-nuclear antibody (ANA) is one of the diagnostic parameters that support a diagnosis of autoimmune disorders in humans, dogs, and horses, particularly the condition systemic lupus erythematosus (SLE). The most commonly used method for detecting ANA in canine serum is the indirect immunofluorescence antibody assay (IFA) that detects dog IgG with reactivity towards mammalian cell nuclei. Interpretation of the IFA results is very subjective and dependent on the source of tissue/cellular substrate. We have developed a flow cytometry based assay to detect canine serum antibodies specific to histones. Histones were chosen as the target antigen because these nuclear proteins are the most common nuclear substrate for ANA in dogs with SLE. Microsphere beads were coated with histones and incubated with canine sera. Bound anti-histone antibodies were detected by FITC-conjugated rabbit F(ab')2 anti-dog IgG. Sera from four groups of dogs (47 dogs total) were tested for anti-histone antibodies and compared with the traditional IFA assay. The groups included 15 healthy dogs, 15 dogs with noninflammatory diseases, 9 dogs with polyarthritis and positive ANA, and 8 German shepherds with perianal fistulas. The microsphere assay results indicated that only one dog in the noninflammatory group and four out of nine dogs in the polyarthritis group had mean fluorescent intensity values above our established cut-off (defined as 2 S.D. above the mean of healthy controls). There was moderate agreement between the anti-histone assay and the traditional ANA (kappa statistic=0.54). Absorption of ANA positive serum with total histones dramatically diminished the fluorescent signal detected by flow cytometry and the speckled nuclear pattern observed by IFA, whereas preabsorption did not change the diffuse nuclear staining pattern. These findings indicate that the anti-histone assay will not replace the ANA test and that other nuclear proteins, such as ribonucleoproteins may contribute to the

  7. Accelerated removal of antibody-coated red blood cells from the circulation is accurately tracked by a biotin label

    PubMed Central

    Mock, Donald M.; Lankford, Gary L.; Matthews, Nell I.; Burmeister, Leon F.; Kahn, Daniel; Widness, John A.; Strauss, Ronald G.

    2013-01-01

    BACKGROUND Safe, accurate methods to reliably measure circulating red blood cell (RBC) kinetics are critical tools to investigate pathophysiology and therapy of anemia, including hemolytic anemias. This study documents the ability of a method using biotin-labeled RBCs (BioRBCs) to measure RBC survival (RCS) shortened by coating with a highly purified monomeric immunoglobulin G antibody to D antigen. STUDY DESIGN AND METHODS Autologous RBCs from 10 healthy D+ subjects were labeled with either biotin or 51Cr (reference method), coated (opsonized) either lightly (n = 4) or heavily (n = 6) with anti-D, and transfused. RCS was determined for BioRBCs and for 51Cr independently as assessed by three variables: 1) posttransfusion recovery at 24 hours (PTR24) for short-term RCS; 2) time to 50% decrease of the label (T50), and 3) mean potential life span (MPL) for long-term RCS. RESULTS BioRBCs tracked both normal and shortened RCS accurately relative to 51Cr. For lightly coated RBCs, mean PTR24, T50, and MPL results were not different between BioRBCs and 51Cr. For heavily coated RBCs, both short-term and long-term RCS were shortened by approximately 17 and 50%, respectively. Mean PTR24 by BioRBCs (84 ± 18%) was not different from 51Cr (81 ± 10%); mean T50 by BioRBCs (23 ± 17 days) was not different from 51Cr (22 ± 18 days). CONCLUSION RCS shortened by coating with anti-D can be accurately measured by BioRBCs. We speculate that BioRBCs will be useful for studying RCS in conditions involving accelerated removal of RBCs including allo- and autoimmune hemolytic anemias. PMID:22023312

  8. Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

    SciTech Connect

    Hovi, T.; Roivainen, M.

    1989-04-01

    We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with /sup 51/Cr, (/sup 3/H)leucine, or, preferentially, with (/sup 3/H)uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30/degree/C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well.

  9. Gold nanoparticle-based enzyme-linked antibody-aptamer sandwich assay for detection of Salmonella Typhimurium.

    PubMed

    Wu, Wenhe; Li, Jun; Pan, Dun; Li, Jiang; Song, Shiping; Rong, Mingge; Li, Zixi; Gao, Jimin; Lu, Jianxin

    2014-10-08

    Enzyme-linked immunosorbent assay (ELISA) provides a convenient means for the detection of Salmonella enterica serovar Typhimurium (STM), which is important for rapid diagnosis of foodborne pathogens. However, conventional ELISA is limited by antibody-antigen immunoreactions and suffers from poor sensitivity and tedious sample pretreatment. Therefore, development of novel ELISA remains challenging. Herein, we designed a comprehensive strategy for rapid, sensitive, and quantitative detection of STM with high specificity by gold nanoparticle-based enzyme-linked antibody-aptamer sandwich (nano-ELAAS) method. STM was captured and preconcentrated from samples with aptamer-modified magnetic particles, followed by binding with detector antibodies. Then nanoprobes carrying a large amount of reporter antibodies and horseradish peroxidase molecules were used for colorimetric signal amplification. Under the optimized reaction conditions, the nano-ELAAS assay had a quantitative detection range from 1 × 10(3) to 1 × 10(8) CFU mL(-1), a limit of detection of 1 × 10(3) CFU mL(-1), and a selectivity of >10-fold for STM in samples containing other bacteria at higher concentration with an assay time less than 3 h. In addition, the developed nanoprobes were improved in terms of detection range and/or sensitivity when compared with two commercial enzyme-labeled antibody signal reporters. Finally, the nano-ELAAS method was demonstrated to work well in milk samples, a common source of STM contamination.

  10. The diagnosis of proventricular dilatation disease: use of a Western blot assay to detect antibodies against avian Borna virus.

    PubMed

    Villanueva, Itamar; Gray, Patricia; Mirhosseini, Negin; Payne, Susan; Hoppes, Sharman; Honkavuori, Kirsi S; Briese, Thomas; Turner, Debra; Tizard, Ian

    2010-07-14

    Avian Borna virus (ABV) has recently been shown to be the causal agent of proventricular dilatation disease (PDD) a lethal neurologic disease of captive psittacines and other birds. An immunoblot assay was used to detect the presence of antibodies against avian Borna virus in the serum of affected birds. A lysate from ABV-infected duck embryo fibroblasts served as a source of antigen. The assay was used to test for the presence of antibodies to ABV in 117 birds. Thirty of these birds had biopsy or necropsy-confirmed proventricular dilatation disease (PDD), while the remaining 87 birds were apparently healthy or were suffering from diseases other than PDD. Sera from 27 of the 30 PDD cases (90%) contained antibodies to ABV. Seventy-three (84%) of the apparently "healthy" birds were seronegative. Additionally, sera from seven macaws and one parrot trapped in the Peruvian Amazon were seronegative. Positive sera recognized the bornaviral nucleoprotein (N-protein). While the presence of antibodies to ABV largely corresponded with the development of clinical PDD, 14 apparently healthy normal birds possessed detectable antibodies to ABV. The existence of a carrier state was confirmed when 13 of 15 apparently healthy cockatiels were shown by PCR to have detectable ABV RNA in their feces. Western blot assays may be of significant assistance in diagnosing proventricular dilatation disease. Many apparently healthy birds may however be seronegative while, at the same time, shedding ABV in their feces.

  11. Development of a simple, accurate SPME-based method for assay of VOCs in column breakthrough experiments.

    PubMed

    Salaices Avila, Manuel Alejandro; Breiter, Roman; Mott, Henry

    2007-01-01

    Solid-phase microextraction (SPME) with gas chromatography is to be used for assay of effluent liquid samples from soil column experiments associated with VOC fate/transport studies. One goal of the fate/transport studies is to develop accurate, highly reproducible column breakthrough curves for 1,2-cis-dichloroethylene (cis-DCE) and trichloroethylene (TCE) to better understand interactions with selected natural solid phases. For SPME, the influences of the sample equilibration time, extraction temperature and the ratio of volume of sample bottle to that of the liquid sample (V(T)/V(w)) are the critical factors that could influence accuracy and precision of the measured results. Equilibrium between the gas phase and liquid phase was attained after 200 min of equilibration time. The temperature must be carefully controlled due to variation of both the Henry's constant (K(h)) and the fibre/gas phase distribution coefficient (K(fg)). K(h) decreases with decreasing temperature while K(fg) increases. Low V(T)/V(w) yields better sensitivity but results in analyte losses and negative bias of the resultant assay. High V(T)/V(w) ratio yields reduced sensitivity but analyte losses were found to be minimal, leading to better accuracy and reproducibility. A fast SPME method was achieved, 5 min for SPME extraction and 3.10 min for GC analysis. A linear calibration function in the gas phase was developed to analyse the breakthrough curve data, linear between a range of 0.9-236 microgl(-1), and a detection limit lower than 5 microgl(-1).

  12. Quantitative Serology Assays for Determination of Antibody Responses to Ebola Virus Glycoprotein and Matrix Protein in Nonhuman Primates and Humans

    PubMed Central

    Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C.; Warfield, Kelly L.; Aman, M. Javad; Holtsberg, Frederick W.

    2016-01-01

    The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA’s), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in E. coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals. PMID:26681387

  13. Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.

    PubMed

    Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C; Warfield, Kelly L; Aman, M Javad; Holtsberg, Frederick W

    2016-02-01

    The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA's), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in Escherichia coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals.

  14. Flow-Through Assay for Detection of Antibodies Using Protein-A Colloidal Gold Conjugate as a Probe.

    PubMed

    Chennuru, Sreedevi; Pavuluri, Panduranga Rao

    2015-01-01

    Flow-through assay (FTA) is a rapid, simple-to-perform, cost-effective, and user-friendly diagnostic test for monitoring infections in non-laboratory settings. It is mostly applied for antibody detection. FTA employing protein-A colloidal gold conjugate to detect antibodies against porcine cysticerci using cyst fluid and whole cyst antigens of Taenia solium metacestode is described here. Antibodies in the serum are captured by an antigen spotted onto a nitrocellulose membrane mounted on a flow-through device that serves as the antigen capture matrix. The bound antibodies are visualized by the addition of protein-A colloidal gold conjugate, which imparts a pink color. The test can be completed within 3 min at room temperature without any instrumentation. The sensitivity and specificity of the FTA are in agreement with ELISA.

  15. Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

    PubMed Central

    Sherwood, Laura J.; Hayhurst, Andrew

    2013-01-01

    Background Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism. Methods and Findings In parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections. Conclusions The C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent

  16. Effect of heat inactivation of serum on Bordetella pertussis antibody determination by enzyme-linked immunosorbent assay.

    PubMed

    Lopez, A L; Pineda, E; Garakian, A; Cherry, J D

    1998-01-01

    The effect of heat inactivation on Bordetella pertussis antibodies determined by enzyme-linked immunosorbent assay (ELISA) was studied. Sera were heated at increasing temperatures (from 30 to 50 degrees C at 5 degrees C increments and from 52 to 70 degrees C at 2 degrees C increments). Between 30 and 50 degrees C, no significant differences were observed in immunoglobin G (IgG) antibodies to pertussis toxin (PT). From 50 to 56 degrees C the antibody values were twofold higher than those of uninactivated sera; at 64 degrees C the values were 3.6- to 9.1-fold higher. The increase in PT IgG antibody values was more pronounced in sera with low antibody values. ELISA antibody values of sera from a vaccine trial were determined in unheated and heat inactivated sera. The geometric mean value (GMV) of the heat inactivated samples was 3.2 times the geometric mean value of the uninactivated sera. ELISA IgG antibodies to filamentous hemagglutinin, fimbriae-2, and pertactin were studied, and the values of heat inactivated sera did not differ significantly from the values of the uninactivated sera. Our findings indicate that heat inactivation of sera leads to higher, variable, and false-positive PT and IgG values.

  17. An optimized assay of specific IgE antibodies to reactive dyes and studies of immunologic responses in exposed workers.

    PubMed

    Wass, U; Nilsson, R; Nordlinder, R; Belin, L

    1990-03-01

    Methods of assaying reactive dye-specific IgE antibodies were investigated with a RAST. Sera from three patients, occupationally exposed to a reactive dye, Remazol black B (Chemical Abstract registry number 17095-24-8), were used. Directly dyed disks, that is, disks without any carrier protein, resulted in poor and unreliable measures of specific IgE. In contrast, optimized preparation of conjugates between the dye and human serum albumin resulted in efficient binding of specific IgE. The patients' RAST results were strongly positive, whereas sera from 36 exposed workers but without symptoms and sera from unexposed subjects with high levels of total IgE were negative. The hapten and carrier specificity of the IgE antibodies was studied by direct RAST and RAST inhibition. In one patient, the antibodies were principally hapten specific, whereas another patient was found to have antibodies with a high degree of specificity to the carrier. The third patient's antibodies were intermediate between the other two patients' antibodies in this respect, suggesting that antibody specificity is dependent not only on the nature of the hapten but also on individual immune response factors. The study demonstrates that it is important to use an optimized preparation of dye-protein conjugates to elicit reliable results and a high degree of specific IgE binding in the RAST.

  18. Antibodies against denatured HLA class II molecules detected in luminex-single antigen assay.

    PubMed

    Grenzi, Patricia C; de Marco, Renato; Silva, Rosemeire Z R; Campos, Erika F; Gerbase-DeLima, Maria

    2013-10-01

    False-positive anti-HLA reactions may occur in Luminex-single antigen (SA) beads assays, and it is important to recognize them to correctly interpret the test. The purpose of this report is to describe a peculiar pattern of reactivity, characterized by positivity with beads coated with HLA-DRB1*09:01, DRB3*01:01, DRB3*02:02, DRB3*03:01, DPB1*02:01, DPB1*20:01 and DPB1*28:01, that was observed in 141 of 8121 serum samples tested in our laboratory with three different lots of the same kit (LABScreen(®) SA, One Lambda). These 141 serum samples came from 56 different patients on the kidney transplant waiting list, corresponding to 1% of the patients. Of these, 10 males had never been transfused or transplanted. About 66% of the patients had positive reactions against self-antigen HLA-DRB3 alleles. No reactions against native HLA-DRB1*09:01 were observed in flow cytometry crossmatch and in absorption/elution experiments, leading to the conclusion that the reactivity was due to antibodies against epitopes present in denatured forms of HLA-class II antigens. The occurrence of this reactivity pattern was associated with female gender and systemic lupus erythematosus (SLE).

  19. The incidence of antinuclear antibodies (ANA) detected by indirect immunofluorescence assay (IFA) method.

    PubMed

    Karamehic, Jasenko; Subasic, Djemo; Gavrankapetanovic, Faris; Zecevic, Lamija; Eminovic, Izet; Memic, Senaid; Seric, Natasa; Drace, Zahida

    2007-01-01

    Human anti-nuclear antibodies (ANA) in Systemic Lupus Erythematosus (SLE) react specificaly with DNA, RNA, several proteins and ribonucleoproteins. Systemic Lupus Erythematosus is the classic type of polysystemic autoimmune disease. The high frequency of ANA is determined in these patients. Actually, all SLE patients are ANA positive. ANA testing by IFA (Indirect Immunofluorescence Assay) is an excellent screening tool for SLE cases, but it is not so highly specific test. Patients with connective tissue diseases, such as rheumatoid arthritis, scleroderma and dermatomyositis are also frequently positive. Results of IFA ANA have relative low specific degree, and for this reason the titration of these specimens to the end point is usually recommended. Indirect immunoflourescence is reference method for ANA testing. Common substrates are thin sections of rodent organs or various types of cell lines. The cell line substrates are preferable to organ sections, because these rapidly dividing cells have higher level for detection of certain clinically relevant antigens such as (e.g., centromere, SSA (Ro) and Scl-70). In this paper we present the results evaluation of ANA incidence, detected by IFA in serum specimens of corresponding clinical patients, during 2005 and 2006.

  20. Comparison of three enzyme-linked immunosorbent assays for detection of immunoglobulin g antibodies to tetanus toxoid with reference standards and the impact on clinical practice.

    PubMed

    van Hoeven, Karen H; Dale, Connie; Foster, Phil; Body, Barbara

    2008-12-01

    Accurate determination of the concentrations of immunoglobulin G (IgG) antibody to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, determine immune competence in individual patients, and measure the prevalence of immunity in populations. The performance of three commercially available enzyme-linked immunosorbent assays (ELISAs) for IgG antibodies to tetanus toxoid were evaluated. Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglobulin international reference standards were analyzed in quadruplicate using ELISAs manufactured by The Binding Site, Inc. (VaccZyme); Scimedx; and Euroimmun. In addition, IgG antibodies to tetanus toxoid were measured in 83 deidentified serum specimens using each manufacturer's ELISA. Each ELISA provided linear results when evaluated with the reference preparations. The Binding Site ELISA provided results that closely corresponded to the reference preparations (y=1.09x-0.08), whereas the Scimedx ELISA gave results that were consistently lower (y=0.21x-0.07) and the Euroimmun ELISA gave results that were consistently higher (y=1.5x+0.30) than the reference preparation concentrations. Using the recommended cutoff for each ELISA (<0.10 IU/ml), the overall agreement of all of the ELISA methods was 78%. Three of eighty-three (3.6%) human serum samples demonstrated inadequate immunity with all three assays. The Binding Site ELISA yielded nonprotective antibody concentrations in only these 3 samples, whereas 19 samples (22.9%) according to the Scimedx ELISA and 6 samples (7.2%) according to the Euroimmun ELISA demonstrated nonprotective concentrations. The performance characteristics of ELISAs for tetanus immunoglobulin titers were manufacturer dependent, and the differences translated into important disparities in reported results.

  1. Detection of BRAF mutation in Chinese tumor patients using a highly sensitive antibody immunohistochemistry assay

    NASA Astrophysics Data System (ADS)

    Qiu, Tian; Lu, Haizhen; Guo, Lei; Huang, Wenting; Ling, Yun; Shan, Ling; Li, Wenbin; Ying, Jianming; Lv, Ning

    2015-03-01

    BRAF mutations can be found in various solid tumors. But accurate and reliable screening for BRAF mutation that is compatible for clinical application is not yet available. In this study, we used an automated immunohistochemistry (IHC) staining coupled with mouse monoclonal anti-BRAF V600E (VE1) primary antibody to screen the BRAF V600E mutation in 779 tumor cases, including 611 colorectal carcinomas (CRC), 127 papillary thyroid carcinomas (PTC) and 41 malignant melanomas. Among the 779 cases, 150 cases were positive for BRAF (V600E) staining, including 38 (of 611, 6%) CRCs, 102 (of 127, 80%) PTCs and 10 (of 41, 24%) malignant melanomas. Sanger sequencing and real-time PCR confirmed the sensitivity and specificity of IHC staining for the V600E mutation are 100% and 99%, respectively. Therefore, our study demonstrates that the fully automated IHC is a reliable tool to determine BRAF mutation status in CRC, PTC and melanoma and can be used for routine clinical screen.

  2. Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

    SciTech Connect

    Perkins, J; Parida, S; Clavijo, A

    2007-05-14

    Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.

  3. Assays for detecting and diagnosing antibody-mediated pure red cell aplasia (PRCA): an assessment of available procedures.

    PubMed

    Thorpe, Robin; Swanson, Steven J

    2005-05-01

    Antibody (Ab)-mediated pure red cell aplasia (PRCA) develops when patients mount a neutralizing Ab response to recombinant erythropoiesis-stimulating agents (ESAs) such as epoetin-alpha (EPO). These neutralizing Abs can also cross-neutralize endogenous EPO, leading to a state of absolute EPO resistance and transfusion dependence. The diagnosis of Ab-mediated PRCA in part relies on the sensitive and specific detection of serum anti-EPO Abs and a confirmatory examination of patients' bone marrow for lack of erythroid precursors. To date, a variety of assays have been used to detect anti-EPO Abs, including radioimmunoprecipitation (RIP) assay, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) and bioassays that measure neutralizing Abs. Each of these assays can yield informative results and possess characteristic benefits and limitations, so it is unclear whether or not a "superior" assay is required or possible. To date, a universal standardized assay has not yet been established that would facilitate the comparison of Ab data derived from different laboratories and retrospective analysis of stored sera. This review evaluates the results of studies measuring anti-EPO Abs with different assays and compares their relative advantages and disadvantages in terms of specificity, sensitivity, ease of use and ability to measure Ab-binding affinities and subclasses. These comparisons provide a basis for determining the optimal assay(s) for screening and/or analysis of patients' serum for anti-EPO Abs during treatment or after onset of Ab-mediated PRCA.

  4. Immunologic diversity among serogroup 1 Legionella pneumophila urinary antigens demonstrated by monoclonal antibody enzyme-linked immunosorbent assays.

    PubMed Central

    Kohler, R B; Wilde, C; Johnson, W; Joly, J; Wheat, L J; Baker, R; Misfeldt, M

    1988-01-01

    We tested urine specimens from 222 patients with serogroup 1 Legionella pneumophila pneumonia in two enzyme-linked immunosorbent assays (ELISAs) which used different monoclonal antibodies (A and B) as detector antibodies. Of 171 specimens which contained enough antigen to be detected in the ELISAs, 169 reacted in only one of the two assays. A total of 25 patients whose infections were acquired in any of three Indianapolis hospitals excreted antigen reactive with monoclonal antibody B, but 18 patients who were treated for infections acquired elsewhere reacted with monoclonal antibody A. The urinary antigen ELISA reactivity patterns correlated with the reactivity patterns of L. pneumophila isolates when a separate panel of seven monoclonal antibodies was used. The isolate patterns, in turn, correlated well with environmental isolate patterns from two of the hospitals with nosocomial cases. We conclude that at least two different epitopes exist on the antigen molecules in urine from patients with serogroup 1 L. pneumophila pneumonia and that the subtyping of urinary antigens can be useful epidemiologically. PMID:2460492

  5. Analysis of antibody responses to Hymenolepis nana infection in mice by the enzyme-linked immunosorbent assay and immunoprecipitation.

    PubMed

    Ito, A; Honey, R D; Scanlon, T; Lightowlers, M W; Rickard, M D

    1988-05-01

    Serum antibody responses in two strains of mice infected with embryonated eggs of Hymenolepis nana were analysed by the enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) using sodium deoxycholate (DOC)-solubilized antigens prepared from embryonated eggs (eggs), mouse-derived cysticercoids (cysts) and adult tapeworms with immature segments only (adults). Highly susceptible dd mice, which harbour mature tapeworms for a long period (greater than 70 days), produced high levels of antibodies to all three different stages of H. nana. BALB/c mice, almost all of which expel adult tapeworms by 30 days after infection, produced high levels of antibody against egg antigens only. The high antibody titres to cyst and adult antigens in dd mice did not lead to expulsion of the worms. However, worms are rejected early in BALB/c mice when there is little or no detectable serum antibody. The antibody responses to eggs seen in BALB/c mice which had long since shed their adult worms were probably due to ingestion of eggs from faeces of other infected mice. Antibodies to eggs were not detected in BALB/c mice which were initially inoculated with eggs (day 0) and then treated with praziquantel on day 6 after the tissue phase of infection only. The different antibody responses to egg antigens and the other two antigens (cyst and adult) in BALB/c mice suggest a difference in antigen specificity between eggs and both cysts and adults. A major antigen component with Mr 32,000 appears to be specific to the egg (or oncosphere) stage of H. nana. Antibody to this major component of eggs was absorbed only with intact eggs, but not with intact cysts nor adults with immature segments only, so that the antigen appears to be on the surface of the oncosphere.

  6. Comparison of different assays to assess human papillomavirus (HPV) type 16- and 18-specific antibodies after HPV infection and vaccination.

    PubMed

    Scherpenisse, Mirte; Schepp, Rutger M; Mollers, Madelief; Mooij, Sofie H; Meijer, Chris J L M; Berbers, Guy A M; van der Klis, Fiona R M

    2013-08-01

    We compared the measurement of human papillomavirus (HPV)-specific serum antibody levels with the virus-like-particle multiplex immunoassay (VLP-MIA), competitive Luminex immunoassay (cLIA), and glutathione S-transferase (GST) L1-based MIA. Using a large panel of serum samples, these assays showed mutually good correlations for both naturally induced and vaccine-derived HPV-specific antibody levels. However, an adaptation of the GST L1-based MIA resulted in an improved correlation with both cLIA and VLP-MIA.

  7. Comparison of Different Assays To Assess Human Papillomavirus (HPV) Type 16- and 18-Specific Antibodies after HPV Infection and Vaccination

    PubMed Central

    Schepp, Rutger M.; Mollers, Madelief; Mooij, Sofie H.; Meijer, Chris J. L. M.; Berbers, Guy A. M.; van der Klis, Fiona R. M.

    2013-01-01

    We compared the measurement of human papillomavirus (HPV)-specific serum antibody levels with the virus-like-particle multiplex immunoassay (VLP-MIA), competitive Luminex immunoassay (cLIA), and glutathione S-transferase (GST) L1-based MIA. Using a large panel of serum samples, these assays showed mutually good correlations for both naturally induced and vaccine-derived HPV-specific antibody levels. However, an adaptation of the GST L1-based MIA resulted in an improved correlation with both cLIA and VLP-MIA. PMID:23740920

  8. Study on the use of an enzyme-linked immunosorbent assay in determining human antibodies to diphtheria toxin as compared with a reference toxin neutralization assay.

    PubMed

    Skoura, L; Efstratiou, A; Tsakris, A; Pournaras, S; George, R C; Douboyas, J

    1999-07-01

    Serum samples from 156 Greek persons were assessed by an IgG-specific enzyme-linked immunosorbent assay (ELISA) and a reference tissue culture toxin-neutralization (TN) assay for the quantitation of diphtheria toxin antibodies. By the reference method, 7.7% of the persons were susceptible to diphtheria (antitoxin < 0.01 IU/ml), 28.8% had basic protection (antitoxin 0.01-0.09 IU/ml) and 63.5% were fully protective (antitoxin > or = 0.1 IU/ ml), while the corresponding figures were 17.9, 36.5 and 45.5% when they were tested by the immunoassay. None of the samples been susceptible by the TN assay were found to have some protection when tested by ELISA. However, three (6.7%) of the 45 samples showing a basic protection with TN, were fully protective when titrated by the immunoassay. In addition, 31 (31.3%) of the 99 samples been fully protective by the bioassay, were found to be either basically protective or susceptible by means of the ELISA. Overall, validity features of the immunoassay were: sensitivity 68.7%, specificity 94.7%, positive predictive value 95.8% and negative predictive value 63.5%. The ELISA tested in our study could be used to determine diphtheria antitoxin in individuals needed a booster immunization (susceptible or basic protective samples), although it might falsely include in the above categories samples that are within the fully protective levels of antibodies.

  9. Enzyme-linked immunosorbent assay for efficient detection of antibody to bluetongue virus in pronghorn (Antilocapra americana).

    PubMed

    Drolet, B S; Mills, K W; Belden, E L; Mecham, J O

    1990-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA), using cell-associated viral antigen, was developed for detection of antibody to bluetongue virus (BTV) in field-collected pronghorn (Antilocapra americana) sera. To test the applicability of the ELISA to seroepizootiologic studies, pronghorn serum samples from three Wyoming counties (USA) were tested. Bluetongue virus ELISA results were compared to those of the bluetongue immunodiffusion assay. Discrepant serum samples were retested for reaction to either BTV or epizootic hemorrhagic disease virus. The pronghorn BTV ELISA gave rapid, quantitative, objective results and should facilitate testing large numbers of sera for BT diagnostic and seroepizootiologic studies.

  10. Modular, Antibody-free Time-Resolved LRET Kinase Assay Enabled by Quantum Dots and Tb3+-sensitizing Peptides

    NASA Astrophysics Data System (ADS)

    Cui, Wei; Parker, Laurie L.

    2016-07-01

    Fluorescent drug screening assays are essential for tyrosine kinase inhibitor discovery. Here we demonstrate a flexible, antibody-free TR-LRET kinase assay strategy that is enabled by the combination of streptavidin-coated quantum dot (QD) acceptors and biotinylated, Tb3+ sensitizing peptide donors. By exploiting the spectral features of Tb3+ and QD, and the high binding affinity of the streptavidin-biotin interaction, we achieved multiplexed detection of kinase activity in a modular fashion without requiring additional covalent labeling of each peptide substrate. This strategy is compatible with high-throughput screening, and should be adaptable to the rapidly changing workflows and targets involved in kinase inhibitor discovery.

  11. EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

    PubMed

    Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A

    2013-01-01

    A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

  12. Enzyme-linked immunosorbent assay employing a recombinant antigen for detection of protective antibody against swine erysipelas.

    PubMed

    Imada, Yumiko; Mori, Yasuyuki; Daizoh, Masaji; Kudoh, Kazuma; Sakano, Tetsuya

    2003-11-01

    The specificities and sensitivities of five recombinant proteins of the surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae were examined by indirect enzyme-linked immunosorbent assay (ELISA) with the aim of developing a reliable serological test for the detection of protective antibody against E. rhusiopathiae. Fully mature protein and the N-terminal 416 amino acids (SpaA416) showed sufficient antigenicities, and further examination was done with SpaA416 because of its higher yield. The antibody titers of pigs experimentally immunized with commercial live vaccine and two types of inactivated vaccines clearly increased after immunization, and all pigs were completely protected against challenge with virulent strains. On the other hand, the antibody titers of nonimmunized control pigs remained very low until they were challenged, and all showed severe symptoms or subsequently died. Interference with the production of antibody against live vaccine by maternal antibody or porcine respiratory and reproductive syndrome virus infection 1 week after vaccination was also clearly detected. Because the ELISA titer correlated well with the protection results, the specificity and sensitivity of the ELISA were further evaluated with sera collected from pigs reared on 1 farm on which animals had acute septicemia, 2 farms on which the animals were infected or free from infection, and 10 farms on which the animals were vaccinated with live vaccine, among others. The ELISA titers clearly revealed the conditions of the herds. These results indicate that the SpaA416 ELISA is an effective method not only for evaluating pigs for the presence of protective antibody levels resulting from vaccination or maternal antibody but also for detecting antibody produced by natural infection. This test has important potential for the effective control of swine erysipelas.

  13. Use of enzyme-labelled protein G assay for the detection of anti Borrelia burgdorferi antibodies in wild animal sera.

    PubMed

    Deruaz, D; Eid, P; Deruaz, J; Sempéré, A; Bourgouin, C; Rodhain, F; Pérez-Eid, C

    1996-10-01

    A modified ELISA was developed for the detection of anti-Borrelia burgdorferi (Bb) IgG antibodies in wild animal sera based on an Enzyme-Labelled-protein G Assay (ELGA). Microplates were coated with an extract of Bb sensu stricto strain (SVI) as antigen. Specific antibodies of the serum samples were detected by a peroxidase-labelled-protein G. Using comparative immunodiagnosis by means of a passive hemagglutination test (HA), ELGA was tested on 82 roe-deer blood samples. A correlation was found between the two methods (r = 0.66). Good reproducibility of titers was observed by ELGA technique. A minimal cross-reactivity was discovered with Leptospira. ELGA could facilitate the recognition of specific antibodies in collections of wild animal sera.

  14. Detection of Antiendothelial Cell Antibodies by an Enzyme-Linked Immunosorbent Assay Using Antigens from Cell Lysate: Minimal Interference with Antinuclear Antibodies and Rheumatoid Factors

    PubMed Central

    Drouet, Christian; Nissou, Marie-France; Ponard, Denise; Arvieux, Josiane; Dumestre-Pérard, Chantal; Gaudin, Philippe; Imbert, Bernard; Massot, Christian; Sarrot-Reynauld, Françoise

    2003-01-01

    The objective of the present work was to set up a routine test adapted to screening for antiendothelial cell antibodies (AECAs) in serum samples with minimal interference from antinuclear antibodies (ANAs) or rheumatoid factors (RFs). We compared the titers of AECAs titrated following two enzyme-linked immunosorbent assays (ELISAs): (i) an ELISA with ethanol-fixed EA.hy926 monolayers as the antigenic substrate and (ii) an ELISA with nucleus-depleted lysates prepared from EA.hy926 cells and normalized for protein (1.0 to 1.7 mg/ml) and DNA (≤0.1 μg/ml) contents as a surrogate substrate (postnuclear supernatant ELISA [PNS-ELISA]). The AECA titers in 51 serum samples, including 28 samples containing ANAs, were compared. A significantly positive correlation (r = 0.77; P < 0.001) between the two series was shown only for the ANA-negative serum samples. Conversely, ANAs or RFs in samples were shown not to interfere in tests for AECAs by the PNS-ELISA. AECAs recognize their antigenic targets in postnuclear supernatants, which is representative of the endothelial antigenic content, with improvement of the reliability of the assay, a prerequisite to application of the assay for their evaluation in clinical practice. PMID:12965929

  15. Minimizing antibody cross-reactivity in multiplex detection of biomarkers in paper-based point-of-care assays

    NASA Astrophysics Data System (ADS)

    Dias, J. T.; Lama, L.; Gantelius, J.; Andersson-Svahn, H.

    2016-04-01

    Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 103-fold (5 pg mL-1) lower than when no ultrasound was applied (50 ng mL-1) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays.Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 103-fold (5 pg mL-1) lower than when no ultrasound was applied (50 ng mL-1) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr09207h

  16. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  17. A direct antigen-binding assay for detection of antibodies against native epitopes using alkaline phosphatase-tagged proteins.

    PubMed

    Baranov, Konstantin; Volkova, Olga; Chikaev, Nikolai; Mechetina, Ludmila; Laktionov, Pavel; Najakshin, Alexander; Taranin, Alexander

    2008-03-20

    We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.

  18. Measuring affinity constants of 1450 monoclonal antibodies to peptide targets with a microarray-based label-free assay platform.

    PubMed

    Landry, J P; Ke, Yaohuang; Yu, Guo-Liang; Zhu, X D

    2015-02-01

    Monoclonal antibodies (mAbs) are major reagents for research and clinical diagnosis. For their inherently high specificities to intended antigen targets and thus low toxicity in general, they are pursued as one of the major classes of new drugs. Yet binding properties of most monoclonal antibodies are not well characterized in terms of affinity constants and how they vary with presentations and/or conformational isomers of antigens, buffer compositions, and temperature. We here report a microarray-based label-free assay platform for high-throughput measurements of monoclonal antibody affinity constants to antigens immobilized on solid surfaces. Using this platform we measured affinity constants of over 1410 rabbit monoclonal antibodies and 46 mouse monoclonal antibodies to peptide targets that are immobilized through a terminal cysteine residue to a glass surface. The experimentally measured affinity constants vary from 10 pM to 200 pM with the median value at 66 pM. We compare the results obtained from the microarray-based platform with those from a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).

  19. Direct replacement of antibodies with molecularly imprinted polymer nanoparticles in ELISA--development of a novel assay for vancomycin.

    PubMed

    Chianella, Iva; Guerreiro, Antonio; Moczko, Ewa; Caygill, J Sarah; Piletska, Elena V; De Vargas Sansalvador, Isabel M Perez; Whitcombe, Michael J; Piletsky, Sergey A

    2013-09-03

    A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop assays similar to the enzyme-linked immunosorbent assay (ELISA) is presented here for the first time. NanoMIPs were synthesized by a solid-phase approach with an immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering, and electron microscopy. Immobilization, blocking, and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a horseradish peroxidase-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range of 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was 3 orders of magnitude better than a previously described ELISA based on antibodies. In these experiments, nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA.

  20. Screening for IgG antinuclear autoantibodies by HEp-2 indirect fluorescent antibody assays and the need for standardization.

    PubMed

    Copple, Susan S; Giles, S Rashelle; Jaskowski, Troy D; Gardiner, Anna E; Wilson, Andrew M; Hill, Harry R

    2012-05-01

    We evaluated 5 commercially available HEp-2 antinuclear antibody (ANA) indirect fluorescent antibody (IFA) assays using patient serum samples from 45 patients with rheumatoid arthritis, 50 with systemic lupus erythematosus (SLE), 35 with scleroderma, 20 with Sjögren syndrome, 10 with polymyositis, and 100 healthy control subjects. In addition, 12 defined serum samples from the Centers for Disease Control and Prevention and 100 patient serum samples sent to ARUP Laboratories (Salt Lake City, UT) for ANA IFA testing were also examined (n = 372). Standardization among the HEp-2 IFA assays occurred when they exhibited the same titer ± 1 doubling dilution. Agreement of the 5 assays was 78%. Within the specific groups of serum samples, agreement ranged from 44% in scleroderma serum samples to 93% in healthy control subjects, with 72% agreement in the SLE group. Variations in slide and substrate quality were also noted (ie, clarity, consistency of fluorescence, cell size, number and quality of mitotic cells). Along with subjectivity of interpretation, HEp-2 IFA assays are also vulnerable to standardization issues similar to other methods for ANA screening.

  1. Profiling of multiple signal pathway activities by multiplexing antibody and GFP-based translocation assays.

    PubMed

    Henriksen, Ulla; Fog, Jacob; Loechel, Frosty; Praestegaard, Morten

    2008-08-01

    Multiplexing of GFP based and immunofluorescence translocation assays enables easy acquisition of multiple readouts from the same cell in a single assay run. Immunofluorescence assays monitor translocation, phosphorylation, and up/down regulation of endogenous proteins. GFP-based assays monitor translocation of stably expressed GFP-fusion proteins. Such assays may be multiplexed along (vertical), across (horizontal), and between (branch) signal pathways. Examples of these strategies are presented: 1) The MK2-GFP assay monitors translocation of MK2-GFP from the nucleus to the cytoplasm in response to stimulation of the p38 pathway. By applying different immunofluorescent assays to the MK2 assay, a multiplexed HCA system is created for deconvolution of p38 pathway activation including assay readouts for MK2, p38, NFkappaB, and c-Jun. 2) A method for evaluating GPCR activation and internalization in a single assay run has been established by multiplexing GFP-based internalization assays with immunofluorescence assays for downstream transducers of GPCR activity: pCREB (cAMP sensor), NFATc1 (Ca(2+) sensor), and ERK (G-protein activation). Activation of the AT1 receptor is given as an example. 3) Cell toxicity readouts can be linked to primary readouts of interest via acquisition of secondary parameters describing cellular morphology. This approach is used to flag cytotoxic compounds and deselect false positives. The ATF6 Redistribution assay is provided as an example. These multiplex strategies provide a unique opportunity to enhance HCA data quality and save time during drug discovery. From a single assay run, several assay readouts are obtained that help the user to deconvolute the mode of action of test compounds.

  2. Evaluation of enzyme-linked immunosorbent and alternative assays for detection of HIV antibodies using panels of Brazilian sera.

    PubMed

    Ivo-Dos-Santos, J; Mello, D L; Couto-Fernandez, J C; Passos, R M; Dias-Carneiro, L A; Castilho, E A; Galvão-Castro, B

    1990-01-01

    Sera from 472 Brazilian subjects, confirmed to be either positive or negative for HIV antibodies and comprising the total clinical spectrum of HIV infection, were utilized in the evaluation of six commercially available enzyme-linked immunosorbent assays (ELISA), as well as of four alternative assays, namely indirect immunofluorescence (IIF), passive hemagglutination (PHA), dot blot and Karpas AIDS cell test. The sensitivities ranged from 100% (Abbott and Roche ELISA) to 84.2% (PHA) and the specificities ranged from 99.3% (IIF) to 80.2% (PHA). The sensitivity and specificity of the PHA and the sensitivity of the Karpas cell test were significantly lower than those of the other tests. Although the IFF and dot blot had good sensitivities and specificities, the six ELISA were more attractive than those tests when other parameters such as ease of reading and duration of assay were considered.

  3. Performance of HerpeSelect and Kalon Assays in Detection of Antibodies to Herpes Simplex Virus Type 2▿

    PubMed Central

    LeGoff, Jérôme; Mayaud, Philippe; Gresenguet, Gérard; Weiss, Helen A.; Nzambi, Khonde; Frost, Eric; Pepin, Jacques; Belec, Laurent

    2008-01-01

    The performances of commercial enzyme-linked immunosorbent assays (ELISAs) in detecting herpes simplex virus type 2 (HSV-2) antibodies have been inconsistent for African and human immunodeficiency virus (HIV)-positive populations. We compared the performances of the HerpeSelect and Kalon glycoprotein G2 ELISAs for patients with genital ulcer disease in Ghana and the Central African Republic. Sera from 434 women were tested with the HerpeSelect assay, and a subsample (n = 199) was tested by the Kalon assay. Ulcer swabs and cervicovaginal lavage samples were tested for HSV-2 DNA by PCR. HSV-2-seronegative women with detectable genital HSV-2 DNA were retested for HSV-2 antibodies 14 and 28 days later by the two ELISAs. A total of 346 (80%) women were positive by HerpeSelect at baseline, and 225 (54%) had detectable genital (lesional or cervicovaginal) HSV-2 DNA. Sixty-six (19%) HerpeSelect-positive samples had low-positive index values (1.1 to 3.5), and 58% of these samples had detectable genital HSV-2 DNA. Global agreement between the two serological assays was 86%. Concordance was high (99%) for sera that were negative by HerpeSelect or had high index values (>3.5). Defining infection detected by HSV-2 DNA PCR and/or Kalon assay as true infection, 71% of sera with low-positive index values were associated with true HSV-2 infection. Twenty-five women were identified as having nonprimary first-episode genital HSV-2 infection. Rates of HSV-2 seroconversion at day 14 were 77% (10/13 patients) by HerpeSelect assay and 23% (3/13 patients) by Kalon assay, with four additional seroconversions detected by Kalon assay at day 28. HIV serostatus did not influence assay performance. Low index values obtained with the HerpeSelect assay may correspond to true HSV-2 infection, in particular to nonprimary first episodes of genital HSV-2 infection, and need to be interpreted in the context of clinical history. PMID:18385443

  4. Performance of HerpeSelect and Kalon assays in detection of antibodies to herpes simplex virus type 2.

    PubMed

    LeGoff, Jérôme; Mayaud, Philippe; Gresenguet, Gérard; Weiss, Helen A; Nzambi, Khonde; Frost, Eric; Pepin, Jacques; Belec, Laurent

    2008-06-01

    The performances of commercial enzyme-linked immunosorbent assays (ELISAs) in detecting herpes simplex virus type 2 (HSV-2) antibodies have been inconsistent for African and human immunodeficiency virus (HIV)-positive populations. We compared the performances of the HerpeSelect and Kalon glycoprotein G2 ELISAs for patients with genital ulcer disease in Ghana and the Central African Republic. Sera from 434 women were tested with the HerpeSelect assay, and a subsample (n = 199) was tested by the Kalon assay. Ulcer swabs and cervicovaginal lavage samples were tested for HSV-2 DNA by PCR. HSV-2-seronegative women with detectable genital HSV-2 DNA were retested for HSV-2 antibodies 14 and 28 days later by the two ELISAs. A total of 346 (80%) women were positive by HerpeSelect at baseline, and 225 (54%) had detectable genital (lesional or cervicovaginal) HSV-2 DNA. Sixty-six (19%) HerpeSelect-positive samples had low-positive index values (1.1 to 3.5), and 58% of these samples had detectable genital HSV-2 DNA. Global agreement between the two serological assays was 86%. Concordance was high (99%) for sera that were negative by HerpeSelect or had high index values (>3.5). Defining infection detected by HSV-2 DNA PCR and/or Kalon assay as true infection, 71% of sera with low-positive index values were associated with true HSV-2 infection. Twenty-five women were identified as having nonprimary first-episode genital HSV-2 infection. Rates of HSV-2 seroconversion at day 14 were 77% (10/13 patients) by HerpeSelect assay and 23% (3/13 patients) by Kalon assay, with four additional seroconversions detected by Kalon assay at day 28. HIV serostatus did not influence assay performance. Low index values obtained with the HerpeSelect assay may correspond to true HSV-2 infection, in particular to nonprimary first episodes of genital HSV-2 infection, and need to be interpreted in the context of clinical history.

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

    PubMed

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

    2014-08-01

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

  6. Comparison of the prognostic impact of serum anti-EBV antibody and plasma EBV DNA assays in nasopharyngeal carcinoma

    SciTech Connect

    Twu, C.-W.; Wang, W.-Y.; Liang, W.-M.; Jan, J.-S.; Jiang, R.-S.; Chao, Jeffrey; Jin, Y.-T.; Lin, J.-C. . E-mail: jclin@vghtc.gov.tw

    2007-01-01

    Purpose: Nasopharyngeal carcinoma (NPC) has been proven as an Epstein-Barr virus (EBV)-associated cancer. Serum anti-EBV antibodies and plasma EBV DNA have been investigated as surrogate markers for NPC. A comparison of the prognostic impacts of both assays has never been reported. Methods and Materials: Paired serum and plasma samples from 114 previously untreated NPC patients were collected and subjected to an immunofluorescence assay for immunoglobulin (Ig)A and IgG antibodies against the viral capsid antigen (VCA) and a real-time quantitative polymerase chain reaction assay for EBV DNA measurement. The effects of both assays on patient prognosis were thoroughly investigated. Results: Relapsed patients had significantly higher pretreatment EBV DNA concentration than patients without relapse (p 0.0006). No associations of VCA-IgA (p = 0.9669) or VCA-IgG (p = 0.6125) were observed between patients with and without relapse. The 4-year overall survival (60.3% vs. 93.1%, p < 0.0001) and relapse-free survival rates (54.4% vs. 77.9%, p = 0.0009) were significantly lower in patients with higher pretreatment EBV DNA load than in those with lower EBV DNA load. Patients with persistently detectable EBV DNA after treatment had significantly worse 4-year overall (30.8% vs. 84.6%, p < 0.0001) and relapse-free survival rates (15.4% vs. 74.0%, p < 0.0001) than those with undetectable EBV DNA. The VCA-IgA and VCA-IgG titer could not predict survivals (all p > 0.1). Cox multivariate analyses also showed the same results. Conclusion: Plasma EBV DNA is superior to serum EBV VCA antibodies in prognostic predictions for NPC.

  7. Combined hepatitis C virus (HCV) antigen-antibody detection assay does not improve diagnosis for seronegative individuals with occult HCV infection.

    PubMed

    Quiroga, Juan A; Castillo, Inmaculada; Pardo, Margarita; Rodríguez-Iñigo, Elena; Carreño, Vicente

    2006-12-01

    A combined hepatitis C virus (HCV) antigen-antibody assay was evaluated for 115 seronegative individuals with occult HCV infection. The assay was reactive in one patient and negative to weakly reactive in three others (all four gave indeterminate results by supplemental assay) but failed to detect HCV in the remaining patients. Despite increased sensitivity the combined assay does not improve serodiagnosis of occult HCV infection.

  8. Development of an Assay to Detect Antibodies to HIV-2 Using Recombinant DNA Derived Antigens

    DTIC Science & Technology

    1990-09-11

    CBre3 EIA is sensitive enough to detect envelope antibodies in seropositive patients before the antibodies are detected in a viral lysate Western blot ...induced E. coli were electrophoresed in polyacrylamide gels and analyzed by Coomassie blue staining and Western blotting (10). Blots were blocked in 1 X... Western blot analysis was used to show that the induced protein is coded for by the HIV-2 DNA. A duplicate gel, as shown in Figure 3, was blotted and

  9. A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination

    PubMed Central

    van der Gaast—de Jongh, Christa E.; Diavatopoulos, Dimitri A.; de Jonge, Marien I.

    2017-01-01

    The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also anti-PspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner. PMID:28288168

  10. A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays.

    PubMed

    Rosas-Arellano, Abraham; Villalobos-González, Juan B; Palma-Tirado, Lourdes; Beltrán, Felipe A; Cárabez-Trejo, Alfonso; Missirlis, Fanis; Castro, Maite A

    2016-10-01

    Immunolocalization techniques are standard in biomedical research. Tissue fixation with aldehydes and cell membrane permeabilization with detergents can distort the specific binding of antibodies to their high affinity epitopes. In immunofluorescence protocols, it is desirable to quench the sample's autofluorescence without reduction of the antibody-dependent signal. Here we show that adding glycine to the blocking buffer and diluting the antibodies in a phosphate saline solution containing glycine, Triton X-100, Tween20 and hydrogen peroxide increase the specific antibody signal in tissue immunofluorescence and immunogold electron microscopy. This defined antibody signal enhancer (ASE) solution gives similar results to the commercially available Pierce Immunostain Enhancer (PIE). Furthermore, prolonged tissue incubation in resin and fixative and application of ASE or PIE are described in an improved protocol for triple immunogold electron microscopy that is used to show co-localization of GABA-A ρ2 and dopamine D2 receptors in GFAP-positive astrocytes in the mouse striatum. The addition of glycine, Triton X-100, Tween20 and hydrogen peroxide during antibody incubation steps is recommended in immunohistochemistry methods.

  11. A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay.

    PubMed

    Sakamoto, Seiichi; Tanizaki, Yusuke; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-05-01

    A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv.

  12. Detection of Klebsiella pneumoniae antibodies in Aotus l. lemurinus (Panamanian owl monkey) using an enzyme linked immunosorbent assay (ELISA) test.

    PubMed

    Obaldia, N

    1991-04-01

    An enzyme linked immunosorbent assay (ELISA), was adapted to detect antibodies against Klebsiella pneumoniae in Aotus l. lemurinus monkeys. It was used to define the prevalence of infection and the immunogenicity of an Al(OH)3 bacterin in a population of laboratory born A. l. lemurinus monkeys. This represents a preliminary step to reduce K. pneumoniae produced mortality. A striking finding during a cross-sectional prevalence study was that none of the babies of less than 2 months old had detectable levels of antibody. The antibody prevalence gradually increased in all other age groups reaching 87.5% in the 8-10-month-old group. These results indicate that infection with K. pneumoniae occurred sometime between 2 and 6 months of age, probably as a result of oral-faecal contamination and a change in the feeding and grooming behaviour. To determine whether infants had maternal antibodies or if they were asymptomatic carriers of the bacterium, a cross-sectional study was done in 15 infants less than 4 months old and their mothers. K. pneumoniae antibodies were detected in 11/15 mothers with serum titers ranging from 1:4 to greater than 1:256 and the bacterium was isolated from 3 babies and one mother and her baby. Results showed that no maternal antibodies remained in babies older than 3 weeks old. A prospective study indicated a reduction in mortality from 20% for the previous 3 years to 3.7% (3/79) in AL(OH)3 K. pneumoniae bacterin vaccinated infants born during 1988-89.

  13. Silicate antibodies in women with silicone breast implants: development of an assay for detection of humoral immunity.

    PubMed Central

    Shen, G Q; Ojo-Amaize, E A; Agopian, M S; Peter, J B

    1996-01-01

    Silicon, in the form of sodium silicate (Na2SiO3), adsorbed onto bovine serum albumin (BSA)-precoated plates served as the solid-phase antigen in an enzyme immunoassay to detect silicate-reactive antibodies in the plasma of 40 symptomatic women with silicone breast implants, 91 asymptomatic women with silicone breast implants, 50 healthy control women, and 52 women with rheumatic diseases and without silicone breast implants, Silicate-reactive antibodies of immunoglobulin G (IgG) or IgM isotypes were detected in the plasma of 30% (12 of 40) of the symptomatic women with silicone breast implants; 9% (8 of 91) of the asymptomatic women with silicone breast implants; 5% (1 of 20) of the women without implants who had systemic lupus erythematosus; and 0% (0 of 32) of the women without implants who had either Sjögren syndrome, scleroderma, or rheumatoid arthritis. Only 2% (1 of 50) of the sera from the healthy control women contained silicate-reactive antibodies. Preincubation of sera with silicate and eight other metal compounds (including SiO2) demonstrated that the IgG and IgM antibodies bound specifically to silicate, because preincubation with Na2SiO3 inhibited more than 90% of the activity, whereas CrO3, Li2SO4, MgSO4, NiSO4, HgCl2, ZrOCl2, BeSO4, and SiO2 failed to inhibit the IgG or IgM antibody binding to the silicate-BSA plates. Furthermore, the F(ab')2 portion and not the Fc portion of the silicate-reactive IgG was reactive with BSA-bound silicate in the enzyme immunoassay. The assay for silicate-reactive antibodies was quantified by assigning arbitrary units to a standard curve composed of serial twofold dilutions of high-positive (ten times higher than the cutoff) silicate antibody sera. This novel assay is a useful method for detecting and quantifying humoral immune response to silicate. PMID:8991630

  14. Competitive detection of influenza neutralizing antibodies using a novel bivalent fluorescence-based microneutralization assay (BiFMA).

    PubMed

    Baker, Steven F; Nogales, Aitor; Santiago, Felix W; Topham, David J; Martínez-Sobrido, Luis

    2015-07-09

    Avian-derived influenza A zoonoses are closely monitored and may be an indication of virus strains with pandemic potential. Both successful vaccination and convalescence of influenza A virus in humans typically results in the induction of antibodies that can neutralize viral infection. To improve long-standing and new-generation methodologies for detection of neutralizing antibodies, we have employed a novel reporter-based approach that allows for multiple antigenic testing within a single sample. Central to this approach is a single-cycle infectious influenza A virus (sciIAV), where a functional hemagglutinin (HA) gene was changed to encode either the green or the monomeric red fluorescent protein (GFP and mRFP, respectively) and HA is complemented in trans by stable HA-expressing cell lines. By using fluorescent proteins with non-overlapping emission spectra, this novel bivalent fluorescence-based microneutralization assay (BiFMA) can be used to detect neutralizing antibodies against two distinct influenza isolates in a single reaction, doubling the speed of experimentation while halving the amount of sera required. Moreover, this approach can be used for the rapid identification of influenza broadly neutralizing antibodies. Importantly, this novel BiFMA can be used for any given influenza HA-pseudotyped virus under BSL-2 facilities, including highly pathogenic influenza HA isolates.

  15. Radial-immunodiffusion assay of human apolipoprotein A-I with use of two monoclonal antibodies combined.

    PubMed

    Marcovina, S; Di Cola, G; Catapano, A L

    1986-12-01

    We produced and characterized several monoclonal antibodies directed toward human plasma apolipoprotein A-I. Two of them, A-I-12 and A-I-57, individually precipitated purified or native high-density lipoprotein in agarose gel by double immunodiffusion. Because radial immunodiffusion performed with a single monoclonal antibody gave faint and diffuse rings of precipitation, we developed and optimized working conditions for using these two monoclonal antibodies combined to determine apolipoprotein A-I in human plasma. This combination gave easy-to-measure, clear, sharp rings, and linear and parallel standard curves for HDL3 (the primary standard) and a reference serum (the secondary standard). Moreover, no pretreatment of samples with dissociating agents or detergents is necessary. The assay was complete after overnight incubation, as compared with two to three days when polyclonal antisera were used. Apolipoprotein A-I concentrations as measured in 128 normolipidemic subjects and in 72 patients with various lipid disorders by the radial immunodiffusion technique with monoclonal antibodies (x) compared well (r = 0.882; y = 1.029x-0.036) with those measured by radial immunodiffusion with polyclonal antisera (y).

  16. Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays.

    PubMed

    Choudhary, Nandlal; Roy, Avijit; Leon, M G; Wei, G; Nakhla, M K; Levy, L; Brlansky, R H

    2017-05-01

    The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.

  17. Complement-fixing donor-specific antibodies identified by a novel C1q assay are associated with allograft loss.

    PubMed

    Sutherland, Scott M; Chen, Ge; Sequeira, Flavia A; Lou, Calvin D; Alexander, Steven R; Tyan, Dolly B

    2012-02-01

    Long-term outcomes following renal transplantation remain disappointing. Recently, interest has focused on the antibody-mediated component of allograft injury and the deleterious effects of DSA. We applied a novel C1q solid-phase assay in parallel with the standard IgG SAB assay to identify DSA with the potential to activate complement by binding C1q. Among 193 consecutive renal transplants at our center, 19.2% developed de novo DSA following transplantation. Of the patients with DSA, 43% had antibodies that bound C1q in vitro [C1q+ DSA]. Patients with C1q+ DSA were more likely to develop allograft loss than patients with DSA that did not bind C1q (46.7% vs. 15%; p = 0.04); patients with C1q+ DSA were nearly six times more likely to lose their transplant than those with C1q- DSA. Additionally, patients with C1q+ DSA who underwent allograft biopsy were more likely to demonstrate C4d deposition (50% vs. 8%; p = 0.03) and meet criteria for acute rejection (60% vs. 17%; p = 0.02) when compared with patients with DSA that did not bind C1q. These data suggest that DSA with the ability to activate complement, as determined by this novel C1q assay, are associated with greater risk of acute rejection and allograft loss.

  18. Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs.

    PubMed

    Ribotta, M J; Higgins, R; Gottschalk, M; Lallier, R

    2000-01-01

    Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources, expertise, or facilities to perform the microscopic agglutination test (MAT). Thus, there is a need for a rapid and simple serological test that could be used in any diagnostic laboratory. In this study, a genus-specific, heat-stable antigenic preparation from Leptospira interrogans serovar pomona was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of leptospiral antibodies in dog sera. This antigenic preparation reacted with rabbit antisera against L. interrogans serovars bratislava, autumnalis, icterohaemorrhagiae and pomona and with rabbit antiserum against L. kirschneri serovar grippotyphosa. The ELISA showed a relative specificity of 95.6% with 158 dog sera which were negative at a dilution of 1:100 in the MAT for serovars pomona, bratislava, icterohaemorrhagiae, autumnalis, hardjo, and grippotyphosa. The relative sensitivity of this assay with 21 dog sera that revealed serovars MAT titres of > or =100 to different serovars was 100%. This assay is easily standardized, technically more advantageous than MAT, and uses an antigenic preparation that can be routinely prepared in large amounts. It was concluded that this ELISA is sufficiently sensitive test to be used as an initial screening test for the detection of leptospiral antibodies in canine sera, with subsequent confirmation of positive test results with the MAT.

  19. Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to Junin virus in rodents.

    PubMed

    Morales, María A; Calderón, Gladys E; Riera, Laura M; Ambrosio, Ana M; Enría, Delia A; Sabattini, Marta S

    2002-05-01

    Junin virus is the etiological agent of Argentine hemorrhagic fever, a serious rodent-borne disease. An enzyme-linked immunosorbent assay (ELISA) to detect Junin virus IgG antibodies in rodents was evaluated using sera from 27 Calomys musculinus and five Calomys laucha, inoculated experimentally with a live attenuated strain of this arenavirus. The test performance was compared against an indirect immunofluorescence assay (IFA). The ELISA had a sensitivity and specificity of 100% and a reproducibility of 87.9% for samples with titers above the selected cut-off value. IFA had lower sensitivity (53%) with the same specificity. The ELISA results were similar, whether carried out on whole blood or serum samples, thus eliminating the need for serum separation. A high correlation (K=0.86) between ELISA and IFA results was obtained from 1011 wild sigmodontine and murine rodents collected within and outside of the Argentine hemorrhagic fever endemic area. These results indicate that Junin virus IgG ELISA is the most suitable assay for detection of Junin virus antibodies in rodent samples.

  20. Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs.

    PubMed Central

    Ribotta, M J; Higgins, R; Gottschalk, M; Lallier, R

    2000-01-01

    Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources, expertise, or facilities to perform the microscopic agglutination test (MAT). Thus, there is a need for a rapid and simple serological test that could be used in any diagnostic laboratory. In this study, a genus-specific, heat-stable antigenic preparation from Leptospira interrogans serovar pomona was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of leptospiral antibodies in dog sera. This antigenic preparation reacted with rabbit antisera against L. interrogans serovars bratislava, autumnalis, icterohaemorrhagiae and pomona and with rabbit antiserum against L. kirschneri serovar grippotyphosa. The ELISA showed a relative specificity of 95.6% with 158 dog sera which were negative at a dilution of 1:100 in the MAT for serovars pomona, bratislava, icterohaemorrhagiae, autumnalis, hardjo, and grippotyphosa. The relative sensitivity of this assay with 21 dog sera that revealed serovars MAT titres of > or =100 to different serovars was 100%. This assay is easily standardized, technically more advantageous than MAT, and uses an antigenic preparation that can be routinely prepared in large amounts. It was concluded that this ELISA is sufficiently sensitive test to be used as an initial screening test for the detection of leptospiral antibodies in canine sera, with subsequent confirmation of positive test results with the MAT. PMID:10680654

  1. Optimization and proficiency testing of a pseudovirus-based assay for detection of HIV-1 neutralizing antibody in China.

    PubMed

    Nie, Jianhui; Wang, Wenbo; Wen, Zhiheng; Song, Aijing; Hong, Kunxue; Lu, Shan; Zhong, Ping; Xu, Jianqing; Kong, Wei; Li, Jingyun; Shang, Hong; Ling, Hong; Ruan, Li; Wang, Youchun

    2012-11-01

    Among the neutralizing antibody evaluation assays, the single-cycle pseudovirus infection assay is high-throughput and can provide rapid, sensitive and reproducible measurements after a single cycle of infection. Cell counts, pseudovirus inoculation levels, amount of diethylaminoethyl-dextran (DEAE-dextran), and the nonspecific effects of serum and plasma were tested to identify the optimal conditions for a neutralizing antibody assay based on pseudoviruses. Optimal conditions for cell counts, pseudovirus inoculation, and amount of DEAE-dextran were 1 × 10(4)cells/well, 200TCID(50)/well, and 15 μg/ml, respectively. Compared with serum samples, high-concentration anticoagulants reduced the relative light unit (RLU) value. The RLU value increased sharply initially but then decreased slowly with dilution of the plasma sample. Test kits containing 10 HIV-1 CRF07/08_BC pseudovirus strains and 10 plasma samples from individuals infected with HIV-1 CRF07/08_BC were assembled into two packages and distributed to nine laboratories with a standard operating procedure included. For the 10 laboratories that evaluated the test, 17 of 44 (37%) laboratory pairs were considered equivalent. A statistical qualification rule was developed based on the testing results from 5 experienced laboratories, where a laboratory qualified if at least 83% of values lied within the acceptable range.

  2. Detection of Leptospira-Specific Antibodies Using a Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Chen, Hua-Wei; Zhang, Zhiwen; Halsey, Eric S.; Guevara, Carolina; Canal, Enrique; Hall, Eric; Maves, Ryan; Tilley, Drake H.; Kochel, Tadeusz J.; Ching, Wei-Mei

    2013-01-01

    We produced three highly purified recombinant antigens rLipL32, rLipL41, and rLigA-Rep (leptospiral immunoglobulin-like A repeat region) for the detection of Leptospira-specific antibodies in an enzyme-linked immunosorbent assay (ELISA). The performance of these recombinant antigens was evaluated using 121 human sera. Among them, 63 sera were microscopic agglutination test (MAT)-confirmed positive sera from febrile patients in Peru, 22 sera were indigenous MAT-negative febrile patient sera, and 36 sera were from patients with other febrile diseases from Southeast Asia, where leptospirosis is also endemic. Combining the results of immunoglobulin M (IgM) and IgG detection from these three antigens, the overall sensitivity is close to 90% based on the MAT. These results suggest that an ELISA using multiple recombinant antigens may be used as an alternative method for the detection of Leptospira-specific antibodies. PMID:24166046

  3. Simple visual assay for determination of Pasteurella haemolytica cytotoxin neutralizing antibody titers in cattle sera.

    PubMed Central

    Gentry, M J; Confer, A W; Kreps, J A

    1985-01-01

    A simple visual assay is described for determining the capacity of bovine serum to neutralize the cytotoxin produced by Pasteurella haemolytica serotype 1. The test was reproducible from day to day with different target cell populations and cytotoxin preparations. Cytotoxin neutralization titers obtained by the visual assay were comparable to those determined by the trypan blue exclusion and 51Cr-release methods. The visual assay was used to measure neutralization titers of bovine sera obtained from vaccination experiments and fractions of purified serum obtained by gel filtration. The major advantages of the visual assay over other assays are that it is rapid, inexpensive, and does not use radioisotopes. It also does not require specialized equipment, making it adaptable to most laboratories. Images PMID:3905853

  4. Comparison of phenotypically indistinguishable but geographically distinct Neisseria meningitidis Group B isolates in a serum bactericidal antibody assay.

    PubMed

    Findlow, Jamie; Holland, Ann; Andrews, Nick; Weynants, Vincent; Sotolongo, Franklin; Balmer, Paul; Poolman, Jan; Borrow, Ray

    2007-11-01

    The "gold standard" assay for measuring serologic protection against Neisseria meningitidis group B (MenB) is the serum bactericidal antibody (SBA) assay. Of vital importance to the outcome of the SBA assay is the choice of the target strain(s), which is often chosen on the basis of phenotype or genotype. We therefore investigated the effect on the results produced by the SBA assay of using phenotypically indistinguishable but geographically distinct MenB isolates. Nine PorA P1.19,15 and 11 PorA P1.7-2,4 MenB isolates were incorporated into the SBA assay using human complement and were assayed against sera obtained either before or after outer membrane vesicle vaccination. Large differences in the results produced by the isolates in the SBA assay were demonstrated. These included differences as great as 5.8-fold in SBA geometric mean titers and in the proportions of subjects with SBA titers of >/=4. Ranges of as many as 9 SBA titers were achieved by individual sera across the panels of isolates. To determine the reasons for the differences observed, investigations into the expression of capsular polysaccharide, PorA, PorB, Opc, and lipooligosaccharide (LOS) and into LOS sialylation were completed. However, minor differences were found between strains, indicating similar expression and no antigen masking. These results have implications for the choice of MenB target strains for inclusion in future studies of MenB vaccines and highlight the requirement for standardization of target strains between laboratories.

  5. Antinuclear antibody prevalence in a general pediatric cohort from Mexico City: discordance between immunofluorescence and multiplex assays

    PubMed Central

    Somers, Emily C; Monrad, Seetha U; Warren, Jeffrey S; Solano, Maritsa; Schnaas, Lourdes; Hernandez-Avila, Mauricio; Tellez-Rojo, Martha Maria; Hu, Howard

    2017-01-01

    Objective To characterize antinuclear antibody (ANA) prevalence according to distinct assay methodologies in a pediatric cohort from Mexico City, and to further examine associations with age and sex. Methods Serum ANA were measured by indirect immunofluorescence assay (IFA) and multiplex immunoassay in 114 children aged 9–17 years. IFA was considered positive at a cutoff titer of ≥1:80. Agreement between assay methods was assessed by kappa statistic. Sensitivity, specificity, and 95% confidence intervals (CIs) of the multiplex were computed with IFA as the reference standard. Results Of the 114 children (mean age 14.7 [standard deviation 2.1] years; 54 [47%] female), 18 of 114 (15.8%) were ANA positive by IFA, and 11 of 114 (9.6%) by 11-antigen multiplex assay. ANA prevalence was higher in females compared with males by both of the methods (ratios 1.6–1.9 to 1). Agreement between tests was classified as slight by kappa (κ=0.177 [95% CI −0.051, 0.406]). The multiplex immunoassay had sensitivity of 22.2% (95% CI 6.4, 47.6) and specificity of 92.7% (95% CI 85.6, 97.0), and failed to capture 3 of 4 (75%) of the high-titer (≥1:1280) IFA-positives. Conclusion Up to 15% of children in this general population cohort were ANA positive, with a higher rate of positivity among females according to both assay methods. Substantial discordance in ANA results was found between IFA and multiplex methods, even for high-titer IFA positives. These findings underscore the need to sufficiently account for assay characteristics when interpreting ANA test results, and support IFA as the more appropriate assay for studies of subclinical autoimmunity. PMID:28053555

  6. Improved performance of Brucella melitensis native hapten over Brucella abortus OPS tracer on goat antibody detection by the fluorescence polarization assay.

    PubMed

    Ramírez-Pfeiffer, C; Díaz-Aparicio, E; Rodríguez-Padilla, C; Morales-Loredo, A; Alvarez-Ojeda, G; Gomez-Flores, R

    2008-06-15

    The current method for goat brucellosis diagnosis is based on the World Organization for Animal Health (OIE) using the screening card test (CT), with antigen at 8% (CT8) or 3% (CT3) of cell concentrations, and the confirmatory complement fixation test (CFT). However, these tests do not differentiate antibodies induced by vaccination from those derived from field infections by Brucella species or other bacterial agents; in places like Mexico, where the prevalence of brucellosis and the vaccination rates are high, there is a considerable percentage of false positive reactions that causes significant unnecessary slaughter of animals. Furthermore, results of the fluorescence polarization assay (FPA) using the Brucella abortus O-polysaccharide (OPS) tracer in goats are poorer than those with cattle. The present study was undertaken to investigate a tracer prepared from the native hapten (NH) of the Rev. 1 strain of Brucella melitensis to improve FPA performance on goat brucellosis diagnosis. Evaluation of 48 positive samples and 96 negative samples showed that the NH tracer was more accurate (p<0.01) than the OPS tracer (97.2% vs. 93.8% accuracy, respectively). On the diagnostic performance evaluation, the NH tracer performed better (87.5% accuracy, 79.5% sensitivity, 84.3% specificity, and 163.8 performance index) than the OPS tracer (83.5%, 75.9%, 81.0%, and 156.9, respectively) using 1009 positive and 2039 negative Mexican field goat sera samples selected by test series approved by the OIE (card test 3% and CFT). We demonstrated a new application for the NH lipopolysaccharide on detecting antibodies against Brucella using the FPA, which may yield faster results (minutes vs. 24-72h) than the immunodiagnosis assays frequently used in bovine brucellosis. In addition, NH tracer produces similar or better performance results than the conventional OPS tracer, using the FPA in goat sera samples.

  7. Detection of HLA class I-specific antibodies by the QuikScreen enzyme-linked immunosorbent assay.

    PubMed Central

    Lucas, D P; Paparounis, M L; Myers, L; Hart, J M; Zachary, A A

    1997-01-01

    The GTI QuikScreen test is an enzyme-linked immunosorbent assay (ELISA) that uses soluble HLA class I antigens as targets. In tests of 5,893 human serum specimens, we evaluated the reliability, sensitivity, and utility of the GTI QuikScreen test for detecting HLA class I-specific antibody. We found that the test could reliably detect HLA-specific antibodies of the immunoglobulin G (IgG) but not the IgM class. The degree of correlation with lymphocytotoxicity testing varied among the different serum sources, with the best correlation achieved with sera from renal transplant candidates (r > 0.7) and the poorest with sera from patients with end-stage liver disease (r = 0.26), possibly because of elevated alkaline phosphatase levels in the liver patients. Test reproducibility was high (96%), and test failure rate was low (1.7%). The test sensitivity is comparable to that of the antiglobulin cytotoxicity and, possibly, even flow cytometric tests. There was a highly significant (P < 0.001) correlation between the optical densities obtained in the ELISA and the percent panel reactive antibody determined by cytotoxicity testing. Therefore, although designed only to determine the presence or absence of HLA-specific antibody, GTI QuikScreen test results also provided an indication of the extent of sensitization. The test is one of the most effective and efficient ways to determine if antibodies producing a positive result in crossmatch tests are specific for HLA class I antigens. As an adjunct to serum screening by cytotoxicity testing, the GTI QuikScreen test can produce a substantial savings of time and effort that reduces the cost to the laboratory and to the patient. PMID:9144358

  8. Modular, Antibody-free Time-Resolved LRET Kinase Assay Enabled by Quantum Dots and Tb3+-sensitizing Peptides

    PubMed Central

    Cui, Wei; Parker, Laurie L.

    2016-01-01

    Fluorescent drug screening assays are essential for tyrosine kinase inhibitor discovery. Here we demonstrate a flexible, antibody-free TR-LRET kinase assay strategy that is enabled by the combination of streptavidin-coated quantum dot (QD) acceptors and biotinylated, Tb3+ sensitizing peptide donors. By exploiting the spectral features of Tb3+ and QD, and the high binding affinity of the streptavidin-biotin interaction, we achieved multiplexed detection of kinase activity in a modular fashion without requiring additional covalent labeling of each peptide substrate. This strategy is compatible with high-throughput screening, and should be adaptable to the rapidly changing workflows and targets involved in kinase inhibitor discovery. PMID:27426233

  9. Array-in-well platform-based multiplex assay for the simultaneous detection of anti-HIV- and treponemal-antibodies, and Hepatitis B surface antigen.

    PubMed

    Talha, Sheikh M; Saviranta, Petri; Hattara, Liisa; Vuorinen, Tytti; Hytönen, Jukka; Khanna, Navin; Pettersson, Kim

    2016-02-01

    Multiplex assays detecting sets of related clinical analytes simultaneously can save considerable amount of time and resources. Array-in-well (AIW) is a powerful platform for the multiplex detection of different analytes where microarrays can be printed at the bottom of microtiter wells, thus combining the potential of microarrays with the ease of handling microtiter wells. We have developed a single-step AIW assay for the simultaneous screening of HIV, Treponema pallidum subspecies pallidum (causing syphilis) and Hepatitis B virus infections targeting the specific detection of anti-HIV- and treponemal-antibodies and Hepatitis B surface antigen (HBsAg), respectively, using two different fluorescent label technologies i.e. DyLight 633 and europium nanoparticle. Double-antigen assay formats were used for anti-HIV- and treponemal-antibody detection that can simultaneously detect both IgG and IgM, and thus reduce the window period of detection. AIW assay was evaluated with well characterized serum/plasma samples (n=111), and the qualitative results were in near complete agreement with those of the reference assays. The AIW assay exhibited 100% sensitivities for all three analytes, and 100% specificities for anti-HIV antibodies and HBsAg, and 98.6% specificity for treponemal antibodies. The limit of detection of HBsAg in AIW assay was 0.18 ng/ml. This high performing AIW assay has the potential to be used as a multiplex screening test for these three infections.

  10. Recent advances in the characterization of HIV-1 neutralization assays for standardized evaluation of the antibody response to infection and vaccination.

    PubMed

    Polonis, Victoria R; Brown, Bruce K; Rosa Borges, Andrew; Zolla-Pazner, Susan; Dimitrov, Dimiter S; Zhang, Mei-Yun; Barnett, Susan W; Ruprecht, Ruth M; Scarlatti, Gabriella; Fenyö, Eva-Maria; Montefiori, David C; McCutchan, Francine E; Michael, Nelson L

    2008-06-05

    In AIDS vaccine development the pendulum has swung towards a renewed emphasis on the potential role for neutralizing antibodies in a successful global vaccine. It is recognized that vaccine-induced antibody performance, as assessed in the available neutralization assays, may well serve as a "gatekeeper" for HIV-1 subunit vaccine prioritization and advancement. As a result, development of a standardized platform for reproducible measurement of neutralizing antibodies has received considerable attention. Here we review current advancements in our knowledge of the performance of different types of antibodies in a traditional primary cell neutralization assay and the newer, more standardized TZM-bl reporter cell line assay. In light of recently revealed differences (see accompanying article) in the results obtained in these two neutralization formats, parallel evaluation with both platforms should be contemplated as an interim solution until a better understanding of immune correlates of protection is achieved.

  11. Differentiation between human coronaviruses NL63 and 229E using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies.

    PubMed

    Sastre, Patricia; Dijkman, Ronald; Camuñas, Ana; Ruiz, Tamara; Jebbink, Maarten F; van der Hoek, Lia; Vela, Carmen; Rueda, Paloma

    2011-01-01

    Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in an Escherichia coli system and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections.

  12. Fabrication of an antibody-aptamer sandwich assay for electrochemical evaluation of levels of β-amyloid oligomers

    PubMed Central

    Zhou, Yanli; Zhang, Huanqing; Liu, Lantao; Li, Congming; Chang, Zhu; Zhu, Xu; Ye, Baoxian; Xu, Maotian

    2016-01-01

    Amyloid β-peptide (Aβ) in its oligomeric form is often considered as the most toxic species in Alzheimer’s disease (AD), and thus Aβ oligomer is a potentially promising candidate biomarker for AD diagnosis. The development of a sensitive and reliable method for monitoring the Aβ oligomer levels in body fluids is an urgent requirement in order to predict the severity and progression at early or preclinical stages of AD. Here, we show a proof of concept for a sensitive and specific detection of Aβ oligomers by an antibody-aptamer sandwich assay. The antibodies of Aβ oligomers and a nanocomposite of gold nanoparticles with aptamer and thionine (aptamer-Au-Th) were used as the recognition element and the detection probe for specifically binding to Aβ oligomers, respectively. The electrochemical signal of Th reduction could provide measurable electrochemical signals, and a low limit of detection (100 pM) was achieved due to the signal amplification by high loading of Th on the gold nanoparticles. The feasibility of the assay was verified by test of Aβ oligomers in artificial cerebrospinal fluid. The proposed strategy presents valuable information related to early diagnosis of AD process. PMID:27725775

  13. Evaluation of enzyme-linked immunosorbent assays for detection of Mycoplasma bovis-specific antibody in bison sera.

    PubMed

    Register, Karen B; Sacco, Randy E; Olsen, Steven C

    2013-09-01

    Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. A method for the detection of M. bovis-specific serum antibodies is needed in order to establish prevalence and transmission patterns. Enzyme-linked immunosorbent assays (ELISAs) validated for the detection of M. bovis-specific serum IgG in cattle are commercially available, but their suitability for bison sera has not been determined. A collection of bison sera, most from animals with a known history of infection or vaccination with M. bovis, was tested for M. bovis-specific IgG using commercially available kits as well as an in-house ELISA in which either cattle or bison M. bovis isolates were used as a source of antigen. Comparison of the results demonstrates that ELISAs optimized for cattle sera may not be optimal for the identification of bison seropositive for M. bovis, particularly those with low to moderate antibody levels. The reagent used for the detection of bison IgG and the source of the antigen affect the sensitivity of the assay. Optimal performance was obtained when the capture antigen was derived from bison isolates rather than cattle isolates and when a protein G conjugate rather than an anti-bovine IgG conjugate was used for the detection of bison IgG.

  14. Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

    PubMed Central

    Kim, Sung-Hee; Cha, Sang-Ho; Karyn, Bischoff; Park, Sung-Won; Son, Seong-Wan

    2011-01-01

    Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1- carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with λ-type light chains. The IC50s of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml (R2 > 0.99) and from 1 to 100 ng/ml (R2 > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed. PMID:24278561

  15. Evaluation of classical swine fever virus antibody detection assays with an emphasis on the differentiation of infected from vaccinated animals.

    PubMed

    Schroeder, S; von Rosen, T; Blome, S; Loeffen, W; Haegeman, A; Koenen, F; Uttenthal, A

    2012-12-01

    The aim of this study was to evaluate the general characteristics of commercially available enzyme-linked immunosorbent assays (ELISAs) to detect antibody against classical swine fever (CSF), as well as to assess their potential use as accompanying marker tests able to differentiate infected from vaccinated animals (DIVA). The Chekit* CSF-Sero and the HerdChek* CSFV Ab, both of which detect antibodies against the E2 protein of classical swine fever virus (CSFV), had the highest sensitivity. Both tests were practicable and showed good reproducibility. Comparable sensitivity was shown by the Chekit* CSF-Marker, an Erns ELISA. However, this test does not allow differentiation between antibodies directed against ruminant pestiviruses and those against CSFV. Therefore, it is not suitable for use with the chimeric marker vaccines tested. The PrioCHECK CSFV Erns was the only ELISA suitable for use in DIVA with marker vaccines containing Erns proteins from ruminant pestiviruses. However, this test was less sensitive and selective than the E2-ELISAs and cannot be recommended.

  16. Evaluation of an enzyme-linked immunosorbent assay for quantitation of antibodies to Junin virus in human sera.

    PubMed

    García Franco, S; Ambrosio, A M; Feuillade, M R; Maiztegui, J I

    1988-01-01

    An enzyme-linked immunosorbent assay (ELISA) was evaluated for the quantitation of anti-Junin virus (JV) antibodies, in 83 selected cases of Argentine haemorrhagic fever (AHF). Serum samples were studied in two groups to facilitate comparative analysis; the first group was ELISA with indirect immunofluorescence (IF) test, in the second ELISA with plaque reduction neutralization test (PRINT). From the results obtained by using ELISA and IF on the same serum samples, a clear tendency of ELISA to demonstrate seroconversion for JV earlier and at higher frequency than IF test was noted. Simultaneous titration of specific antibodies by ELISA and PRNT tests rendered significantly correlated titers (r = 0.81), both methods being equivalently specific (100%). The demonstration of specific antibodies by ELISA in two cases that were undetected by the PRNT test resulted in a higher sensitivity index for ELISA than for PRNT (100% vs 97%). It is concluded that ELISA could efficiently replace IF and PRNT tests for the diagnosis of AHF.

  17. Effect of anti-thyroid peroxidase (TPO) antibodies on TPO activity measured by chemiluminescence assay.

    PubMed

    Kaczur, V; Vereb, G; Molnár, I; Krajczár, G; Kiss, E; Farid, N R; Balázs, C

    1997-08-01

    A chemiluminescence method was developed to measure thyroid peroxidase (TPO) activity and the inhibitory effect of anti-TPO antibodies in purified porcine TPO. The TPO preparation was characterized kinetically and controlled by Western-blotting technique. The chemiluminescence method proved to be reproducible and much more sensitive than the widely used guaiacol method, being able to detect TPO concentrations of 2.21 x 10(-5) g/L vs 6.63 x 10(-2) g/L with the latter. Otherwise, the determinations with the two methods correlated well (r = 0.76). Investigating the effect of IgGs from 23 hypothyroid patients on measured TPO activity, we detected inhibition in 19 cases with the chemiluminescence technique (15 with the guaiacol method). Anti-TPO antibodies showed competitive inhibition of TPO activity with respect to the substrate guaiacol. In both systems, the inhibition is present in the IgG F(ab')2 fragment. We conclude that the high sensitivity of chemiluminescence detection allows routine determination of the inhibition of TPO activity by anti-TPO antibodies.

  18. An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis.

    PubMed Central

    Nicholson, W L; Comer, J A; Sumner, J W; Gingrich-Baker, C; Coughlin, R T; Magnarelli, L A; Olson, J G; Childs, J E

    1997-01-01

    An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent. PMID:9163471

  19. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes.

    PubMed

    Bartosch, Birke; Bukh, Jens; Meunier, Jean-Christophe; Granier, Christelle; Engle, Ronald E; Blackwelder, William C; Emerson, Suzanne U; Cosset, François-Loïc; Purcell, Robert H

    2003-11-25

    Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C.

  20. In vitro assay for neutralizing antibody to hepatitis C virus: Evidence for broadly conserved neutralization epitopes

    PubMed Central

    Bartosch, Birke; Bukh, Jens; Meunier, Jean-Christophe; Granier, Christelle; Engle, Ronald E.; Blackwelder, William C.; Emerson, Suzanne U.; Cosset, François-Loïc; Purcell, Robert H.

    2003-01-01

    Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C. PMID:14617769

  1. Detection of antibody to staphylococcal lipoteichoic acid with a microenzyme-linked immunosorbent assay.

    PubMed Central

    Carruthers, M M; Jenkins, K E; Kabat, W J; Buranosky, T

    1984-01-01

    Sera from individuals with Staphylococcus aureus endocarditis and osteomyelitis and from some individuals with other forms of gram-positive endocarditis yielded higher readings in a microenzyme-linked immunosorbent assay against lipoteichoic acid from S. aureus than did sera from individuals with other types of serious staphylococcal infection or non-staphylococcal osteomyelitis, or from unselected inpatients. PMID:6715523

  2. Understanding antinuclear antibodies.

    PubMed

    Pertusi, R M; Rubin, B R

    1991-06-01

    The American College of Rheumatology (formerly the American Rheumatism Association) diagnostic criteria for connective tissue disorders frequently include positive antinuclear antibody (ANA) assays. Proper interpretation of these tests requires an understanding of the principles governing ANA assays. Assay results are reported in two ways: as titers and as descriptions of fluorescent patterns. A titer is a quantitative measure of ANAs in serum. Different patterns of immunofluorescence are associated with different subsets of collagen vascular disease. Positive results can occur in the absence of connective tissue disease. Accurate diagnosis of connective tissue disorders requires judicious use of ANA assays as well as skillful interpretation of the results.

  3. Monoclonal antibody production and the development of an indirect competitive enzyme-linked immunosorbent assay for screening spiramycin in milk.

    PubMed

    Jiang, Wenxiao; Zhang, Huiyan; Li, Xiangmei; Liu, Xinxin; Zhang, Suxia; Shi, Weimin; Shen, Jianzhong; Wang, Zhanhui

    2013-11-20

    To monitor spiramycin (SP) residue in milk, a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. This study described the preparation of three immunogens and the production of a high-affinity mAb. After optimization, the 50% inhibition concentration (IC50) for the developed icELISA was estimated as 0.97 ng/mL in the assay buffer, and the limit of detection and limit of quantitation were 2.51 and 4.40 μg/L in the milk matrix. The newly developed assay demonstrated negligible cross-reactivity with 15 other macrolide antibiotics, but not with kitasamycin (23.4%). The mean recoveries ranged from 81 to 103% for the spiked samples (5, 10, and 50 μg/L), and the coefficient of variation ranged from 5.4 to 9.6%. The icELISA was validated by LC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting SP residue in milk without requiring a cleanup process.

  4. Accurate, quantitative assays for the hydrolysis of soluble type I, II, and III /sup 3/H-acetylated collagens by bacterial and tissue collagenases

    SciTech Connect

    Mallya, S.K.; Mookhtiar, K.A.; Van Wart, H.E.

    1986-11-01

    Accurate and quantitative assays for the hydrolysis of soluble /sup 3/H-acetylated rat tendon type I, bovine cartilage type II, and human amnion type III collagens by both bacterial and tissue collagenases have been developed. The assays are carried out at any temperature in the 1-30/sup 0/C range in a single reaction tube and the progress of the reaction is monitored by withdrawing aliquots as a function of time, quenching with 1,10-phenanthroline, and quantitation of the concentration of hydrolysis fragments. The latter is achieved by selective denaturation of these fragments by incubation under conditions described in the previous paper of this issue. The assays give percentages of hydrolysis of all three collagen types by neutrophil collagenase that agree well with the results of gel electrophoresis experiments. The initial rates of hydrolysis of all three collagens are proportional to the concentration of both neutrophil or Clostridial collagenases over a 10-fold range of enzyme concentrations. All three assays can be carried out at collagen concentrations that range from 0.06 to 2 mg/ml and give linear double reciprocal plots for both tissue and bacterial collagenases that can be used to evaluate the kinetic parameters K/sub m/ and k/sub cat/ or V/sub max/. The assay developed for the hydrolysis of rat type I collagen by neutrophil collagenase is shown to be more sensitive by at least one order of magnitude than comparable assays that use rat type I collagen fibrils or gels as substrate.

  5. Novel Monoclonal Antibody-Based Immunodiagnostic Assay for Rapid Detection of Deamidated Gluten Residues.

    PubMed

    Masiri, Jongkit; Benoit, Lora; Katepalli, Madhu; Meshgi, Mahzad; Cox, David; Nadala, Cesar; Sung, Shao-Lei; Samadpour, Mansour

    2016-05-11

    Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food-processing measures can affect epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, and rye and deamidated gluten down to 2 ppm in food as well as its performance in food testing.

  6. Standardization of Assays That Detect Anti-Rubella Virus IgG Antibodies

    PubMed Central

    Grangeot-Keros, Liliane; Vauloup-Fellous, Christelle

    2015-01-01

    SUMMARY Rubella virus usually causes a mild infection in humans but can cause congenital rubella syndrome (CRS). Vaccination programs have significantly decreased primary rubella virus infection and CRS; however, vaccinated individuals usually have lower levels of rubella virus IgG than those with natural infections. Rubella virus IgG is quantified with enzyme immunoassays that have been calibrated against the World Health Organization (WHO) international standard and report results in international units per milliliter. It is recognized that the results reported by these assays are not standardized. This investigation into the reasons for the lack of standardization found that the current WHO international standard (RUB-1-94) fails by three key metrological principles. The standard is not a pure analyte but is composed of pooled human immunoglobulin. It was not calibrated by certified reference methods; rather, superseded tests were used. Finally, no measurement uncertainty estimations have been provided. There is an analytical and clinical consequence to the lack of standardization of rubella virus IgG assays, which leads to misinterpretation of results. The current approach to standardization of rubella virus IgG assays has not achieved the desired results. A new approach is required. PMID:26607813

  7. Qualification of a free ligand assay in the presence of anti-ligand antibody Fab fragments.

    PubMed

    Hansen, Ryan J; Brown, Robin M; Lu, Jirong; Wroblewski, Victor J

    2013-01-01

    The aim of this work was to develop and characterize an ELISA to measure free ligand concentrations in rat serum in the presence of a Fab to the same ligand. A variety of experiments were conducted to understand optimal assay conditions and to verify that only free ligand was detected. The parameters explored included sample incubation time on plate, the initial concentrations of Fab and ligand, and the pre-incubation time required for the Fab-ligand complex concentrations to reach equilibrium. We found the optimal experimental conditions to include a 10-minute on-plate incubation of ligand-containing samples, with a 24-hour pre-incubation time for test samples of Fab and ligand to reach equilibrium. An alternative approach, involving removal of Fab-ligand complexes from the solution prior to measuring concentrations of the ligand, was also used to verify that the assay only measured free ligand. Rats were dosed subcutaneously with Fab and the assay was used to demonstrate dose-dependent suppression of endogenous free ligand levels in vivo.

  8. Human anti-mouse antibodies: pitfalls in tumor marker measurement and strategies for enhanced assay robustness; including results with Elecsys CEA.

    PubMed

    Nussbaum, S; Roth, H J

    2000-01-01

    Therapies using monoclonal antibodies may have undesirable consequences for the diagnostic use of tumor markers. These effects can be minimised by employing chimeric antibodies as well as special interference eliminating reagents. Human Anti-Mouse Antibodies (HAMA) are produced as a result of the immune response of a patient to treatment with murine monoclonal antibodies. The interaction of HAMA with the murine monoclonal antibodies of a tumor marker assay can simulate (false) positive or negative results leading to misdiagnosis and to inadequate disease management of a patient. To avoid HAMA-interferences "Roche Diagnostics" established a three-component-system: The use of chimeric antibodies, the interference elimination, which is realised in the parameters most frequently used like CEA and TSH. By employing such a chimeric antibody, Elecsys CEA proved to be extremely robust against HAMA-interferences. With 20 clinical relevant samples from different Mab-approaches, no HAMA-interference was observed. By fragmentation of the antibodies, i.e., elimination of the constant region and using monovalent fab-fragments (antigen binding fragment) combined with the addition of special blocking reagents all not-chimerized, Elecsys assays showed comparable results to chimerisation. This could also be shown with 20 clinical relevant samples.

  9. Stable isotope dilution assay for the accurate determination of mycotoxins in maize by UHPLC-MS/MS.

    PubMed

    Varga, Elisabeth; Glauner, Thomas; Köppen, Robert; Mayer, Katharina; Sulyok, Michael; Schuhmacher, Rainer; Krska, Rudolf; Berthiller, Franz

    2012-03-01

    A fast, easy-to-handle and cost-effective analytical method for 11 mycotoxins currently regulated in maize and other cereal-based food products in Europe was developed and validated for maize. The method is based on two extraction steps using different acidified acetonitrile–water mixtures. Separation is achieved using ultrahigh-performance liquid chromatography (UHPLC) by a linear water–methanol gradient. After electrospray ionisation, tandem mass spectrometric detection is performed in dynamic multiple reaction monitoring mode. Since accurate mass spectrometric quantification is hampered by matrix effects, uniformly [(13)C]-labelled mycotoxins for each of the 11 compounds were added to the sample extracts prior to UHPLC-MS/MS analysis. Method performance parameters were obtained by spiking blank maize samples with mycotoxins before as well as after extraction on six levels in triplicates. The twofold extraction led to total recoveries of the extraction steps between 97% and 111% for all target analytes, including fumonisins. The [(13)C]-labelled internal standards efficiently compensated all matrix effects in electrospray ionisation, leading to apparent recoveries between 88% and 105% with reasonable additional costs. The relative standard deviations of the whole method were between 4% and 11% for all analytes. The trueness of the method was verified by the measurement of several maize test materials with well-characterized concentrations. In conclusion, the developed method is capable of determining all regulated mycotoxins in maize and presuming similar matrix effects and extraction recovery also in other cereal-based foods.

  10. Comparison of antibody assays for detection of autoantibodies to Ro 52, Ro 60 and La associated with primary Sjögren's syndrome.

    PubMed

    Trier, Nicole Hartwig; Nielsen, Inger Ødum; Friis, Tina; Houen, Gunnar; Theander, Elke

    2016-06-01

    Anti-Ro(52/60) and anti-La constitute the hallmark autoantibodies in primary Sjögren's syndrome, being present in 40-70% of sera. Several anti-Ro/La assays exist, but antibody detection appears to be assay-specific, thus the aim of this study was to compare several anti-Ro/La assays. In total, 96 sera from individuals with primary Sjögren's syndrome and 114 healthy controls were tested for anti-Ro 52/60 and anti-La in 17 immunoassays. Especially the immunoassays used for detection of anti-Ro 52 differed in their sensitivity (48-79%), while only small differences in sensitivities were observed for the anti-Ro 60 (69-77%) anti-La (39-44%) assays. Concordances of 65%, 79% and 73% for the anti-Ro 52, anti-Ro 60 and anti-La assays were found, respectively. The majority of the assays yielded high specificities, primarily ranging from 97 to 100%, except from a single anti-Ro 60 assay, which yielded a specificity of 79%. Occasionally, reactivity levels were increased in a few assays, indicating that false-positive results can be obtained when applying assays of reduced specificity. In general, the commercial assays appeared to perform better than the in-house analyses. When correcting the in-house assays for background reactivity, sensitivities were reduced by approximately 7%, 17%, and 19% for anti-Ro 52, anti-Ro 60 and anti-La assays, respectively, illustrating the pitfalls when applying immunoassays for detection of autoantibodies, which in theory may apply to commercial assays as well. Finally, increased total sensitivities were obtained when combining assays. These studies contribute to clarify the clinical utility of immunoassays for detection of autoantibodies of Ro 52, Ro 60 and La and illustrate that the most efficient strategy to maximize antibody sensitivity is to combine several assays.

  11. Development of a high-throughput β-Gal-based neutralization assay for quantitation of herpes simplex virus-neutralizing antibodies in human samples.

    PubMed

    Baccari, Amy; Cooney, Michael; Blevins, Tamara P; Morrison, Lynda A; Larson, Shane; Skoberne, Mojca; Belshe, Robert B; Flechtner, Jessica B; Long, Deborah

    2016-07-19

    Measurement of neutralizing antibodies against herpes simplex virus (HSV) is important for evaluation of candidate vaccines. The established plaque-reduction neutralization assay is time consuming, labor intensive, and difficult to validate and transfer. Here, we describe the characterization of a HSV-neutralization assay based on the expression of a reporter gene, β-galactosidase (β-Gal). Using previously constructed HSV-β-Gal recombinant viruses, HSV-2/Gal and HSV-1/tk12, we developed a colorimetric β-Gal-based neutralization assay that is sensitive and highly reproducible, and performed in less than 48h. HSV-1 and HSV-2 neutralizing titers measured by the β-Gal-based neutralization assay were equivalent to those obtained by a plaque reduction neutralization assay. Intra- and inter-assay precision studies demonstrated that the β-Gal-based assay was repeatable and yielded low and acceptable variation. In addition, comparison of HSV-2 neutralizing antibody (NAb) titers measured in two independent laboratories by two unique β-Gal-based assays showed a highly significant correlation (r=0.9499, p<0.0001) between the two assays. The new assay will serve as an important tool both for preclinical and clinical trials of new HSV vaccines.

  12. Recommendations for the design, optimization, and qualification of cell-based assays used for the detection of neutralizing antibody responses elicited to biological therapeutics.

    PubMed

    Gupta, Shalini; Indelicato, Stephen R; Jethwa, Vijay; Kawabata, Thomas; Kelley, Marian; Mire-Sluis, Anthony R; Richards, Susan M; Rup, Bonita; Shores, Elizabeth; Swanson, Steven J; Wakshull, Eric

    2007-04-10

    The administration of biological therapeutics can evoke some level of immune response to the drug product in the receiving subjects. An immune response comprised of neutralizing antibodies can lead to loss of efficacy or potentially more serious clinical sequelae. Therefore, it is important to monitor the immunogenicity of biological therapeutics throughout the drug product development cycle. Immunoassays are typically used to screen for the presence and development of anti-drug product antibodies. However, in-vitro cell-based assays prove extremely useful for the characterization of immunoassay-positive samples to determine if the detected antibodies have neutralizing properties. This document provides scientific recommendations based on the experience of the authors for the development of cell-based assays for the detection of neutralizing antibodies in non-clinical and clinical studies.

  13. Indirect immunofluorescence assay for the simultaneous detection of antibodies against clinically important old and new world hantaviruses.

    PubMed

    Lederer, Sabine; Lattwein, Erik; Hanke, Merle; Sonnenberg, Karen; Stoecker, Winfried; Lundkvist, Åke; Vaheri, Antti; Vapalahti, Olli; Chan, Paul K S; Feldmann, Heinz; Dick, Daryl; Schmidt-Chanasit, Jonas; Padula, Paula; Vial, Pablo A; Panculescu-Gatej, Raluca; Ceianu, Cornelia; Heyman, Paul; Avšič-Županc, Tatjana; Niedrig, Matthias

    2013-01-01

    In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n=97); SEOV (China, n=5); DOBV (Romania, n=7); SNV (Canada, n=23); ANDV (Argentina and Chile, n=52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n=177; 96%) and/or IgM (n=131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).

  14. Toward the development of monoclonal antibody-based assays to probe virion-like epitopes in hepatitis B vaccine antigen

    PubMed Central

    Zhu, Yibin; Zhang, Tianying; Zhao, Jinghua; Weng, Zusen; Yuan, Quan; Li, Shaowei; Zhang, Jun; Xia, Ning-Shao; Zhao, Qinjian

    2014-01-01

    Prophylactic vaccines against hepatitis B Virus (HBV) infection were produced in different expression systems under different processing conditions. Since the recombinant HBV surface antigen (HBsAg) in these vaccines is a cysteine-rich protein with 14 cysteines among a total of 226 amino acids, the epitopes are dependent on the formation of intra- and intermolecular disulfide bonds. A panel of 22 monoclonal antibodies (mAbs) were developed and evaluated with respect to their sensitivity to disulfide reduction treatment of recombinant HBsAg. Not surprisingly, different mAbs showed different degree of sensitivity to controlled HBsAg disulfide reduction. With a view to exploring the functionality of anti-HBsAg mAbs to be used in HBsAg quality analysis, in vitro neutralization activity for the mAbs was assessed. One of the mAbs tested, 5F11, which showed high sensitivity to the disulfide integrity in HBsAg, was shown also to be highly effective in neutralizing HBV in vitro. Conversely, 42B6, while exhibiting similar neutralization activity, showed comparable binding HBsAg with or without reduction treatment. Based on these mAb characteristics, a sandwich ELISA with 42B6 being the capture Ab and detection Ab was developed to quantify HBsAg (like a “mass” assay) during antigen bioprocessing or in vaccine products. In parallel, when 5F11 was used as the detection Ab (with the same capture Ab), the assay can be used to probe disulfide-dependent and virion-like epitopes in intermediates or final products of hepatitis B vaccine, serving as a surrogate marker for vaccine efficacy to elicit neutralizing antibodies. This approach enables the comparative epitope specific antigenicity analysis of HBsAg antigen preparations from different sources. PMID:24499806

  15. Antigenic features of human follicle stimulating hormone delineated by monoclonal antibodies and construction of an immunoradiomometric assay

    SciTech Connect

    Berger, P.; Panmoung, W.; Khaschabi, D.; Mayregger, B.; Wick, G.

    1988-11-01

    The characterization of human (h) FSH with 181 monoclonal antibodies (MCA) allowed the elucidation of its antigenic topography. One- and two-site, limited as well as excess reagent type radioimmuno- and enzymoimmunoassays revealed three main categories of MCA molecular binding specificities; two thirds of all antibodies were directed against the alpha-subunit and one fourth toward the beta-chain, and less than one tenth recognized the conformationally (c) intact holohormone. With high frequency immunization schedules these specificities were shifted toward a higher proportion of beta-MCA. On the basis of intra- and interspecies cross-reaction studies as well as epitope contiguity analyses by sandwich assays, the three main categories could be further subdivided into nine epitopes: 1) five epitopes associated with the alpha-subunit, two of which were suprisingly shared by other species, and two being iodination sensitive, 2) two evolutionary conserved structures on the beta-subunit, adjacent to each other, and 3) two c-determinants, one of these present also on hTSH. The epitopes were arranged in three major antigenic domains, which seems to be a common homologous construction principle of the four human glycoprotein hormones: a central domain, consisting of three identically arranged alpha- and similarly located c-epitopes, is flanked by a single spatially distinct domain on each subunit. The establishment of an epitope map was followed by the construction of an immunoradiometric assay with a sensitivity of 0.25 ng hFSH/ml and an apparent cross-reactivity vs. hLH, hTSH, and hCG of less than 1%.

  16. Comparison of seven commercial enzyme-linked immunosorbent assays for the detection of anti-diphtheria toxin antibodies.

    PubMed

    Zasada, A A; Rastawicki, W; Śmietańska, K; Rokosz, N; Jagielski, M

    2013-07-01

    Determination of immune status of patients to diphtheria toxin is based mainly on the results of commercially available ELISA kits. The aim of the present study was to compare the results obtained by ELISAs from seven different manufacturers: Mikrogen, Immunolab, Sekisui Virotech, NovaTec, Virion\\Serion, IBL International and Euroimmun. All assays were performed according to the manufacturers' instructions. The concentrations of the anti-diphtheria toxin antibodies in 72 serum samples were calculated on the basis of curves constructed from standards supplied by manufacturers and the new reference material-International Standard for Diphtheria Antitoxin (10/262). The repeatability and reproducibility of all the ELISA kits tested were good. Number of sera with concentrations of the anti-diphtheria toxin antibodies below the WHO-recommended level of protection (0.1 IU/ml) were dependent on the ELISA used: Mikrogen, 20/72 samples (27.7%); Immunolab, 11/72 samples (15.3%); Sekisui Virotech, 0/72 samples (0%); NovaTec 18/72 samples (25.0%); Serion 12/72 samples (16.7%); IBL International, 7/72 samples (9.7 %); and Euroimmun, 17/72 samples (23.6%). However, the results obtained in particular ELISAs, with the exception of Sekisui Virotech, were much more consistent when the concentrations of the anti-diphtheria toxin antibodies in 72 sera measured by using curves constructed from the International Standard 10/262. The data obtained clearly demonstrated that manufacturer-dependent differences between anti-diphtheria IgG ELISA kits exist. The differences in recommendations accepted by the individual manufacturers together with differences shown in our studies in sensitivity greatly affect the clinical interpretation of results.

  17. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.

    PubMed

    Takeda, Hiroyuki; Ogasawara, Tomio; Ozawa, Tatsuhiko; Muraguchi, Atsushi; Jih, Pei-Ju; Morishita, Ryo; Uchigashima, Motokazu; Watanabe, Masahiko; Fujimoto, Toyoshi; Iwasaki, Takahiro; Endo, Yaeta; Sawasaki, Tatsuya

    2015-06-10

    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

  18. Indirect enzyme-linked immunosorbent assay for detection of Brucella melitensis-specific antibodies in goat milk.

    PubMed

    Funk, N D; Tabatabai, L B; Elzer, P H; Hagius, S D; Martin, B M; Hoffman, L J

    2005-02-01

    Brucella melitensis is the cause of brucellosis in sheep and goats, which often results in abortion. Few cases of B. melitensis infection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection of B. melitensis-specific antibodies in goat milk. Brucella salt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of <200 and 50 animals, respectively. Based on this estimation, it is recommended that herds be sampled in groups of 50 animals or less for bulk milk testing. The iELISA developed for this study was found to be sensitive and specific and shows potential for use as a bulk milk test for the detection of B. melitensis-specific antibodies in goat milk.

  19. A novel monoclonal antibody-based immunoenzymatic assay for epidemiological surveillance of the vector snails of Fasciola hepatica (Trematoda: Digenea).

    PubMed

    Alba, Annia; Hernández, Hilda M; Marcet, Ricardo; Vázquez, Antonio A; Figueredo, Mabel; Sánchez, Jorge; Otero, Oscar; Sarracent, Jorge

    2015-02-01

    Fasciolosis is a globally distributed snail-borne disease which requires economic consideration due to its enormous impact on veterinary medicine. During recent decades, this parasitosis has also shown increasing prevalence in human populations worldwide. The dissemination and successful transmission of fasciolosis ultimately depends on the existence of susceptible snails that act as intermediate hosts. Therefore, to accomplish effective control of this disease, surveillance and detection of the infected intermediate host would be essential. The screening of trematodes within snails using classical parasitological examination of the larvae can be unreliable (sensitivity and specificity vary depending on the time of infection and the experience of the observer) and relatively costly when using molecular biological methods during large-scale monitoring. Here we propose a novel monoclonal antibody-based immunoenzymatic assay to detect ongoing Fasciola hepatica infection in lymnaeid snails. Anti-F. hepatica rediae mouse monoclonal antibodies were generated and used to develop a double monoclonal antibody-based ELISA for parasite detection. Fasciola hepatica-infected and uninfected laboratory-reared Galba cubensis and Pseudosuccinea columella were used for assessment of the developed ELISA. Experimentally infected snails were dissected and examined for parasite larvae as the "gold standard" method. Sensitivity results were 100% for both snail species, while specificity was 98% for G. cubensis and 100% for P. columella. No cross-reactivity was detected in lymnaeids infected with Trichobilharzia sp. or Cotylophoron sp. The ELISA enabled detection of the infection from day 8 p.i. in G. cubensis while in P. columella it was noted as early as day 4. To our knowledge no previous immunoassays have been reported to detect helminth-infected snails and the developed sandwich ELISA method is therefore suggested for infection status validation in natural populations of lymnaeid

  20. Detection of specific antibody by enzyme-linked immunosorbent assay and antigenemia by counterimmunoelectrophoresis in humans infected with Pneumocystis carinii.

    PubMed

    Maddison, S E; Hayes, G V; Slemenda, S B; Norman, L G; Ivey, M H

    1982-06-01

    A urea-soluble extract of cyst-rich material from rat lung heavily infected with Pneumocystis carinii was evaluated in an enzyme-linked immunosorption assay for antibody in 461 human sera. The highest level of reactivity occurred in sera submitted for serodiagnosis from proved or highly suspect cases. However, the range of reactivities in these groups, many of whom were on immunosuppressive therapy, was very wide. A more restricted lower range of reactivity was observed in both hospital-family contacts and healthy Serum Bank donors. Because of the overlap in levels of reactivity between the pneumocystosis and control groups, no concise cutoff value to separate infected from noninfected individuals could be made. Specificity of the reactions was shown by absorption of patients' and control sera with uninfected and P. carinii-infected human and rat lung tissue. The data support the concept that P. carinii is highly prevalent as a latent agent in the general population and is provoked to cause clinically manifest disease in the compromised host. Detection of circulating antigen appeared to be specific and possibly a useful adjunct to diagnosis, as 10 of the 14 proved or highly suspect patients with antigenemia did not have measurable antibody to P. carinii.

  1. Use of monoclonal antibodies in an epidemiological marker system: a retrospective study of lung specimens from the 1976 outbreak of Legionnaires disease in Philadelphia by indirect fluorescent-antibody and enzyme-linked immunosorbent assay methods.

    PubMed Central

    Brown, S L; Bibb, W F; McKinney, R M

    1985-01-01

    Autopsy specimens of lung tissues from 15 patients that contracted legionellosis during the 1976 Philadelphia outbreak of Legionnaires disease were examined for the presence of Legionella organisms and soluble antigens by indirect fluorescent-antibody (IFA) testing and by an enzyme-linked immunosorbent assay (ELISA) with both polyclonal and monoclonal antibodies. In all 15 cases, at least one specimen was positive for Legionella pneumophila serogroup 1 (Lp-1) antigens by a polyclonal antibody ELISA system. Of the 15 cases tested for Lp-1, 9 were positive by a polyclonal antibody IFA test. Nine mouse monoclonal antibodies to Lp-1 gave essentially the same reactivity pattern with extracts from lung tissue homogenates as that obtained with a Philadelphia 1 culture extract by using a monoclonal antibody ELISA system. The same monoclonal antibody panel gave similar results when used in the IFA system with tissue homogenates. Monoclonal antibodies can be used as epidemiological marker systems with both IFA and ELISA testing. This study provides evidence that the 1976 common source outbreak in Philadelphia was probably caused by a single Lp-1 strain. ELISA testing of the soluble antigen of Lp-1 from lung tissue homogenate supernatants was more sensitive than IFA testing of the homogenates and should be extremely useful as either a primary test or as an adjunct to fluorescent antibody testing for legionellosis. Images PMID:3881470

  2. Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum

    PubMed Central

    Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M.; Nguyen, Chelsea; Stevenson, Mark A.; Wilks, Colin R.; Firestone, Simon M.

    2016-01-01

    Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants. PMID:27122484

  3. Serodiagnosis of pulmonary tuberculosis in Argentina by enzyme-linked immunosorbent assay (ELISA) of IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative

    PubMed Central

    Balestrino, E. A.; Daniel, T. M.; de Latini, M. D. S.; Latini, O. A.; Ma, Y.; Scocozza, J. B.

    1984-01-01

    IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative (PPD) was measured, by enzyme-linked immunosorbent assay (ELISA), in serum samples from 86 patients with active pulmonary tuberculosis and 91 non-tuberculous control subjects from Santa Fé, Argentina. The geometric mean titre for the tuberculosis patients was 74.6 with antigen 5 and 99.5 with PPD. In 91 control subjects the geometric mean titres were 3.6 and 15.6 respectively. Titres were not related to tuberculin reactor status or prior BCG vaccination. At a serum dilution end-point of 1:40, ELISA with antigen 5 had a sensitivity of 81.4% and a specificity of 93.4% for tuberculosis. At 1:40, ELISA with PPD showed a sensitivity of 82.6% and a specificity of 54.9% for tuberculosis. Applied at a serum dilution of 1:40 to a hypothetical model population with a tuberculosis prevalence of 2%, ELISA using antigen 5 would correctly classify 93.2% of persons and ELISA with PPD, 55.5%. At a dilution of 1:80, accuracy is increased to 99.3% with antigen 5 and 83.3% with PPD, but sensitivity decreases to 64.0% with antigen 5 and 72.1% with PPD. Thus, antigen 5 is more accurate than PPD for the diagnosis of tuberculosis using ELISA. PMID:6439426

  4. An integrated microfluidic system for measurement of glycated hemoglobin levels by using an aptamer-antibody assay on magnetic beads.

    PubMed

    Chang, Ko-Wei; Li, Jinglun; Yang, Ching-Hsuan; Shiesh, Shu-Chu; Lee, Gwo-Bin

    2015-06-15

    Blood glycated hemoglobin (HbA1c), reflecting the average blood glucose level in the proceeding 2-3 months, is recommended for screening/diagnosing and patient management of diabetes. However, accurate measurement of the HbA1c level at the point of care is hampered by costly, large-scale instruments (such as high-performance liquid chromatography) or reagent instability of classical immunologic methods, which involve antibody-based immunoturbidimetry. In this work, an integrated microfluidic system using aptamer-based testing to measure HbA1c in blood samples is therefore presented. This measuring system used nucleic-acid aptamers that exhibited high sensitivity and high specificity for hemoglobin and HbA1c to perform a stable and robust testing. The compact microfluidic system consumed less samples and reagents and significantly shortened the detection time. Combining the advantages of microfluidics and aptamers, this integrated microsystem presents a promising tool for accurate and point-of-case HbA1c detection. To demonstrate its clinical utility, whole blood samples with clinically-relevant concentrations of HbA1c and Hb were automatically measured on the integrated microfluidic system. Experimental data showed that the developed aptamer-based microfluidic system is capable of detecting HbA1c and Hb with a good linear response. The entire process was completed within 25 min. The aptamer-antibody on-chip sandwich immunoassay may be further refined to allow diabetes screening and diagnosis at lower cost and earlier phase to minimize the risk of diabetic complications.

  5. Comparison of Diagnostic Accuracy of Microscopy and Flow Cytometry in Evaluating N-Methyl-D-Aspartate Receptor Antibodies in Serum Using a Live Cell-Based Assay

    PubMed Central

    Ramberger, Melanie; Peschl, Patrick; Schanda, Kathrin; Irschick, Regina; Höftberger, Romana; Deisenhammer, Florian; Rostásy, Kevin; Berger, Thomas; Dalmau, Josep; Reindl, Markus

    2015-01-01

    N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune neurological disease, diagnosed by a specific autoantibody against NMDAR. Antibody testing using commercially available cell-based assays (CBA) or immunohistochemistry on rat brain tissue has proven high specificity and sensitivity. Here we compare an immunofluorescence live CBA to a flow cytometry (FACS) based assay to detect NMDAR antibodies by their binding to the surface of HEK293A cells functionally expressing NMDAR. Both assays were first established using a discovery group of 76 individuals and then validated in a group of 32 patients in a blinded manner. In the CBA, 23 of 23 patients with NMDAR encephalitis were positive for NMDAR antibodies and 0 of 85 controls (32 healthy controls and 53 patients with other neurological diseases), resulting in a sensitivity and specificity of 100% (95% confidence intervals (CI) 85.1–100.0 and 95.7–100.0, respectively). The FACS based assay detected NMDAR antibodies in 20 of 23 patients and in 0 of 85 controls. Therefore, with an equally high specificity (95% CI 95.7–100.0) the sensitivity of the FACS based assay was 87% (95% CI 66.4–97.2). Comparing antibody titers from CBA with delta median fluorescence intensities from FACS showed a high concordance (kappa = 0.943, p<0.0001) and correlation (r = 0.697, p<0.0001). In conclusion, evaluation of the FACS based assay revealed a lower sensitivity and high inter-assay variation, making the CBA a more reliable detection method. PMID:25815887

  6. Comparison of diagnostic accuracy of microscopy and flow cytometry in evaluating N-methyl-D-aspartate receptor antibodies in serum using a live cell-based assay.

    PubMed

    Ramberger, Melanie; Peschl, Patrick; Schanda, Kathrin; Irschick, Regina; Höftberger, Romana; Deisenhammer, Florian; Rostásy, Kevin; Berger, Thomas; Dalmau, Josep; Reindl, Markus

    2015-01-01

    N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune neurological disease, diagnosed by a specific autoantibody against NMDAR. Antibody testing using commercially available cell-based assays (CBA) or immunohistochemistry on rat brain tissue has proven high specificity and sensitivity. Here we compare an immunofluorescence live CBA to a flow cytometry (FACS) based assay to detect NMDAR antibodies by their binding to the surface of HEK293A cells functionally expressing NMDAR. Both assays were first established using a discovery group of 76 individuals and then validated in a group of 32 patients in a blinded manner. In the CBA, 23 of 23 patients with NMDAR encephalitis were positive for NMDAR antibodies and 0 of 85 controls (32 healthy controls and 53 patients with other neurological diseases), resulting in a sensitivity and specificity of 100% (95% confidence intervals (CI) 85.1-100.0 and 95.7-100.0, respectively). The FACS based assay detected NMDAR antibodies in 20 of 23 patients and in 0 of 85 controls. Therefore, with an equally high specificity (95% CI 95.7-100.0) the sensitivity of the FACS based assay was 87% (95% CI 66.4-97.2). Comparing antibody titers from CBA with delta median fluorescence intensities from FACS showed a high concordance (kappa = 0.943, p<0.0001) and correlation (r = 0.697, p<0.0001). In conclusion, evaluation of the FACS based assay revealed a lower sensitivity and high inter-assay variation, making the CBA a more reliable detection method.

  7. Comparison of GD2 Binding Capture ELISA Assays for Anti-GD2-Antibodies Using GD2-Coated Plates and a GD2-Expressing Cell-Based ELISA

    PubMed Central

    Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Mitra, Gautam

    2011-01-01

    Two assay methods for quantification of the disialoganglioside (GD2)-specific binding activities of anti-GD2 monoclonal antibodies and antibody immunofusion proteins, such as ch14.18 and hu14.18-IL2, were developed. The methods differed in the use of either microtiter plates coated with purified GD2 or plates seeded with GD2-expressing cell lines to bind the anti-GD2 molecules. The bound antibodies were subsequently detected using the reactivity of the antibodies to an HRP-labeled anti-IgG Fc or antibodies recognizing the conjugate IL-2 part of the Hu 14.18IL-2 fusion protein. The bound HRP was detected using reagents such as orthophenylene diamine, 2, 2’-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] or tetramethylbenzidine. The capture ELISA using GD2-coated plates was developed earlier in assay development and used to demonstrate assay specificity and to compare lot-to-lot consistency and stability of ch14.18, and Hu14.18 IL-2 in clinical development. During this study, we found a number of issues related to plate-to-plate variability, GD2 lot variability, and variations due to GD2 storage stability, etc., that frequently lead to assay failure in plates coated with purified GD2. The cell-based ELISA (CbELISA) using the GD2 expressing melanoma cell line, M21/P6, was developed as an alternative to the GD2-coated plate ELISA. The results on the comparability of the capture ELISA on GD2-coated plates and the cell-based assay show that both assays give comparable results. However, the cell-based assay is more consistent and reproducible. Subsequently, the anti-GD2 capture ELISA using the GD2-coated plate was replaced with the CbELISA for product lot release testing and stability assessment. PMID:21893062

  8. Protein A radio-assay of H-Y antigen on human leukocytes using mouse and rat antisera and monoclonal antibodies.

    PubMed

    Savikurki, H; Andersson, L C; Wachtel, S S; de la Chapelle, A

    1983-01-01

    The presence of H-Y antigen on human leukocytes was investigated using a protein A radio-assay. H-Y antigen could be demonstrated on male cells using either conventional H-Y antisera produced in mice and rats, or monoclonal H-Y antibodies. With mouse antiserum and IgG-type monoclonal antibody the reaction was male-specific using a single antibody. The reaction obtained with rat antiserum was enhanced by the application of a second antibody (rabbit anti-mouse IgG). This technique provides a rapid, simple, objective, and semiquantitative method for the determination of cellular H-Y antigen, the results being expressed as radioactivity bound to the test cells and thus being independent of human observation. It requires only 10-20 ml of blood and small quantities of antiserum or antibody.

  9. The enzyme-linked immunosorbent assay (ELISA) as a serological test for detecting antibodies against Leptospira interrogans serovar hardjo in sheep.

    PubMed

    Adler, B; Faine, S; Gordon, L M

    1981-09-01

    The enzyme-liked immunosorbent assay (ELISA) was compared with the standard microscopic agglutination test (MAT) as a method for detecting antibodies against Leptospira interrogans serovar hardjo in sheep. Peak antibody levels detected by the 2 tests occurred at different times following experimental infection of sheep. In serums from flocks of sheep with naturally acquired infection there was a 95% correlation between MAT and ELISA with respect to the presence or absence of antibody to serovar hardjo, although the levels of correlation of the titres of the 2 tests was low. The 2 tests appeared to measure different antigen-antibody systems. The ELISA would be a useful test for screening large numbers of serums for antibodies to L. interrogans serovar hardjo.

  10. SPECT assay of radiolabeled monoclonal antibodies. Progress report, September 1, 1992--August 24, 1993

    SciTech Connect

    Jaszczak, R.J.

    1993-08-20

    The overall goal of this project is to improve the effectiveness of single photon emission computed tomography (SPECT) to image and quantify radiolabeled monoclonal antibodies. During the past year, we have made significant progress toward this goal, and this report summarizes that work. Our efforts have been mainly directed along three fronts. First, we have developed and tested new reconstruction methods including three-dimensional iterative algorithms that model non-uniform attenuation and distance-dependent detector response. Both fan beam and parallel beam collimator geometries have been modeled and novel ways of improving the efficiency of the computationally intensive methods have been introduced. Second, an ultra-high resolution, small field-of-view pinhole collimator has been constructed and evaluated. Reconstructed spatial resolution of 1 to 3 mm (FWHM) has been achieved in phantom scans with a useful field-of-view of 9 to 10 cm. Finally, we have investigated the ability of SPECT to image and quantify astatine-211 distributions. Reconstructed images of phantom data demonstrated quantitative accuracy to within 10% with proper attenuation and scatter compensation.

  11. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    PubMed Central

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  12. Detection of Cronobacter Genus in Powdered Infant Formula by Enzyme-linked Immunosorbent Assay Using Anti-Cronobacter Antibody

    PubMed Central

    Song, Xinjie; Shukla, Shruti; Lee, Gibaek; Park, Sunhyun; Kim, Myunghee

    2016-01-01

    Cronobacter species (Cronobacter spp.) are hazardous foodborne pathogens associated with baby food, powdered infant formula (PIF). To develop a rapid and sensitive method for simultaneous detection of seven Cronobacter spp. in PIF, an indirect non-competitive enzyme-linked immunosorbent assay (INC-ELISA) was developed based on a novel immunoglobulin G (IgG), anti-Cronobacter IgG. The developed INC-ELISA was able to detect seven Cronobacter spp. at concentrations ranging from (5.6 ± 0.30) × 103 to (2.1 ± 0.01) × 105 colony forming unit (CFU)/mL in pure culture. Further, INC-ELISA employing anti-Cronobacter IgG was applicable for analysis of PIF samples contaminated with less than <10 cells of Cronobacter spp. per 25 g of PIF in 36 h. The developed antibody showed slight cross-reactivity with Franconibacter pulveris (LMG 24057) at high concentration (108 CFU/mL). The INC-ELISA method displayed excellent specificity without compromising cross-reactivity with other foodborne pathogens. The INC-ELISA assay method developed in this study using a novel anti-Cronobacter IgG facilitated highly sensitive, efficient, and rapid detection of Cronobacter spp. in baby food. PMID:27493642

  13. Binding Assays for the Quantitative Detection of P. Brevis Polyether Neurotoxins in Biological Samples and Antibodies as Therapeutic Aids for Polyether Marine Intoxication

    DTIC Science & Technology

    1990-03-15

    Recommendations 27 VII. Literature Cited 28 I!. [i• Figure 1. Structures of the Brevetoxins 5 Figure 2. Non-Competitive Brevetoxin-Antibody- Protein A Urease ...Barracuda. 20 Figure 9. Toxin- Urease Assay 23 Figure 10. Toxin-Peroxidase Assay 23 Figure 11. Non-Competitive Peroxidase-Linked Sandwich Immuunoassay 24...8217. Thus, the greatest flexibility is gained employing such techniques, and many different variations may be developed to oset defined :riteria. 9 i Urease 2

  14. Enzyme-linked immunosorbent assay using monoclonal antibodies for identification of mycobacteria from early cultures.

    PubMed Central

    Verstijnen, C P; Ly, H M; Polman, K; Richter, C; Smits, S P; Maselle, S Y; Peerbooms, P; Rienthong, D; Montreewasuwat, N; Koanjanart, S

    1991-01-01

    A simple enzyme-linked immunosorbent assay (ELISA) for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii has been developed (R. Schöningh, C. P. H. J. Verstijnen, S. Kuijper, and A. H. J. Kolk. J. Clin. Microbiol. 28:708-713, 1990). The test for the routine identification of cultured mycobacteria was introduced in five clinical laboratories located in Tanzania, Thailand, Vietnam, and The Netherlands. The ELISA can be conducted without an ELISA reader since the test can be read visually. The results of identification of 255 strains of the M. tuberculosis complex by microbiological means and by ELISA were compared; the specificity and the sensitivity were 100%. For the M. avium complex, the specificity was 100% and the sensitivity was 64%. All 26 M. kansasii strains tested could be identified as M. kansasii. The ELISA described here proved to be useful in both well- and modestly equipped laboratories and may replace the microbiological method of identification of M. tuberculosis and M. kansasii. PMID:1909344

  15. Adaptations of a competitive enzyme-linked immunosorbent assays for the detection of antibodies to influenza a virus in horse sera for use in wild aquatic birds.

    PubMed

    Hoque, M A; Skerratt, L F; Garland, S; Burgess, G W; Selleck, P

    2012-12-01

    We applied a competitive enzyme-linked immunosorbent assay for the detection of antibodies for influenza A in equine sera to their detection in sera from wild aquatic birds. Suboptimal results were obtained for the optical density (OD) of the monoclonal antibody (MAb) control and reproducibility between duplicate analyses in the initial assessment. It was therefore necessary to modify the assay to deliver increased reliability and reproducibility while maintaining adequate sensitivity. We optimized reagent concentrations to obtain optimal OD values (close to 2) for the monoclonal antibody control and used 2, 2'-Azino-bis: 3-Benzthiazoline-6-Sulphonic Acid as an alternative chromogen to potentially reduce variability in duplicate analyses. The original assay was compared with the optimized versions, with and without post coating, for the detection of avian influenza viral antibodies in 240 sera obtained from wild plumed whistling ducks. A separate analytical sensitivity study on diluted positive field sera of plumed whistling ducks and a test of antigen stability after post coating were also performed. Some quantitative differences were detected between the original and modified assays. The original assay recorded higher percentage inhibition results which were potentially indicative of increased sensitivity. However, when reagent concentrations were increased in the original assay to the same levels as used in the modified versions, there were no quantitative differences for practical purposes. The original assay produced a median (OD) value of 0.81 for the (MAb) controls that is at the limit of acceptability. By contrast, the modified assays always produced acceptable optical density values for MAb controls. Our overall results indicated the modified assays were potentially more reliable (OD values close to 2), and of adequate sensitivity compared to the original assay in the detection of avian influenza viral antibodies in wild bird sera. Although further

  16. Broad-specificity immunoassay for O,O-diethyl organophosphorus pesticides: Application of molecular modeling to improve assay sensitivity and study antibody recognition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A monoclonal antibody (MAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was...

  17. Identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets by polyclonal antibody-based enzyme-linked immunosorbent assay.

    PubMed

    Asensio, Luis; González, Isabel; Rodríguez, Miguel A; Mayoral, Belén; López-Calleja, Inés; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2003-02-26

    An indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the species identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. The assay was performed in two different formats, microtiter plates and immunostick tubes, and uses polyclonal antibodies raised in rabbits against muscle-soluble proteins of grouper (anti-GSP), wreck fish (anti-WSP), and Nile perch (anti-PSP). The antibodies were made species-specific by blocking them with the heterologous soluble muscle proteins. Immunorecognition of polyclonal antibodies adsorbed to their specific fish samples was made with swine antirabbit immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymatic conversion of the substrate allowed unequivocal identification of the species studied.

  18. [Comparative study on the assay for IgE antibodies specific for Chamaecyparis obtusa pollen between AlaSTAT and CAP-RAST].

    PubMed

    Nohara, O; Imai, T; Saneyoshi, K; Endo, T; Nagakura, H; Ono, M; Moriyama, H

    1996-06-01

    Titers of IgE antibody specific for the pollen of Chamaecyparis obtusa (C. obtusa) were determined by AlaSTAT and CAP-RAST in 221 patients with Japanese cedar pollinosis. IgE antibody to C. obtusa tested positive by CAP-RAST at a higher rate (80.5%) than by AlaSTAT (52.6%). The results obtained from the two assays were compared with those from intradermal skin test. CAP-RAST had a higher sensitivity than that of AlaSTAT. Because the two methods showed no differences in the determination of IgE antibody specific for Cryptomeria japonica, the above differences between AlaSTAT and CAP-RAST are surmised to be ascribable to the differences of C. obtusa antigen used in the both assays.

  19. An Efficient Multiplex PCR-Based Assay as a Novel Tool for Accurate Inter-Serovar Discrimination of Salmonella Enteritidis, S. Pullorum/Gallinarum and S. Dublin

    PubMed Central

    Xiong, Dan; Song, Li; Tao, Jing; Zheng, Huijuan; Zhou, Zihao; Geng, Shizhong; Pan, Zhiming; Jiao, Xinan

    2017-01-01

    Salmonella enterica serovars Enteritidis, Pullorum/Gallinarum, and Dublin are infectious pathogens causing serious problems for pig, chicken, and cattle production, respectively. Traditional serotyping for Salmonella is costly and labor-intensive. Here, we established a rapid multiplex PCR method to simultaneously identify three prevalent Salmonella serovars Enteritidis, Pullorum/Gallinarum, and Dublin individually for the first time. The multiplex PCR-based assay focuses on three genes tcpS, lygD, and flhB. Gene tcpS exists only in the three Salmonella serovars, and lygD exists only in S. Enteritidis, while a truncated region of flhB gene is only found in S. Pullorum/Gallinarum. The sensitivity and specificity of the multiplex PCR assay using three pairs of specific primers for these genes were evaluated. The results showed that this multiplex PCR method could accurately identify Salmonella Enteritidis, Pullorum/Gallinarum, and Dublin from eight non-Salmonella species and 27 Salmonella serovars. The least concentration of genomic DNA that could be detected was 58.5 pg/μL and the least number of cells was 100 CFU. Subsequently, this developed method was used to analyze clinical Salmonella isolates from one pig farm, one chicken farm, and one cattle farm. The results showed that blinded PCR testing of Salmonella isolates from the three farms were in concordance with the traditional serotyping tests, indicating the newly developed multiplex PCR system could be used as a novel tool to accurately distinguish the three specific Salmonella serovars individually, which is useful, especially in high-throughput screening. PMID:28360901

  20. Development of an enzyme-linked immunosorbent assay to detect chicken serum antibody to glycoprotein G of infectious laryngotracheitis virus.

    PubMed

    Shil, Niraj K; Markham, Philip F; Noormohammadi, Amir H; O'Rourke, Denise; Devlin, Joanne M

    2012-09-01

    Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens and has a worldwide distribution. Diagnostic enzyme-linked immunosorbent assays (ELISAs) are commonly used in ILT disease control programs. These ELISAs generally detect serum antibody to infectious laryngotracheitis virus (ILTV) and frequently utilize whole virus as the ELISA antigen. This study investigated the use of recombinant glycoprotein G (gG) of ILTV as an alterative to the use of whole virus antigen. Codon-optimized ILTV gG was expressed in Escherichia coli as a fusion protein with a maltose binding protein tag (gG-MBP). Another gG fusion protein with a 6-histidine tag (gG-His) was expressed in a baculovirus expression system. Following purification, the proteins were assessed for their suitability to be used as an antigen in an ELISA to detect ILTV-specific antibodies in sera from commercial and specific-pathogen-free (SPF) birds. The gG-MBP antigen showed some nonspecific reactions with chicken sera, but the gG-HIS antigen was found to be suitable for differentiating between sera collected from ILTV-vaccinated and unvaccinated chickens. The highest levels of agreement between the results from the gG-HIS ELISA and the commercial Trop-ILT ELISA were achieved using a cut-off value for positivity equal to the geometric mean antibody concentration of the sera from the unvaccinated birds plus 1 SD. This produced a very good level of agreement (kappa [kappa] value of 0.821) using sera from commercial birds and a moderate level of agreement (kappa value of 0.506) using sera from SPF birds. Importantly, this ELISA was also tested for its ability to discriminate between sera collected from SPF chickens vaccinated with a gG deletion mutant candidate vaccine strain of ILTV (gG-ve ILTV) and sera collected from SPF chickens vaccinated with other ILTV strains. The results showed that the gG-His ELISA has the potential to serve as a companion diagnostic tool in conjunction

  1. A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice.

    PubMed

    Gomes-Neto, João Carlos; Mantz, Sara; Held, Kyler; Sinha, Rohita; Segura Munoz, Rafael R; Schmaltz, Robert; Benson, Andrew K; Walter, Jens; Ramer-Tait, Amanda E

    2017-04-01

    Changes in the gastrointestinal microbial community are frequently associated with chronic diseases such as Inflammatory Bowel Diseases. However, understanding the relationship of any individual taxon within the community to host physiology is made complex due to the diversity and individuality of the gut microbiota. Defined microbial communities such as the Altered Schaedler Flora (ASF) help alleviate the challenges of a diverse microbiota by allowing one to interrogate the relationship between individual bacterial species and host responses. An important aspect of studying these relationships with defined microbial communities is the ability to measure the population abundance and dynamics of each member. Herein, we describe the development of an improved ASF species-specific and sensitive real-time quantitative polymerase chain reaction (qPCR) for use with SYBR Green chemistry to accurately assess individual ASF member abundance. This approach targets hypervariable regions V1 through V3 of the 16S rRNA gene of each ASF taxon to enhance assay specificity. We demonstrate the reproducibility, sensitivity and application of this new method by quantifying each ASF bacterium in two inbred mouse lines. We also used it to assess changes in ASF member abundance before and after acute antibiotic perturbation of the community as well as in mice fed two different diets. Additionally, we describe a nested PCR assay for the detection of lowly abundant ASF members. Altogether, this improved qPCR method will facilitate gnotobiotic research involving the ASF community by allowing for reproducible quantification of its members under various physiological conditions.

  2. Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato.

    PubMed

    Tavares, Eliandro Reis; Azevedo, Caroline Souza; Panagio, Luciano Aparecido; Pelisson, Marsileni; Pinge-Filho, Phileno; Venancio, Emerson José; Barros, Tânia Fraga; Yamada-Ogatta, Sueli Fumie; Yamauchi, Lucy Megumi

    2016-01-01

    In this work, two accurate and sensitive real-time polymerase chain reaction (PCR) assays to differentiate pathogenic Cryptococcus gattii sensu lato (s.l.) and C. neoformans sensu lato (s.l.) targeting the intergenic spacer 1 (IGS1) region from rDNA locus were developed. Specific primers were designed based on their IGS1 sequence analyses and the optimal real-time PCR assays showed that the dissociation curves generated two different melting peaks, at 82.8 and 84.2ºC for C. gattii s.l. and C. neoformans s.l., respectively. No amplifications were observed in the negative template control. The minimum limit of detection of both primers was 100 plasmid copies per reaction, and they were highly specific when tested with a range of fungal DNAs. Overall, the results showed that the designed primers completely differentiated C. gattii s.l. and C. neoformans s.l. from clinical and environmental sources with great accuracy when compared to phenotypic identification, with no cross-reactivity to other fungal DNA.

  3. Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

    PubMed

    Shang, Yuqin; Mernaugh, Ray; Zeng, Xiangqun

    2012-10-02

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35-220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV-vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10(13) M(-2). The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.

  4. In-solution virus capture assay helps deconstruct heterogeneous antibody recognition of human immunodeficiency virus type 1.

    PubMed

    Leaman, Daniel P; Kinkead, Heather; Zwick, Michael B

    2010-04-01

    Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) on whole virions is heterogeneous, so molecular analysis of Env with monoclonal antibodies (MAbs) is challenging. Virus capture assays (VCAs) involving immobilized MAbs are typically used, but these assays suffer from immobilization artifacts and do not provide binding constants. Furthermore, we show here that certain HIV-1 neutralizing MAbs, including 2G12, 4E10, 2F5, Z13e1, and D5, will capture virion particles completely devoid of Env. We modified the VCA such that MAbs and virions are incubated in solution, and unbound MAbs are removed prior to the capture step. This modification nearly eliminated evidence of Env-independent binding by MAbs to virions and allowed determination of apparent affinity constants in solution. Three important qualitative observations were further revealed. First, neutralizing MAbs 2F5, 4E10, and Z13e1 against the membrane-proximal external region (MPER) of HIV-1 gp41 were found to capture virions efficiently only if a significant amount of uncleaved gp160 or synthetic MPER peptide was present. Second, we show how non-native forms of Env vary by Env genotype and that Env from HIV-1(JR-FL) is more homogeneously trimeric than that from HIV-1(JR-CSF). Third, we determined that Env containing all or parts of gp41, including uncleaved gp160, binds spontaneously to free virions. This exogenous Env is an indiscriminate molecular "bridge" between Env-specific Ab and virions and can affect VCA analyses, particularly using pseudotyped virions. Heterogeneity in Env from endogenous and exogenous sources might also subvert humoral immunity to HIV-1, so in-solution VCAs may help to dissect this heterogeneity for vaccine design purposes.

  5. Development and implementation of an international proficiency testing program for a neutralizing antibody assay for HIV-1 in TZM-bl cells

    PubMed Central

    Todd, Christopher A.; Greene, Kelli M.; Yu, Xuesong; Ozaki, Daniel A.; Gao, Hongmei; Huang, Yunda; Wang, Maggie; Li, Gary; Brown, Ronald; Wood, Blake; D’Souza, M. Patricia; Gilbert, Peter; Montefiori, David C.; Sarzotti-Kelsoe, Marcella

    2011-01-01

    Recent advances in assay technology have led to major improvements in how HIV-1 neutralizing antibodies are measured. A luciferase reporter gene assay performed in TZM-bl (JC53bl-13) cells has been optimized and validated. Because this assay has been adopted by multiple laboratories worldwide, an external proficiency testing program was developed to ensure data equivalency across laboratories performing this neutralizing antibody assay for HIV/AIDS vaccine clinical trials. The program was optimized by conducting three independent rounds of testing, with an increased level of stringency from the first to third round. Results from the participating domestic and international laboratories improved each round as factors that contributed to inter-assay variability were identified and minimized. Key contributors to increased agreement were experience among laboratories and standardization of reagents. A statistical qualification rule was developed using a simulation procedure based on the three optimization rounds of testing, where a laboratory qualifies if at least 25 of the 30 ID50 values lie within the acceptance ranges. This ensures no more than a 20% risk that a participating laboratory fails to qualify when it should, as defined by the simulation procedure. Five experienced reference laboratories were identified and tested a series of standardized reagents to derive the acceptance ranges for pass–fail criteria. This Standardized Proficiency Testing Program is the first available for the evaluation and documentation of assay equivalency for laboratories performing HIV-1 neutralizing antibody assays and may provide guidance for the development of future proficiency testing programs for other assay platforms. PMID:21968254

  6. Development and implementation of an international proficiency testing program for a neutralizing antibody assay for HIV-1 in TZM-bl cells.

    PubMed

    Todd, Christopher A; Greene, Kelli M; Yu, Xuesong; Ozaki, Daniel A; Gao, Hongmei; Huang, Yunda; Wang, Maggie; Li, Gary; Brown, Ronald; Wood, Blake; D'Souza, M Patricia; Gilbert, Peter; Montefiori, David C; Sarzotti-Kelsoe, Marcella

    2012-01-31

    Recent advances in assay technology have led to major improvements in how HIV-1 neutralizing antibodies are measured. A luciferase reporter gene assay performed in TZM-bl (JC53bl-13) cells has been optimized and validated. Because this assay has been adopted by multiple laboratories worldwide, an external proficiency testing program was developed to ensure data equivalency across laboratories performing this neutralizing antibody assay for HIV/AIDS vaccine clinical trials. The program was optimized by conducting three independent rounds of testing, with an increased level of stringency from the first to third round. Results from the participating domestic and international laboratories improved each round as factors that contributed to inter-assay variability were identified and minimized. Key contributors to increased agreement were experience among laboratories and standardization of reagents. A statistical qualification rule was developed using a simulation procedure based on the three optimization rounds of testing, where a laboratory qualifies if at least 25 of the 30 ID50 values lie within the acceptance ranges. This ensures no more than a 20% risk that a participating laboratory fails to qualify when it should, as defined by the simulation procedure. Five experienced reference laboratories were identified and tested a series of standardized reagents to derive the acceptance ranges for pass-fail criteria. This Standardized Proficiency Testing Program is the first available for the evaluation and documentation of assay equivalency for laboratories performing HIV-1 neutralizing antibody assays and may provide guidance for the development of future proficiency testing programs for other assay platforms.

  7. Quantitative relationship between anticapsular antibody measured by enzyme-linked immunosorbent assay or radioimmunoassay and protection of mice against challenge with Streptococcus pneumoniae serotype 4.

    PubMed Central

    Musher, D M; Johnson, B; Watson, D A

    1990-01-01

    We have recently shown that a substantial proportion of antibody to pneumococcal polysaccharide as measured by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay is removed by adsorption with pneumococcal cell wall polysaccharide (CWPS). The present study was undertaken to validate the hypothesis that only serotype-specific antibody that remains after adsorption with CWPS provides protection against pneumococcal infection. Serum samples were obtained from human subjects before and after they had been vaccinated with pneumococcal polysaccharide vaccine. Antibody to Streptococcus pneumoniae serotype 4 was measured by ELISA without adsorption or after adsorption of serum with CWPS. Groups of mice were injected with graded doses of serum and then challenged intraperitoneally with 10, 100, or 1,000 50% lethal doses (LD50) of S. pneumoniae serotype 4. Without adsorption, prevaccination sera from five healthy adults appeared to contain up to 33 micrograms of antibody to S. pneumoniae serotype 4 antigen per ml; adsorption with CWPS removed all detectable antibody, and pretreating mice with up to 0.1 ml of these sera (less than or equal to 3.3 micrograms of antibody) failed to protect them against challenge with 100 LD50. In contrast, postvaccination sera contained 2.9 to 30 micrograms of antibody per ml that was not removed by adsorption. Diluting sera to administer desired amounts of serotype-specific immunoglobulin G showed a significant relationship between protection and antibody remaining after adsorption (P less than 0.05 by linear regression analysis); 150 ng was uniformly protective against 1,000 LD50, and 50 ng was protective against 100 LD50. These studies have, for the first time, quantitated the amount of serotype-specific antibody that protects mice against challenge with S. pneumoniae type 4. In light of these observations, it is necessary to reassess current concepts regarding the presence of antipneumococcal antibody in the unvaccinated

  8. A whole blood in vitro cytokine release assay with aqueous monoclonal antibody presentation for the prediction of therapeutic protein induced cytokine release syndrome in humans.

    PubMed

    Wolf, Babette; Morgan, Hannah; Krieg, Jennifer; Gani, Zaahira; Milicov, Adriana; Warncke, Max; Brennan, Frank; Jones, Stewart; Sims, Jennifer; Kiessling, Andrea

    2012-12-01

    The administration of several monoclonal antibodies (mAbs) to humans has been associated with acute adverse events characterized by clinically significant release of cytokines in the blood. The limited predictive value of toxicology species in this field has triggered intensive research to establish human in vitro assays using peripheral blood mononuclear cells or blood to predict cytokine release in humans. A thorough characterization of these assays is required to understand their predictive value for hazard identification and risk assessment in an optimal manner, and to highlight potential limitations of individual assay formats. We have characterized a whole human blood cytokine release assay with only minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation format, an assay which has so far received less attention in the scientific world with respect to the evaluation of its suitability to predict cytokine release in humans. This format was compared with a human PBMC assay with immobilized mAbs presentation already well-characterized by others. Cytokine secretion into plasma or cell culture supernatants after 24h incubation with the test mAbs (anti-CD28 superagonist TGN1412-like material (TGN1412L), another anti-CD28 superagonistic mAb (ANC28.1), a T-cell depleting mAb (Orthoclone™), and a TGN1412 isotype-matched control (Tysabri™) not associated with clinically-relevant cytokine release) was detected by a multiplex assay based on electrochemiluminescent excitation. We provide proof that this whole blood assay is a suitable new method for hazard identification of safety-relevant cytokine release in the clinic based on its ability to detect the typical cytokine signatures found in humans for the tested mAbs and on a markedly lower assay background and cytokine release with the isotype-matched control mAb Tysabri™ - a clear advantage over the PBMC assay. Importantly, quantitative and qualitative differences in the relative cytokine

  9. Nationwide survey of leptospira antibodies in dogs in Japan: results from microscopic agglutination test and enzyme-linked immunosorbent assay.

    PubMed

    Iwamoto, Emiko; Wada, Yuko; Fujisaki, Yuka; Umeki, Saori; Jones, Miyuki Y; Mizuno, Takuya; Itamoto, Kazuhito; Maeda, Ken; Iwata, Hiroyuki; Okuda, Masaru

    2009-09-01

    Leptospirosis is an infectious disease caused by Leptospira interrogans sensu lato and is common in both humans and animals. In the present study, serum samples were collected from 801 dogs across all 47 prefectures in Japan, and evaluated with a microscopic agglutination test (MAT), using 5 major L. interrogans serovars (Icterohaemorrhagiae, Canicola, Autumnalis, Hebdomadis, and Australis) as antigens, and an enzyme-linked immunosorbent assay (ELISA) using recombinant OmpL1 protein as the antigen. Across all dogs tested, 217 (27.0%) and 29 (3.6%) were MAT- and ELISA-positive, respectively. However, evidence strongly suggests that MAT also detected antibodies produced by vaccination. Of 243 dogs never inoculated with any canine vaccine, 41 (16.9%) from 23 prefectures were MAT and/or ELISA positive. The most commonly detected serovar was Icterohaemorrhagiae (22 dogs, 19 prefectures). Our results suggest that there are dogs with subclinical Leptospira infection throughout Japan. To the best of our knowledge, the present study is the first nationwide survey of Leptospira infection in dogs, and the findings are relevant not only for clinical veterinary medicine but also for public health.

  10. Enzyme-linked immunosorbent assay for detecting antibodies in cattle in a herd in which anaplasmosis was diagnosed.

    PubMed Central

    Thoen, C O; Blackburn, B; Mills, K; Lomme, J; Hopkins, M P

    1980-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies in anaplasmosis with an antigen prepared from infected bovine erythrocytes. Protein A labeled with horseradish peroxidase was used as conjugate. A comparison was made of results of ELISA, complement fixation test (CFT), and card test (CT) on sera from 97 cows in a herd in which anaplasmosis had been diagnosed. Positive ELISA reactions were observed in serum dilutions of 1:20 or greater in each of 26 cows positive by CFT and in 18 of 22 cows suspected by CFT. Of 49 cows negative by CFT, 31 were negative by ELISA. Positive ELISA reactions were observed in 45 cows positive by CT; 27 of 44 cows negative by ct were negative by ELISA. No ELISA reactions were detected in the sera of 23 cows found negative in the CFT and CT or in 8 cows negative in CFT and positive in CT. No positive CFT, CT, or ELISA reactions were observed in sera of cattle in a noninfected herd. PMID:7381017

  11. A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay

    PubMed Central

    Mohan, Anju; Saxena, Hari Mohan; Malhotra, Puneet

    2016-01-01

    Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT), microtiter plate agglutination test (MAT), indirect hemagglutination assay (IHA), and indirect enzyme-linked immunosorbent assay (iELISA) as per standard protocols. Results: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (p<0.0001). The mean MAT titers were 2.244±0.727 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was very significant (p<0.005). The mean IHA titers in infected cattle were 2.284±0.574, and those in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002). However, the difference in mean iELISA titers of infected cattle (1.3678±0.014) and healthy vaccinated cattle (1.367±0.014) was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. However, it cannot be ascertained whether these antibodies are due to vaccine or response to infection. Since the infected animals had been vaccinated earlier, the current infection may suggest that vaccination was unable to induce protective levels of antibody. The heightened antibody response after infection may also indicate a secondary immune response to the antigens common to the vaccine strain and wild Brucella organisms. Conclusion: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. PMID:27536032

  12. Complement dependent cytotoxicity (CDC) activity of a humanized anti Lewis-Y antibody: FACS-based assay versus the 'classical' radioactive method -- qualification, comparison and application of the FACS-based approach.

    PubMed

    Nechansky, A; Szolar, O H J; Siegl, P; Zinoecker, I; Halanek, N; Wiederkum, S; Kircheis, R

    2009-05-01

    The fully humanized Lewis-Y carbohydrate specific monoclonal antibody (mAb) IGN311 is currently tested in a passive immunotherapy approach in a clinical phase I trail and therefore regulatory requirements demand qualified assays for product analysis. To demonstrate the functionality of its Fc-region, the capacity of IGN311 to mediate complement dependent cytotoxicity (CDC) against human breast cancer cells was evaluated. The "classical" radioactive method using chromium-51 and a FACS-based assay were established and qualified according to ICH guidelines. Parameters evaluated were specificity, response function, bias, repeatability (intra-day precision), intermediate precision (operator-time different), and linearity (assay range). In the course of a fully nested design, a four-parameter logistic equation was identified as appropriate calibration model for both methods. For the radioactive assay, the bias ranged from -6.1% to -3.6%. The intermediate precision for future means of duplicate measurements revealed values from 12.5% to 15.9% and the total error (beta-expectation tolerance interval) of the method was found to be <40%. For the FACS-based assay, the bias ranged from -8.3% to 0.6% and the intermediate precision for future means of duplicate measurements revealed values from 4.2% to 8.0%. The total error of the method was found to be <25%. The presented data demonstrate that the FACS-based CDC is more accurate than the radioactive assay. Also, the elimination of radioactivity and the 'real-time' counting of apoptotic cells further justifies the implementation of this method which was subsequently applied for testing the influence of storage at 4 degrees C and 25 degrees C ('stability testing') on the potency of IGN311 drug product. The obtained results demonstrate that the qualified functional assay represents a stability indicating test method.

  13. Comparison of Two Assays to Determine Anti-Citrullinated Peptide Antibodies in Rheumatoid Arthritis in relation to Other Chronic Inflammatory Rheumatic Diseases: Assaying Anti-Modified Citrullinated Vimentin Antibodies Adds Value to Second-Generation Anti-Citrullinated Cyclic Peptides Testing

    PubMed Central

    Díaz-Toscano, Miriam Lizette; Olivas-Flores, Eva Maria; Zavaleta-Muñiz, Soraya Amali; Gamez-Nava, Jorge Ivan; Cardona-Muñoz, Ernesto German; Ponce-Guarneros, Manuel; Castro-Contreras, Uriel; Nava, Arnulfo; Salazar-Paramo, Mario; Celis, Alfredo; Fajardo-Robledo, Nicte Selene; Corona-Sanchez, Esther Guadalupe; Gonzalez-Lopez, Laura

    2014-01-01

    Determination of anti-citrullinated peptide antibodies (ACPA) plays a relevant role in the diagnosis of rheumatoid arthritis (RA). To date, it is still unclear if the use of several tests for these autoantibodies in the same patient offers additional value as compared to performing only one test. Therefore, we evaluated the performance of using two assays for ACPA: second-generation anti-citrullinated cyclic peptides antibodies (anti-CCP2) and anti-mutated citrullinated vimentin (anti-MCV) antibodies for the diagnosis of RA. We compared three groups: RA (n = 142), chronic inflammatory disease (CIRD, n = 86), and clinically healthy subjects (CHS, n = 56) to evaluate sensitivity, specificity, predictive values, and likelihood ratios (LR) of these two assays for the presence of RA. A lower frequency of positivity for anti-CCP2 was found in RA (66.2%) as compared with anti-MCV (81.0%). When comparing RA versus other CIRD, sensitivity increased when both assays were performed. This strategy of testing both assays had high specificity and LR+. We conclude that adding the assay of anti-MCV antibodies to the determination of anti-CCP2 increases the sensitivity for detecting seropositive RA. Therefore, we propose the use of both assays in the initial screening of RA in longitudinal studies, including early onset of undifferentiated arthritis. PMID:25025037

  14. A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry.

    PubMed

    Su, Jui-Lan; Fornwald, Jim; Rivers, Philip; Goldsworthy, Susan; Looney, Noeleen A; Hanvey, Jeff; Plumpton, Chris; Parham, Janet; Romanos, Michael; Kost, Thomas A; Kull, Frederick C

    2004-08-01

    A cell-based time-resolved fluorescence (celTRF) immunoassay is described for pre-screening antibodies to G protein-coupled receptor (GPCR) peptides that predicts suitability for immunohistochemistry (IHC). Rat GPCRs were expressed in Saos-2 human osteosarcoma cells via recombinant baculoviruses designed for mammalian cell expression, i.e., the transduced cells were used as a "screening lawn". The lawn was fixed and permeabilized similarly to IHC tissue. The celTRF, a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), employed Eu-labelled goat anti-rabbit IgG. It exhibited a broad dynamic range upon which enzyme-linked immunosorbant assay (ELISA)-positive affinity-purified anti-peptide antibody reagents were examined for specificity and potency. Over 150 anti-peptide reagents to 27 GPCRs were characterized. All celTRF-positive antibodies were found to be suitable for IHC, whereas ELISA alone did not predict IHC utility. Examples are illustrated with five rabbit anti-neuropeptide FF receptor 1 (NPFF1) antibodies, where a strong correlation between celTRF potency and IHC utility was observed in both applications. In contrast, two high anti-peptide ELISA titer but celTRF-negative antibodies failed to recognize the NPFF1 receptor in IHC. The celTRF assay was performed manually and in an automated fashion, in our case, using a Biomek FX station and Sami scheduling software. The celTRF is the first in vitro automated assay that offers confident pre-selection of antibodies for IHC and the versatility to accommodate the rapid screening of large numbers of GPCRs. The celTRF is readily applicable to other protein target classes.

  15. Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase.

    PubMed

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; Barr, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2012-01-01

    Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.

  16. A liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay for the detection of antibodies against Newcastle disease virus in serum of free-ranging pigeons.

    PubMed

    de Oliveira, Elisabete Schirato; Silva, Ketherson Rodrigues; Fernando, Filipe Santos; Gonçalves, Mariana Costa Mello; Fernandes, Camila Cesário; Borzi, Mariana Monezi; dos Santos, Romeu Moreira; Tamanini, Maria de Lourdes Feres; Montassier, Maria de Fátima da Silva; Montassier, Helio José

    2013-11-01

    A competitive liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay (LPB-ConA-ELISA) was developed in the current study. The assay used ConA as a capture reagent, and the sera of specific pathogen-free chickens immunized with nonpurified Newcastle disease virus (NDV) suspension as detector antibodies, to detect and quantify specific antiviral antibodies in serum samples from free-ranging pigeons. The comparison between the LPB-ConA-ELISA and the hemagglutination inhibition (HI) test for the detection of antibodies in serum samples from 107 pigeons showed significant correlation between the assays (r = 0.875), a high sensitivity (100%), specificity (95.8%), accuracy (96.3%) for the ELISA, and good agreement (κ = 0.83) between the 2 assays. The results of this study suggest that the LPB-ConA-ELISA could be a useful alternative to HI test in the serodiagnosis of NDV in pigeons, or other species of birds.

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

    PubMed Central

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

    2015-01-01

    For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2332-452 protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2332-452 ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2332-452 ELISA may be a preferable method for detecting antibodies against AMDV. PMID:26582828

  18. Quantitative estimation of diphtheria and tetanus toxoids. 6. Use of different antibody titration methods for evaluation of immunogenicity in animals during potency assay of diphtheria toxoid.

    PubMed

    Lyng, J; Heron, I

    1992-06-01

    Two diphtheria toxoid preparations were compared in potency assays in guinea-pigs using different methods for evaluation of the responses to vaccination. The methods used were the direct skin challenge (Schick test) and ELISA and VERO cell titration of antibodies. The different evaluation methods resulted in the same relative potencies between the toxoids. It was observed that when first-vaccination sera were compared with a second-vaccination serum, the relative antibody concentration depended on whether ELISA or VERO cell titration was used.

  19. Comparison of inhibitory and binding characteristics of an antibody causing acquired von Willebrand syndrome: an assay for von Willebrand factor binding by antibody.

    PubMed

    Fricke, W A; Brinkhous, K M; Garris, J B; Roberts, H R

    1985-09-01

    An acquired inhibitor of von Willebrand factor (vWF) activity occurring in a patient with benign gammopathy and von Willebrand syndrome (vWS) has been partially characterized. The inhibitor-induced syndrome resulted in low to undetectable plasma levels of vWF/ristocetin, vWF/botrocetin, FVIIIR:Ag, and FVIII:C with a normal to slightly prolonged bleeding time. Platelet vWF was normal. Intensive and continuous infusion of a heat-treated factor VIII concentrate (Hemofil-T, Hyland, Glendale, Calif) elevated the FVIII:C plasma levels to about 100%, with an increase in FVIIIR:Ag levels to about 340% and vWF/ristocetin levels to about 40%, much lower than expected based on the dose of Hemofil-T and its content of vWF and FVIII:C activities. The inhibitor bound to staphylococcal protein A (SpA) with high affinity, indicating an IgG antibody (Ab). An assay for the vWF-binding capacity was developed on the basis of absorption of the Ab from serially diluted plasma by SpA and removal of vWF and FVIII:C activities from normal plasma by the SpA-Ab complex. The Ab-binding site was on the vWF component of the factor VIII complex. The Ab was unable to bind isolated FVIII:C. The combined use of the new vWF-binding assay and a battery of tests for inhibition of vWF-dependent platelet aggregation with ristocetin (which detects high molecular weight vWF), with botrocetin (which detects high and low molecular weight vWF), and with platelet-aggregating factor (which detects high molecular weight vWF) provided a means of analysis of Ab effect on in vitro vWF function. Using these tests, a comparison was made of the effects of the vWS Ab with those of an Ab inhibitor occurring in homozygous von Willebrand's disease. The Ab of the vWS patient had weak inhibitory action on vWF/ristocetin without having an effect on vWF/botrocetin and platelet-aggregating factor, a high titer vWF-binding capacity, and no anamnestic response following concentrate therapy. These findings contrasted with those

  20. Technical note: Hapten synthesis, antibody production and development of an enzyme-linked immunosorbent assay for detection of the natural steroidal alkaloid Dendrogenin A.

    PubMed

    de Medina, Philippe; Paillasse, Michael R; Segala, Gregory; Al Saati, Talal; Boyes, Jeannine; Delsol, Georges; Allal, Cuider; Marsili, Sabrina; Silvente-Poirot, Sandrine; Poirot, Marc

    2013-03-01

    We have recently discovered the existence of 5α-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-ol, called Dendrogenin A (DDA), as the first endogenous steroidal alkaloid ever described in mammals. We found that the DDA content of tumors and cancer cell lines was low or absent compared with normal cells showing that a deregulation in DDA biosynthesis was associated with cancer and therefore suggesting that DDA could represent a metabolomic cancer biomarker. This prompted us to produce antibodies that selectively recognize DDA. For this purpose, the hapten 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-o-hemisuccinate with a carboxylic spacer arm attached to the 3β-hydroxyl group of DDA was synthesized. The hapten was coupled to bovine serum albumin and keyhole limpet hemocyanin for antibody production to develop an enzyme-linked immunosorbent assay (ELISA). The protein conjugates were injected into BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the hybridoma cell lines established were assessed with indirect ELISA by competitive assays using dilutions of a DDA standard. The antibodies from the selected hybridomas had an IC(50) value ranging from 0.8 to 425 ng/ml. Three antibodies showed no cross-reactivity with structurally related compounds including histamine, cholesterol, ring B oxysterols and a regio-isomer of DDA. In this study, high-affinity and selective antibodies against DDA were produced for the first time, and a competitive indirect ELISA was developed.

  1. Accuracy of a Stick-Type Kit and Enzyme-Linked Immunosorbent Assay in Detecting Helicobacter pylori Antibodies in Urine of People Living in the Japan Sea Region of Northern Japan.

    PubMed

    Shimoyama, Tadashi; Sawada, Yoshihiko; Sawada, Naoya; Chinda, Daisuke; Fukuda, Shinsaku

    2017-03-24

    In Japan, both a stick-type kit and an enzyme-linked immunosorbent assay (ELISA) kit are available for the detection of antibodies to Helicobacter pylori in urine. However, the accuracy of these tests has not been fully examined in northern Japanese populations. Urine samples from 359 subjects were tested using a stick-type H. pylori-antibody detection kit (RAPIRUN), and urine samples from 201 subjects were tested using an ELISA-based test (URINELISA). The prevalence of H. pylori infection was determined by the (13)C-urea breath test (UBT) and a monoclonal antibody-based stool antigen test (TPAg). Subjects were considered to have the infection if either the UBT or rapid TPAg results were positive. The percentage of positive test results for RAPIRUN and URINELISA was 54.0% and 40.8%, respectively. Sensitivity and specificity were 83.3% and 67.0%, respectively, for RAPIRUN and 86.5% and 85.8% for URINELISA. Nineteen subjects had cut-off index values of between 0.4 and 0.9 by URINELISA, and 4 of these subjects (21.1%) were found to be infected with H. pylori. The urine-based ELISA was more accurate than the rapid stick-type kit in these patients. If negative ELISA results are near the cut-off value, subjects should receive an additional test to determine whether they are infected with H. pylori.

  2. Development and evaluation of indirect enzyme-linked immunosorbent assay for a screening test to detect antibodies against classical swine fever virus.

    PubMed

    Sakoda, Yoshihiro; Wakamoto, Hiroaki; Tamura, Tehpin; Nomura, Takushi; Naito, Michiko; Aoki, Hiroshi; Morita, Hiroshi; Kida, Hiroshi; Fukusho, Akio

    2012-08-01

    An indirect enzyme-linked immunosorbent assay (ELISA) was developed for a screening test to detect antibodies against classical swine fever virus (CSFV). Viral glycoproteins, which were purified from swine kidney cells infected with CSFV ALD/A76 strain by the immunoaffinity purification using monoclonal antibody against E2 protein, were adsorbed on a microtiter plate as the antigen for the antibody detection. Each antibody titer of serum sample was expressed as a sample per positive value calculated with optical absorbance of each sample and that of a positive control. The advantage of this ELISA is its higher sensitivity: most sera containing more than 4 neutralization titers were determined to be positive. This ELISA is unable to discriminate between antibodies against CSFV and those against other ruminant pestiviruses, therefore positive sera in this ELISA should be evaluated by a cross-neutralization test using CSFV, bovine viral diarrhea virus, and border disease virus. Taken together, the indirect ELISA developed in this study is useful screening tool to detect antibodies against CSFV for the large-scale monitoring of classical swine fever.

  3. Rheumatoid arthritis and its association with HLA-DR antigens. II. Antibodies to native connective tissue antigens detected by enzyme linked immunosorbent assay.

    PubMed

    Pesoa, S A; Vullo, C M; Onetti, C M; Riera, C M

    1989-01-01

    The distribution of frequencies of HLA-DR alloantigens in HLA-DR4 negative subjects was determined in patients with Rheumatoid arthritis (RA) and normal individuals. An increased incidence of HLA-DR1 alloantigen in DR4 negative RA patients (45.9%) compared with DR4 negative healthy controls (23.6%) was found. The difference became significant when the incidence of DR1 was compared between patients with severe disease stages (III-IV) (75%) in contrast to 32% of incidence in patients of the milder stages (I-II) (p less than 0.05). Using Enzyme Linked Immunosorbent Assay we have determined the incidence of serum antibodies to native bovine type I and type II collagens and proteoglycans in patients with RA. Presence of serum antibodies to native type I collagen was detected in 59% of patients with RA, 60% of sera exhibited reactivity to type II collagen and 12% had antibodies to proteoglycans. There was no correlation between the presence of antibodies to type I and II collagens and disease stages, however, the incidence of serum antibodies to proteoglycans was increased in severe disease stages. On the other hand, the presence of high levels of antibodies to type I collagen was associated to HLA-DR1 antigen, (p less than 0.05).

  4. Detection of anti-U3-RNP/fibrillarin IgG antibodies by line immunoblot assay has comparable clinical significance to immunoprecipitation testing in systemic sclerosis.

    PubMed

    Peterson, Lisa K; Jaskowski, Troy D; Mayes, Maureen D; Tebo, Anne E

    2016-04-01

    The aim of this study was to evaluate the performance and clinical relevance of a commercially available line immunoblot assay (LIA) for detecting anti-U3-RNP/fibrillarin (anti-U3-RNP), against immunoprecipitation (gold standard). This study involved a multi-ethnic cohort of 1000 American systemic sclerosis (SSc) patients and 50 healthy controls. Antinuclear antibodies and centromere antibodies were detected by indirect immunofluorescent antibody test, anti-topo I by immunodiffusion and anti-RNAP III by ELISA. The presence of anti-U3-RNP in select serum samples was detected by immunoprecipitation (IP) and LIA. By IP, U3-RNP antibody was detected in 75 (7.5 %) patients with SSc. Overall agreement between LIA and IP was very good (κ = 0.966). Analytic sensitivity and specificity of the U3-RNP LIA was 100 and 94.7 %, respectively. Clinical features associated with positivity for the anti-U3-RNP antibody include diffuse cutaneous SSc and increased prevalence of renal crisis, consistent with previous studies that used IP. Testing for U3-RNP antibodies is only performed by a small number of laboratories due to the complexity of both performance and interpretation of the IP. LIA is faster and less complex than IP. Excellent agreement between IP and LIA demonstrates that LIA is an acceptable and attractive alternative to IP for anti-U3-RNP detection.

  5. Development of a monoclonal antibody against deoxynivalenol for magnetic nanoparticle-based extraction and an enzyme-linked immunosorbent assay.

    PubMed

    Lee, Hyuk-Mi; Song, Sung-Ok; Cha, Sang-Ho; Wee, Sung-Bok; Bischoff, Karyn; Park, Sung-Won; Son, Seong-Wan; Kang, Hwan-Goo; Cho, Myung-Haing

    2013-01-01

    Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 mg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 mg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.

  6. Development of a cell-based qualitative assay for detection of neutralizing anti-human interleukin-1 receptor antagonist (hIL-1Ra) antibodies in rats.

    PubMed

    Gao, Jin; Li, Jingjing; Yang, Minmin; Wu, Mingyuan; Tu, Ping; Yu, Yan; Han, Wei

    2015-01-01

    To determine the incidence of the positive neutralizing anti-human interleukin receptor antagonist (anti-IL-1Ra), a novel assay based on the proliferation of human melanoma A375.S2 cells was developed and validated. In the presence of a growth-limiting concentration of IL-1β, A375.S2 cells were able to regain proliferation following the addition of IL-1Ra in a concentration-dependent manner. This dose-response effect enabled the validation of a standard curve for calculation of the concentration of IL-1Ra or, inversely, the concentration of neutralizing anti-IL-1Ra antibodies in cell culture medium or sera. The assay used CCK-8 as an indicator of proliferation. The dose-response relationship between rhIL-1Ra (dose range of 5-75 ng/ml rhIL-1Ra) and A375.S2 cell proliferation was sigmoidal and fitted a four-parameter logistic model. The percent coefficients of variation (%CVs) of quality control samples were 12.5 and 11.9% for intra-assay repeatability and 14.5 and 19.5% for inter-assay repeatability, while the total accuracy was in the range of 97.2-103.6%. For the neutralization assay, the optimal sample dilution factor was found to be 40-fold and the reasonable standard for positive and negative decision was calculated to be 59.4% neutralization rate. The %CVs of quality control samples were 12.7 and 24.0% for intra-assay repeatability and 11.6 and 30.0% for inter-assay repeatability. Analysis using the assay showed that rats could produce neutralizing anti-IL-1Ra antibodies after repeated intramuscular injection with rhIL-1Ra, and this response was not significantly dependent on the dose injected.

  7. Time-Resolved Fluorescent Resonance Energy Transfer Assay for Simple and Rapid Detection of Anti-Brucella Antibodies in Ruminant Serum Samples▿

    PubMed Central

    McGiven, John A.; Thompson, Iain J.; Commander, Nicola J.; Stack, Judy A.

    2009-01-01

    Brucellosis is a globally significant zoonosis, the control of which is difficult and resource intensive. Serological tests form a vital part of a multifactorial approach to control and are often performed in large numbers. The aim of the present study was to develop a new assay to improve the efficiency, ease, and effectiveness of serological testing. An existing competitive enzyme-linked immunosorbent assay (cELISA) was adapted to a completely homogeneous time-resolved fluorescent resonance energy transfer (TR-FRET) assay. This was achieved by labeling an anti-Brucella monoclonal antibody with a long-lifetime donor fluorophore and Brucella smooth lipopolysaccharide with a compatible acceptor and optimizing the reading conditions. The assay was performed in a 96-well plate with a single 30-min incubation period and no separation (wash) steps and was concluded by a single plate-reading step. The performance of the assay was evaluated with a panel of serum samples from infected (n = 73) and uninfected (n = 480) sources and compared to the performance of the parent cELISA, an indirect ELISA (iELISA), and fluorescence polarization assay (FPA). The performance of the TR-FRET assay matched the performance of the iELISA, which had 100% diagnostic sensitivity and specificity, and surpassed the performance of the cELISA and the FPA. The results also demonstrated that the TR-FRET technique is effective with poor-quality serum samples from the field. To the knowledge of the authors, this is the first homogeneous TR-FRET assay to detect antibodies raised against an infectious disease. The technique appears to be sufficiently adaptable to meet the needs of many other similar testing requirements to identify infectious diseases. PMID:19656980

  8. Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

    PubMed Central

    Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathy L.; Marks, James D.; Varnum, Susan M.

    2012-01-01

    Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A–G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3 fM (0.2 pg/ml) to 14.7 fM (2.2 pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples. PMID:22935296

  9. An Improved Ultrasensitive Enzyme-Linked Immunosorbent Assay Using Hydrangea-Like Antibody-Enzyme-Inorganic Three-in-One Nanocomposites.

    PubMed

    Wei, Tianxiang; Du, Dan; Zhu, Mei-Jun; Lin, Yuehe; Dai, Zhihui

    2016-03-01

    Protein-inorganic nanoflowers, composed of protein and copper(II) phosphate (Cu3(PO4)2), have recently grabbed people's attention. Because the synthetic method requires no organic solvent and because of the distinct hierarchical nanostructure, protein-inorganic nanoflowers display enhanced catalytic activity and stability and would be a promising tool in biocatalytical processes and biological and biomedical fields. In this work, we first coimmobilized the enzyme, antibody, and Cu3(PO4)2 into a three-in-one hybrid protein-inorganic nanoflower to enable it to possess dual functions: (1) the antibody portion retains the ability to specifically capture the corresponding antigen; (2) the nanoflower has enhanced enzymatic activity and stability to produce an amplified signal. The prepared antibody-enzyme-inorganic nanoflower was first applied in an enzyme-linked immunosorbent assay to serve as a novel enzyme-labeled antibody for Escherichia coli O157:H7 (E. coli O157:H7) determination. The detection limit is 60 CFU L(-1), which is far superior to commercial ELISA systems. The three-in-one antibody (anti-E. coli O157:H7 antibody)-enzyme (horseradish peroxidase)-inorganic (Cu3(PO4)2) nanoflower has some advantages over commercial enzyme-antibody conjugates. First, it is much easier to prepare and does not need any complex covalent modification. Second, it has fairly high capture capability and catalytic activity because it is presented as aggregates of abundant antibodies and enzymes. Third, it has enhanced enzymatic stability compared to the free form of enzyme due to the unique hierarchical nanostructure.

  10. [Anti-deamidated gliadin peptides antibodies and coeliac disease: state of art and analysis of false-positive results from five assays].

    PubMed

    Lutteri, Laurence; Sagot, Clémence; Chapelle, Jean-Paul

    2010-01-01

    Recently, anti-deamidated gliadin antibodies were proposed for the serological diagnosis of celiac disease. We evaluate the specificity of different anti-deamidated gliadin antibodies ELISA in comparison with conventional anti-native gliadin kits. Serum samples from 46 non celiac patients were analyzed by five different quantitative ELISA for anti-native gliadin, anti-deamidated gliadin and anti-transglutaminase neo-epitope antibodies together with a screening ELISA. Twenty-four percent of the patients demonstrated anti-native gliadin IgA and 63% IgG antibodies. Using anti-deamidated gliadin antibodies, the number of false positive IgA and, particularly, IgG results, markedly decreased in the non celiac patients: 21 and 24% respectively with anti-Gliadin (GAF-3X) Euroimmun kit, 7 and 26% with Bindazyme Human Anti-Gliadin (MGP) The Binding Site kit and 0 and 41% with Celiac G+ Immco kit. The new assay which makes use of the physiological complex of tissue transglutaminase cross-linked with deamidated gliadin peptides, called neo-epitope, did not improve the differential diagnosis of celiac disease with 30% of false positive results in IgG (2% in IgA). Using the Inova screening kit, a positive result for IgA and/or IgG anti-deamidated gliadin and/or anti-tissue transglutaminase antibodies was obtained in 24% of the non celiac patients. In conclusion, our study assessed the superiority, in terms of specificity, of anti-deamidated gliadin antibodies, over the conventional anti-gliadin antibodies for the differential diagnosis of celiac disease.

  11. Development and Application of a Saccharomyces cerevisiae-Expressed Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Infectious Bronchitis Virus

    PubMed Central

    Gibertoni, Aliandra M.; Montassier, Maria de Fátima S.; Sena, Janete A. D.; Givisiez, Patrícia E. N.; Furuyama, Cibele R. A. G.; Montassier, Hélio J.

    2005-01-01

    A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA. PMID:15815038

  12. Rapid detection of serum antibody by dual-path platform VetTB assay in white-tailed deer infected with Mycobacterium bovis.

    PubMed

    Lyashchenko, Konstantin P; Greenwald, Rena; Esfandiari, Javan; O'Brien, Daniel J; Schmitt, Stephen M; Palmer, Mitchell V; Waters, W Ray

    2013-06-01

    Bovine tuberculosis (TB) in cervids remains a significant problem affecting farmed herds and wild populations. Traditional skin testing has serious limitations in certain species, whereas emerging serological assays showed promising diagnostic performance. The recently developed immunochromatographic dual-path platform (DPP) VetTB assay has two antigen bands, T1 (MPB83 protein) and T2 (CFP10/ESAT-6 fusion protein), for antibody detection. We evaluated the diagnostic accuracy of this test by using serum samples collected from groups of white-tailed deer experimentally inoculated with Mycobacterium bovis, M. avium subsp. paratuberculosis, or M. bovis BCG Pasteur. In addition, we used serum samples from farmed white-tailed deer in herds with no history of TB, as well as from free-ranging white-tailed deer culled during field surveillance studies performed in Michigan known to have bovine TB in the wild deer population. The DPP VetTB assay detected antibody responses in 58.1% of experimentally infected animals within 8 to 16 weeks postinoculation and in 71.9% of naturally infected deer, resulting in an estimated test sensitivity of 65.1% and a specificity of 97.8%. The higher seroreactivity found in deer with naturally acquired M. bovis infection was associated with an increased frequency of antibody responses to the ESAT-6 and CFP10 proteins, resulting in a greater contribution of these antigens, in addition to MPB83, to the detection of seropositive animals, compared with experimental M. bovis infection. Deer experimentally inoculated with either M. avium subsp. paratuberculosis or M. bovis BCG Pasteur did not produce cross-reactive antibodies that could be detected by the DPP VetTB assay. The present findings demonstrate the relatively high diagnostic accuracy of the DPP VetTB test for white-tailed deer, especially in the detection of naturally infected animals.

  13. Rapid Detection of Panton-Valentine Leukocidin in Staphylococcus aureus Cultures by Use of a Lateral Flow Assay Based on Monoclonal Antibodies

    PubMed Central

    Müller, Elke; Buechler, Joseph; Rejman, John; Stieber, Bettina; Akpaka, Patrick Eberechi; Bandt, Dirk; Burris, Rob; Coombs, Geoffrey; Hidalgo-Arroyo, G. Aida; Hughes, Peter; Kearns, Angela; Abós, Sonia Molinos; Pichon, Bruno; Skakni, Leila; Söderquist, Bo; Ehricht, Ralf

    2013-01-01

    Panton-Valentine leukocidin (PVL) is a virulence factor of Staphylococcus aureus, which is associated with skin and soft-tissue infections and necrotizing pneumonia. To develop a rapid phenotypic assay, recombinant PVL F component was used to generate monoclonal antibodies by phage display. These antibodies were spotted on protein microarrays and screened using different lukF-PV preparations and detection antibodies. This led to the identification of the optimal antibody combination that was then used to establish a lateral flow assay. This test was used to detect PVL in S. aureus cultures. The detection limit of the assay with purified native and recombinant antigens was determined to be around 1 ng/ml. Overnight cultures from various solid and liquid media proved suitable for PVL detection. Six hundred strains and clinical isolates from patients from America, Europe, Australia, Africa, and the Middle East were tested. Isolates were genotyped in parallel by DNA microarray hybridization for confirmation of PVL status and assignment to clonal complexes. The sensitivity, specificity, and positive and negative predictive values of the assay in this trial were 99.7, 98.3, 98.4, and 99.7%, respectively. A total of 302 clinical isolates and reference strains were PVL positive and were assigned to 21 different clonal complexes. In summary, the lateral flow test allows rapid and economical detection of PVL in a routine bacteriology laboratory. As the test utilizes cultures from standard media and does not require sophisticated equipment, it can be easily integrated into a laboratory's workflow and might contribute to timely therapy of PVL-associated infections. PMID:23175260

  14. Production of Furin-cleaved Papillomavirus Pseudovirions and their use for in vitro neutralization assays of L1 or L2-specific antibodies

    PubMed Central

    Wang, Joshua W; Matsui, Ken; Pan, Yuanji; Kwak, Kihyuck; Peng, Shiwen; Kemp, Troy; Pinto, Ligia; Roden, Richard B.S

    2015-01-01

    Immunization with Human Papillomavirus (HPV) L1 virus-like particles or L2 capsid protein elicits neutralizing antibodies that mediate protection. A high throughput and sensitive in vitro neutralization assay is therefore valuable for prophylactic HPV vaccine studies. Over several hours during infection of the genital tract, virions take on a distinct intermediate conformation, including a required furin cleavage of L2 at its N-terminus. This intermediate is an important target for neutralization by L2-specific antibody, but it is very transiently exposed during in vitro infection of most cell lines resulting in insensitive measurement for L2, but not L1-specific neutralizing antibodies. To model this intermediate, we describe a protocol to generate furin-cleaved HPV pseudovirions (fc-PsV) which deliver an encapsidated reporter plasmid to facilitate infectivity measurements. We also describe a protocol for use of fc-PsV in a high throughput in vitro neutralization assay for the sensitive measurement of both L1 and L2-specific neutralizing antibodies. PMID:26237105

  15. Gold magnetic nanoparticle conjugate-based lateral flow assay for the detection of IgM class antibodies related to TORCH infections.

    PubMed

    Li, Xingxing; Zhang, Qinlu; Hou, Peng; Chen, Mingwei; Hui, Wenli; Vermorken, Alphons; Luo, Zhiyi; Li, Hong; Li, Qin; Cui, Yali

    2015-11-01

    In this study, a lateral flow immunochromatographic assay (LFIA) system for the detection of immunoglobulin M (IgM) antibodies, related to TORCH [(T)oxoplasmosis, (O)ther agents, (R)ubella (also known as German Measles), (C)ytomegalovirus, and (H)erpes simplex virus infections], based on gold magnetic nanoparticles, was established. Following modification with poly(methacrylic acid), the gold magnetic nanoparticles conjugated with an anti‑human IgM antibody (μ‑chain specific) to construct a probe. A lateral flow assay device was constructed based on these conjugates. IgM antibodies to four types of pathogens, notably toxoplasmosis, rubella virus, cytomegalovirus and herpes simplex virus type 2, were detected using this device. Compared with commercial colloidal gold‑based LFIA strips, our method exhibited higher sensitivity. No interference with triglycerides, hemoglobin and bilirubin occurred, and no cross‑reactivity was noted among the four pathogens. The gold magnetic nanoparticle‑LFIA strips were used to assess 41 seropositive and 121 seronegative serum samples. The sensitivity was 100% (162/162) and the specificity was 100% (162/162). This method cannot only be used for the detection of TORCH IgM-specific antibodies, but it can potentially be developed for use in the diagnosis of other acute or recently identified autoimmune diseases.

  16. Production of Furin-Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1- or L2-Specific Antibodies.

    PubMed

    Wang, Joshua W; Matsui, Ken; Pan, Yuanji; Kwak, Kihyuck; Peng, Shiwen; Kemp, Troy; Pinto, Ligia; Roden, Richard B S

    2015-08-03

    Immunization with Human Papillomavirus (HPV) L1 virus-like particles or L2 capsid protein elicits neutralizing antibodies that mediate protection. A high-throughput and sensitive in vitro neutralization assay is therefore valuable for prophylactic HPV vaccine studies. Over several hours during infection of the genital tract, virions take on a distinct intermediate conformation, including a required furin cleavage of L2 at its N-terminus. This intermediate is an important target for neutralization by L2-specific antibody, but it is very transiently exposed during in vitro infection of most cell lines resulting in insensitive measurement for L2, but not L1-specific neutralizing antibodies. To model this intermediate, we describe a protocol to generate furin-cleaved HPV pseudovirions (fc-PsV), which deliver an encapsidated reporter plasmid to facilitate infectivity measurements. We also describe a protocol for use of fc-PsV in a high-throughput in vitro neutralization assay for the sensitive measurement of both L1 and L2-specific neutralizing antibodies.

  17. A novel cell-based assay for inhibitory anti-muscarinic type 3 receptor antibodies in primary Sjögren's syndrome.

    PubMed

    Bastian, Isabell; Gordon, Tom P; Jackson, Michael W

    2015-12-01

    Inhibitory autoantibodies acting at the muscarinic acetylcholine receptor type 3 (M3R) are postulated to mediate autonomic dysfunction, including decreased salivary and lacrimal gland output and extra-glandular manifestations, in patients with primary Sjögren's syndrome. However, the contention that anti-M3R antibodies are pathogenic in patients remains untested, due to a lack of assays both sophisticated enough to detect inhibitory anti-M3R antibodies yet suitable for screening large patient cohorts. In the current study, we have established a cell-based bioassay of M3R activity, based on dual transfection of the M3R and a luciferase reporter gene. The bioassay is capable of capturing real-time agonist-mediated signalling of the M3R, which is inhibited specifically by patient IgG that have previously been demonstrated to have anti-M3R activity. The assay can be run in multi-well culture plates, and analysed using simple luminescence readers. As such, the new bioassay incorporating M3R-mediated luciferase transduction is the first assay adaptable to common diagnostic platforms that is capable of determining the presence in patient serum of functionally active anti-M3R autoantibodies. The new bioassay should prove useful for large cohort screening studies aiming to correlate the presence in patients of inhibitory anti-M3R antibodies with symptoms of both glandular and extra-glandular autonomic dysfunction.

  18. Optimization of fixed-permeabilized cell monolayers for high throughput micro-neutralizing antibody assays: application to the zebrafish/viral hemorrhagic septicemia virus (vhsv) model.

    PubMed

    Chinchilla, Blanca; Encinas, Paloma; Estepa, Amparo; Coll, Julio; Gomez-Casado, Eduardo

    2013-11-01

    A new high throughput centrifugation-free method to estimate viral neutralizing antibody levels in low volumes and large numbers of plasma blood samples is described. Cell monolayers were, (i) plated on poly-d-Lys coated 96-wells, (ii) infected with viruses previously incubated with fish plasma containing antibodies, (iii) fixed with formaldehyde to increase cell recovery and avoid centrifugation steps, (iv) permeabilized with Saponin, (v) immunostained in the presence of Saponin by using a monoclonal antibody (MAb) to viral protein, (vi) digested with trypsin to detach cells from the monolayer, in the absence of Saponin to reduce damage of intracellular MAb-antigen complexes, and (vii) gated by flow cytometry using automatic 96-well batch analysis. The method was applied to the determination of plasma neutralizing antibodies from zebrafish (Danio rerio) surviving infections with viral hemorrhagic septicemia virus (VHSV) (an important rhabdovirus of salmonids). This semi-automatic, rapid and practical assay detected anti-VHSV neutralizing antibodies in the plasma (∼3 μl per fish) of 95.1% of the zebrafish surviving VHSV infections. The fixed-permeabilized monolayer (FIXPERM) micro-neutralization method might help to analyze sera/plasma from small fish under standarized high throughput conditions.

  19. Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

    PubMed

    Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K

    2015-10-21

    A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples.

  20. Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

    SciTech Connect

    Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.; Marks, James D.; Varnum, Susan M.

    2012-11-15

    Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL) for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.

  1. Most proteinase3- and myeloperoxidase-antineutrophil cytoplasmic antibodies enzyme-linked immunosorbent assays perform less well in treated small-vessel vasculitis than in active disease.

    PubMed

    Savige, Judy; Trevisin, Michelle; Hayman, Matthew; Pollock, Wendy

    2009-06-01

    Antineutrophil cytoplasmic antibodies (ANCA) levels have been thought to follow disease activity, with levels being high at presentation, declining with treatment and increasing just before relapse. However, we have shown that ANCA often persist for many years in patients with clinically inactive Wegener's granulomatosis. ANCA assays are less sensitive for treated disease than for active disease, and the levels in treated patients produce different results in different assay systems. ANCA often persist for years without relapse, and the risk of relapse probably depend on levels that are critical for any individual patient. The capture enzyme-linked immunosorbent assays may be more sensitive in detecting early relapse. Relapse is more common when ANCA levels are high but, although elevated, ANCA levels are lower in relapse than at presentation. Standardized ANCA levels for the definitions of remission and relapse may not be possible, and the optimal ANCA testing protocol for treated disease remains unclear.

  2. Evaluation of fully automated assays for the detection of Rubella IgM and IgG antibodies by the Elecsys(®) immunoassay system.

    PubMed

    van Helden, Josef; Grangeot-Keros, Liliane; Vauloup-Fellous, Christelle; Vleminckx, Renaud; Masset, Frédéric; Revello, Maria-Grazia

    2014-04-01

    Screening for acute rubella infection in pregnancy is an important element of antenatal care. This study compared the sensitivity, specificity and reproducibility of two new, fully automated Elecsys(®) Rubella IgM and IgG immunoassays designed for the Elecsys 2010, Modular Analytics E170, COBAS e-411 and COBAS e-601 and e602 analytical platforms, with current assays using serum from patients with primary rubella infections, vaccinated patients, patients with potentially cross-reacting infections and on routine samples in clinical laboratories in France, Germany and Italy. Both assays showed good within-run and within-laboratory precision. A sensitivity of 79.8-96.0% was demonstrated for Elecsys IgM in primary, early acute infection, consistent with existing assays. In samples obtained from routine antenatal screening, the Elecsys Rubella IgM assay revealed high specificity (98.7-99.0%). A significantly (p<0.0001) lower reactivity was demonstrated in samples from previously infected patients where acute rubella infection was excluded, and the incidence of false positives in patients with potentially cross-reacting infections was lower with Elecsys Rubella IgM compared with other. The Elecsys Rubella IgG assay exhibited a relative sensitivity of 99.9-100.0% and specificity of 97.4-100.0% in samples from routine antenatal screening. The Elecsys Rubella IgM and IgG assays allow convenient, rapid and reliable determination of anti-rubella antibodies. Sensitivity, specificity and reproducibility were comparable with existing assay systems. Assay results were available in approximately half the time required for currently employed methods and the assays are compatible with widely used analytical platforms.

  3. Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

    PubMed Central

    Parma, Y. R.; Chacana, P. A.; Lucchesi, P. M. A.; Rogé, A.; Granobles Velandia, C. V.; Krüger, A.; Parma, A. E.; Fernández-Miyakawa, M. E.

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx2EDL933, stx2vha, stx2vhb, stx2g, stx1EDL933, and stx1d were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli. PMID:22919675

  4. Detection of antibodies to hepatitis B core antigen using the Abbott ARCHITECT anti-HBc assay: analysis of borderline reactive sera.

    PubMed

    Ollier, Laurence; Laffont, Catherine; Kechkekian, Aurore; Doglio, Alain; Giordanengo, Valérie

    2008-12-01

    Routine use of the automated chemiluminescent microparticle immunoassay Abbott ARCHITECT anti-HBc for diagnosis of hepatitis B is limited in case of borderline reactive sera with low signal close to the cut-off index. In order to determine the significance of anti-HBc detection when borderline reactivity occurs using the ARCHITECT anti-HBc assay, a comparative study was designed. 3540 serum samples collected over a 2-month period in the hospital of Nice were examined for markers of HBV infection (HBsAg, anti-HBs and anti-HBc). One hundred seven samples with sufficient volume and with borderline reactivity by the ARCHITECT assay were tested by two other anti-HBc assays, a microparticle enzyme immunoassay (MEIA, AxSYM Core, Abbott Laboratories, IL, USA) and an enzyme linked fluorescent assay (ELFA, VIDAS Anti-HBc Total II, bioMérieux, Lyon, France). Only 46 samples were confirmed by the AxSYM and the VIDAS assays. Additional serological information linked to patient history showed that the remaining samples (61) were false positives (11), had low titer of anti-HBc antibodies (13), or were inconclusive (37). This comparative study highlighted the existence of a grey zone around the cut-off index. Confirmative results through a different immunoassay are needed to confirm the diagnosis of HBV on borderline reactive sera using the ARCHITECT anti-HBc assay.

  5. Enzyme-linked immunosorbent assay for monitoring toxic dioxin congeners in milk based on a newly generated monoclonal anti-dioxin antibody.

    PubMed

    Okuyama, Mitsunobu; Kobayashi, Norihiro; Takeda, Wakako; Anjo, Takako; Matsuki, Yasuhiko; Goto, Junichi; Kambegawa, Akira; Hori, Shinjiro

    2004-04-01

    To develop an enzyme-linked immunosorbent assay (ELISA) for monitoring the toxicity due to polychlorinated dibenzo-p-dioxins and dibenzofurans contaminated in human breast milk, we have generated novel monoclonal antibodies using some haptenic derivatives linked to bovine serum albumin via the C-1 or C-2 position on the dioxin skeleton. BALB/c or A/J mice were repeatedly immunized with the immunogen, and spleen cells were fused with P3/NS1/1-Ag4-1 myeloma cells. After five fusion experiments, a hybridoma clone was established that secretes an antibody D9-36 group specifically recognizing the major toxic congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 2,3,4,7,8-pentachlorodibenzofran. An ELISA is developed on the basis of the competitive and labeled-antigen format. The toxic congeners extracted from butter or milk specimens by a novel extraction cartridge and a peroxidase-labeled dioxin analogue were sequentially reacted with a fixed amount of D9-36 in the presence of Triton X-100. The bound fraction was captured on a microtiter plate, immobilizing a second antibody, and the enzyme activity was colorimetrically determined. This ELISA afforded a practical sensitivity (measurable range, 1-100 pg/assay; detection limit, 1.0 pg/assay as 2,3,7,8-TCDD equivalent). The assay values for milk and butter samples were in reasonable accordance with the sum of the toxicity-equivalent quantity of each congener, which had been determined by a high-resolution gas chromatography/high-resolution mass spectrometry method.

  6. Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay.

    PubMed Central

    Ng, S P; Tsui, C O; Roberts, D; Chau, P Y; Ng, M H

    1996-01-01

    We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples. PMID:8779567

  7. Rapid competitive enzyme-linked immunosorbent assay using a monoclonal antibody reacting with a 15-kilodalton tegumental antigen of Schistosoma mansoni for serodiagnosis of schistosomiasis.

    PubMed Central

    Da Silva, A J; Piuvezam, M R; de Moura, H; Maddison, S; Peralta, J M

    1993-01-01

    A competitive enzyme-linked immunosorbent assay (CELISA) for antibody detection was developed by using a monoclonal antibody which reacts with a 15-kDa tegumental antigen of the adult worm of Schistosoma mansoni. This monoclonal antibody was not able to react with antigens of Schistosoma japonicum or Schistosoma haematobium in enzyme-linked immunoelectrotransfer blot (EITB) and indirect immunofluorescence tests. The assay was performed in a period of 1 h using an adult worm crude extract antigen. To evaluate the CELISA, a total of 73 serum samples was analyzed: 35 were from S. mansoni-infected patients, 23 were from individuals with parasitic infections other than schistosomiasis, and 14 were from healthy individuals. All serum samples from healthy individuals and from patients infected with other parasites were negative, as were two (6%) samples from patients infected with S. mansoni. EITB analysis showed that 32 of 33 CELISA-positive samples were positive in the EITB but with different patterns of reactivity. A 15-kDa protein reacted with 60% of serum samples, and a 60-kDa protein showed the highest level of reactivity (85%). The two samples from patients infected with S. mansoni that were negative in the CELISA reacted with 70-, 60-, 50-, 47-, and 38-kDa proteins. One sample, positive in CELISA, did not react with proteins of the antigenic extract. Images PMID:8408548

  8. Detection of antibodies specific for sheeppox and goatpox viruses using recombinant capripoxvirus antigens in an indirect enzyme-linked immunosorbent assay.

    PubMed

    Bowden, Timothy R; Coupar, Barbara E; Babiuk, Shawn L; White, John R; Boyd, Victoria; Duch, Christine J; Shiell, Brian J; Ueda, Norihito; Parkyn, Geoff R; Copps, John S; Boyle, David B

    2009-10-01

    Viruses in the genus Capripoxvirus, family Poxviridae, cause sheeppox, goatpox and lumpy skin disease, which are the most serious poxvirus diseases of production animals. Despite the considerable threat that these viruses pose to livestock production and global trade in sheep, goats, cattle and their products, convenient and effective serodiagnostic tools are not readily available. To develop a more effective antibody detection capability, selected open reading frames from capripoxvirus DNA were amplified and expressed in Escherichia coli as His-tagged fusion proteins. By screening 42 candidate antigens, two sheeppox virus virion core proteins that were expressed efficiently, purified readily using affinity chromatography and reactive against capripoxvirus immune sera in an indirect enzyme-linked immunosorbent assay (ELISA) were identified. The ELISA performed favourably when sera from sheep and goats infected experimentally with virulent capripoxvirus isolates were tested, with sensitivity and diagnostic specificity ranging between 95 and 97%, but it was unable to detect antibodies reliably in vaccinated sheep or goats. Furthermore, no cross-reactivity with antibodies against orf virus was detected. This assay offers the prospect of a convenient and standardised ELISA-based serodiagnostic test, with no requirement for infectious reagents, that is well suited to high-throughput capripoxvirus surveillance on a flock or herd basis.

  9. Development of an enzyme-linked immunosorbent assay for the detection of isomiroestrol, an identical marker, in White Kwao Krua using a monoclonal antibody.

    PubMed

    Kitisripanya, Tharita; Jutathis, Kamonthip; Inyai, Chadathorn; Komaikul, Jukrapun; Udomsin, Orapin; Tanaka, Hiroyuki; Putalun, Waraporn

    2017-04-15

    Pueraria candollei var. mirifica or White Kwao Krua (WKK) is a phytoestrogen-rich plant widely used among women to improve climacteric symptoms. Additionally, the tuberous roots of this plant have been added as an active ingredient for skin rejuvenation and breast enlargement effects in various functional foods. However, most of the products on the market containing WKK have not been sufficiently standardized with respect to the active compound or identical marker. To control the quality of these plant materials, an enzyme-linked immunosorbent assay (ELISA) using anti-isomiroestrol antibodies was established for the determination of isomiroestrol, an identical marker in WKK. Polyclonal and monoclonal antibodies against isomiroestrol were generated and their specificity characterized in this study. Monoclonal antibody 12C1 showed higher specificity to isomiroestrol and was thus selected to develop the ELISA. Based on the validation analysis and the tested performance of the developed ELISA in variably sourced WKK samples, the assay can provide an alternative approach that is reliable and highly sensitive for the quantitative analysis of isomiroestrol in plant.

  10. Noninfectious virus-like particle antigen for detection of swine vesicular disease virus antibodies in pigs by enzyme-linked immunosorbent assay.

    PubMed

    Ko, Young-Joon; Choi, Kang-Seuk; Nah, Jin-Ju; Paton, David J; Oem, Jae-Ku; Wilsden, Ginette; Kang, Shien-Young; Jo, Nam-In; Lee, Joo-Ho; Kim, Jae-Hong; Lee, Hee-Woo; Park, Jong-Myeong

    2005-08-01

    An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n=1,041). When tested using sera (n=186) collected periodically from pigs (n=19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.

  11. Detection of antibodies against epidermodysplasia verruciformis-associated canine papillomavirus 3 in sera of dogs from Europe and Africa by enzyme-linked immunosorbent assay.

    PubMed

    Lange, C E; Tobler, K; Favrot, C; Müller, M; Nöthling, J O; Ackermann, M

    2009-01-01

    The role of papillomaviruses (PVs) in the development of canine cancers is controversial. However, recently a novel canine PV (CPV3) was detected in a dog affected with a condition reminiscent of epidermodysplasia verruciformis (EV). The aim of the present study was to investigate the seroprevalence of CPV3 by using generic enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against either canine oral PV (COPV) or CPV3. Therefore, the capsid proteins of both PV types were expressed as glutathione S-transferase fusion protein antigens and adsorbed to glutathione-casein-coated ELISA plates. After showing that PV type-specific antibodies could be detected in the sera from dogs with confirmed COPV or CPV3 infection, CPV3- and COPV-seropositive samples were detected in two sets of canine sera collected in Switzerland and South Africa, respectively. We found specific antibodies against COPV and CPV3 among the tested sera and also a large number that were positive for both antigens. The seroprevalences of PV antibodies of 21.9% (COPV) and 26.9% (CPV3) among the tested dogs from South Africa were higher than those among the dogs from Switzerland at 10.5% (COPV) and 1.3% (CPV3). Our data suggest a need for further CPV-related seroepidemiological surveys in different countries, especially in the context of clinical manifestations and possible breed predispositions. For this purpose, the newly developed ELISAs can be a useful tool.

  12. Development of polyclonal antibody based enzyme-linked immunosorbent assay for the analysis of the agricultural insecticide imidacloprid: food quality and safety.

    PubMed

    Li, Zhixiong; Liu, Yingli; Sun, Yuanming; Wu, Qing; Lei, Hongtao; Wang, Hong; Xiao, Zhili

    2007-01-01

    In order to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the insecticide imidacloprid, the anti-imidacloprid polyclonal antibody was produced and the affinity of three different coating antigens with the polyclonal antibody was compared. The antigenic determinant in antigen aiming at the polyclonal antibody was raised and analyzed. The standard curve for imidacloprid had been developed. The effects of organic solvents and buffer ionic strength on the ELISA for insecticide were studied and conditions of analysis were also optimized in this research. Experiment result showed that the coating antigen IMMP-OVA ,whose binding ratio was 14:1, had stronger affinity with the anti-imidacloprid polyclonal antibody than IMMB-OVA and IMEB-OVA ; the IC50 value in the standard curve was 995.4 ng.mL-1 and the limit of detection (LOD) was 30 ng.mL-1; the cross reaction ratios with nitenpyram and acetamiprid were 5.73% and 11.31% respectively.

  13. Development of a monoclonal antibody-based competitive indirect enzyme-linked immunosorbent assay for furaltadone metabolite AMOZ in fish and shrimp samples.

    PubMed

    Shen, Yu-Dong; Xu, Zhen-Lin; Zhang, Shi-Wei; Wang, Hong; Yang, Jin-Yi; Lei, Hong-Tao; Xiao, Zhi-Li; Sun, Yuan-Ming

    2012-11-07

    A monoclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ELISA) with improved sensitivity and specificity for the determination of furaltadone metabolite 5-methylamorpholino-3-amino-2-oxazolidone (AMOZ) was described. AMOZ was derivatized with 2-(3-formylphenoxy)acetic acid and coupled with bovine serum albumin to form a novel immunogen. BABL/c mice were immunized and monoclonal antibody specific to the nitrophenyl derivative of AMOZ (NP-AMOZ) was produced and characterized. Four other haptens with different heterology to the immunizing hapten were synthesized and coupled to ovalbumin as coating antigens to study the effect of heterologous coating on assay sensitivity. Under the optimized heterologous coating format, the competitive indirect ELISA showed very high sensitivity to NP-AMOZ, with an IC(50) of 0.14 μg/L and limit of detection of 0.01 μg/L. The assay showed high specificity toward NP-AMOZ, and negligible cross-reactivity with analogous compounds was observed. The average recoveries of AMOZ from spiked fish and shrimp samples were estimated to range from 81.0 to 104.0%, with coefficients of variation below 20%. Good correlation was obtained between the results of ELISA analysis and of standard liquid chromatography-tandem mass spectrometry analysis. These results indicated that the proposed ELISA is ideally suited as a monitoring method for AMOZ residues at trace level.

  14. Specific Capture of Peptide-Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide-Binding Assay in Live Cells.

    PubMed

    Dirscherl, Cindy; Palankar, Raghavendra; Delcea, Mihaela; Kolesnikova, Tatiana A; Springer, Sebastian

    2017-02-02

    Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H-2K(b) , into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H-2K(b) and hold it at the cell surface. This patterned receptor enables a novel peptide-binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.

  15. Implementation of an Enzyme Linked Immunosorbent Assay for the Quantification of Allergenic Egg Residues in Red Wines Using Commercially Available Antibodies

    PubMed Central

    Koestel, Carole; Simonin, Céline; Belcher, Sandrine

    2016-01-01

    Abstract Since the early 2000s, labeling of potentially allergenic food components to protect people who suffer from food allergies is compulsory in numerous industrialized countries. In Europe, milk and egg components used during the winemaking process must be indicated on the label since July 1, 2012. Several ELISA procedures have been developed to detect allergenic residues in wines. However, the complexity of the wine matrix can inhibit the immunoenzymatic reaction. The aim of this study was to implement an ELISA assay for the detection of ovalbumin in red wines using commercially available antibodies. The specificity of the acquired antibodies and the absence of cross reactivity were assessed by immunoblotting and ELISA. An ELISA assay with a LOD of 14.2 μg/L and a LOQ of 56.4 μg/L of ovalbumin in aqueous solution was obtained. Differences in ELISA signals were observed when analyzing various fining agents, although reproducible conformation of the antigen could be reached for the comparison of ovalbumin and Ovicolle. The differences between samples in terms of pH could be leveled but the inhibition of the ELISA signal, positively correlated to the tannin content of the wines, could not be suppressed. Thus, standard curves of ovalbumin in several wines were obtained by relative quantification. The control steps and the difficulties encountered presented in this study should be considered by anybody working toward the development of ELISA assays for the detection of allergenic residues in complex food matrices. PMID:27356183

  16. Initial development and preliminary evaluation of a multiplex bead assay to detect antibodies to Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis outer membrane peptides in naturally infected dogs from Grenada, West Indies.

    PubMed

    Wilkerson, Melinda J; Black, Kelley E; Lanza-Perea, Marta; Sharma, Bhumika; Gibson, Kathryn; Stone, Diana M; George, Anushka; Nair, Arathy D S; Ganta, Roman R

    2017-01-01

    Tick-borne bacteria, Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis are significant pathogens of dogs worldwide, and coinfections of E. canis and A. platys are common in dogs on the Caribbean islands. We developed and evaluated the performance of a multiplex bead-based assay to detect antibodies to E. canis, A. platys, and E. chaffeensis peptides in dogs from Grenada, West Indies, where E. canis and A. platys infections are endemic. Peptides from outer membrane proteins of P30 of E. canis, OMP-1X of A. platys, and P28-19/P28-14 of E. chaffeensis were coupled to magnetic beads. The multiplex peptide assay detected antibodies in dogs experimentally infected with E. canis and E. chaffeensis, but not in an A. platys experimentally infected dog. In contrast, the multiplex assay and an in-house enzyme-linked immunosorbent assay (ELISA) detected A. platys antibodies in naturally infected Grenadian dogs. Following testing of 104 Grenadian canine samples, multiplex assay results had good agreement with commercially available ELISA and immunofluorescent assay for E. canis antibody-positive dogs ( K values of 0.73 and 0.84), whereas A. platys multiplex results had poor agreement with these commercial assays ( K values of -0.02 and 0.01). Prevalence of seropositive E. canis and A. platys Grenadian dogs detected by the multiplex and commercial antibody assays were similar to previous reports. Although the multiplex peptide assay performed well in detecting the seropositive status of dogs to E. canis and had good agreement with commercial assays, better antigen targets are necessary for the antibody detection of A. platys.

  17. Comparison of two immunochromatographic assays and the indirect immunofluorescence antibody test for diagnosis of Trypanosoma cruzi infection in dogs in south central Louisiana.

    PubMed

    Nieto, Prixia D; Boughton, Roger; Dorn, Patricia L; Steurer, Frank; Raychaudhuri, Syamal; Esfandiari, Javan; Gonçalves, Edson; Diaz, James; Malone, John B

    2009-11-12

    Two rapid tests evaluated in dogs considered to be of high risk of infection with the Chagas parasite Trypanosoma cruzi using two immunochromatographic assays: Trypanosoma Detect for canine, InBios, Seattle, WA and CHAGAS STAT-PAK assay, Chembio Diagnostic Systems, Medford, NY, in south central Louisiana. For this purpose a serological survey was carried out in a total of 122 dogs and a serum bank was created. These 122 animals were first tested by IFAT that was used as the standard test. From the serum bank 50 samples were tested using the two rapid Chagas assays and results compared to the standard test IFAT. The serological survey using IFAT showed a prevalence of T. cruzi infection in 22.1% of the tested dogs. In the immunochromatographic assays, 13 and 11 animals were positive on rapid assay: Trypanosoma Detect for canine, InBios and CHAGAS STAT-PAK, Chembio Diagnostic Systems, respectively compared to 11 positive by IFAT. These two immunochromatographic tests have shown high susceptibility and specificity compared to our standard method IFAT. The rapid, easy and accurate screening assays used in conjunction with confirmatory tests, would be an excellent tool for veterinarians to diagnose T. cruzi infection. Early detection of T. cruzi infection may prevent complications through an effective treatment. Greater awareness by veterinarians of the risk, clinical findings, history along with diagnostic methods will contribute greatly to an understanding of the true prevalence of Chagas disease in dogs in Louisiana.

  18. Three cases of cat scratch disease diagnosed by indirect immunofluorescence antibody assay and/or polymerase chain reaction of 16S rRNA gene of Bartonella henselae.

    PubMed

    Maruyama, S; Kabeya, H; Nogami, S; Sakai, H; Suzuki, J; Suzuki, H; Sugita, H; Katsube, Y

    2000-12-01

    Three suspected cases of cat scratch disease were diagnosed by indirect immunofluorescence antibody assay and/or polymerase chain reaction. Patient 1 was a 10-year-old female who presented swelling of the right axillary [corrected] lymph nodes with pain and fever. She kept a kitten, and many scratches were observed on her both legs and dorsum manus. Antibody titers against Bartonella (B.) henselae were 1:32 for IgM 3 weeks after the onset of the symptoms and 1:64 for IgG 8 weeks after the onset. The DNA for 16S rRNA type I of B. henselae was detected from the blood sample obtained 3 weeks after the onset of symptoms by polymerase chain reaction for the first time in Japan. Patient 2 was a 22-year-old female veterinary student with a cat scratch at the bottom of her neck by a male kitten. She developed a papule at the scratch, slight fever, and neck pain. Although both Bartonella-specific IgG and IgM antibodies were negative before the scratch, the IgG antibody titer rose to 1:512 14 weeks after the onset. B. henselae was isolated from the kitten and its DNA found to be for 16S rRNA type I by PCR. Patient 3 was a 23-year-old female veterinary student with a cat scratch on her left forearm. A small reddish papule developed on the scratch, and she experienced swelling of the left axillary [corrected] lymph node and pain. Both the IgG and IgM antibodies against B. henselae were negative before the cat scratch, and the IgG titer rose significantly to 1:128 and 1:1,024 in 2 and 5 weeks, respectively, after the onset of the symptoms.

  19. Development and application of an enzyme-linked immunosorbent assay for detection of bovine viral diarrhea antibody based on Erns glycoprotein expressed in a baculovirus system.

    PubMed

    Grego, E; Uslenghi, F; Strasser, M; Luzzago, C; Frigerio, M; Peletto, S; Rosati, S

    2007-01-01

    The BVDV envelope glycoprotein E(rns)/gp48 and the C terminal 79 amino acids of the capsid protein coding region were expressed in a baculovirus system and antigenically characterized. Western blot assay was used to detect recombinant E(rns) (r-E(rns)) in infected insect cells using specific monoclonal antibodies. The r-E(rns) was then used in an indirect ELISA to detect BVDV specific antibodies in a panel of 540 well-characterized sera. Results of the r-E(rns) ELISA were compared to those obtained with a commercially available competitive ELISA targeting anti-NS2/3 antibodies. A good correlation was observed between the 2 ELISA (kappa = 0.916, 95% C.I.: 0.876, 0.956). Using the commercial NS2/3 ELISA as the reference test, the relative sensitivity of r-E(rns) ELISA was 97.5% (95% C.I.: 94.3%, 99.1%) and the relative specificity was 93.9% (95% C.I.: 89.4%, 96.9%), while relative specificity was 100% (95% C.I.: 97%, 100%) using true negative sera (derived from a negative herd). All but 1 antigen positive animals (n = 36) tested negative in the r-E(rns) ELISA; among them all 22 confirmed PI animals were negative by r-E(rns) ELISA. The ability of r-E(rns) ELISA to identify cattle immunized with inactivated vaccine was also demonstrated in a small group of cattle, compared to an NS2/3 antibody ELISA. Results suggest that r-E(rns) ELISA represents an alternative test for antibody generated by natural infection or BVDV vaccination.

  20. Development of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against the 3B Protein of Foot-and-Mouth Disease Virus

    PubMed Central

    Yang, Ming; Parida, Satya; Salo, Tim; Hole, Kate; Velazquez-Salinas, Lauro

    2015-01-01

    Foot-and-mouth disease (FMD) is one of the most highly contagious and economically devastating diseases, and it severely constrains the international trade of animals. Vaccination against FMD is a key element in the control of FMD. However, vaccination of susceptible animals raises critical issues, such as the differentiation of infected animals from vaccinated animals. The current study developed a reliable and rapid test to detect antibodies against the conserved, nonstructural proteins (NSPs) of the FMD virus (FMDV) to distinguish infected animals from vaccinated animals. A monoclonal antibody (MAb) against the FMDV NSP 3B was produced. A competitive enzyme-linked immunosorbent assay (cELISA) for FMDV/NSP antibody detection was developed using a recombinant 3ABC protein as the antigen and the 3B-specific MAb. Sera collected from naive, FMDV experimentally infected, vaccinated carrier, and noncarrier animals were tested using the 3B cELISA. The diagnostic specificity was 99.4% for naive animals (cattle, pigs, and sheep) and 99.7% for vaccinated noncarrier animals. The diagnostic sensitivity was 100% for experimentally inoculated animals and 64% for vaccinated carrier animals. The performance of this 3B cELISA was compared to that of four commercial ELISA kits using a panel of serum samples established by the World Reference Laboratory for FMD at The Pirbright Institute, Pirbright, United Kingdom. The diagnostic sensitivity of the 3B cELISA for the panel of FMDV/NSP-positive bovine serum samples was 94%, which was comparable to or better than that of the commercially available NSP antibody detection kits. This 3B cELISA is a simple, reliable test to detect antibodies against FMDV nonstructural proteins. PMID:25651918

  1. Evaluation of enzyme-linked immunosorbent assays for detection of Mycoplasma bovis-Specific antibody in bison sera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. This report demonstrates that ELISAs for detection of M. bovis-specific antibody in cattle are not optimal for identification of seropositive bison. An ELISA optimized for use with bison sera is ...

  2. Population-based Tay-Sachs screening among Ashkenazi Jewish young adults in the 21st century: Hexosaminidase A enzyme assay is essential for accurate testing.

    PubMed

    Schneider, Adele; Nakagawa, Sachiko; Keep, Rosanne; Dorsainville, Darnelle; Charrow, Joel; Aleck, Kirk; Hoffman, Jodi; Minkoff, Sherman; Finegold, David; Sun, Wei; Spencer, Andrew; Lebow, Johannah; Zhan, Jie; Apfelroth, Stephen; Schreiber-Agus, Nicole; Gross, Susan

    2009-11-01

    Tay-Sachs disease (TSD) carrier screening, initiated in the 1970s, has reduced the birth-rate of Ashkenazi Jews with TSD worldwide by 90%. Recently, several nationwide programs have been established that provide carrier screening for the updated panel of Jewish genetic diseases on college campuses and in Jewish community settings. The goals of this study were to determine the performance characteristics of clinical TSD testing in college- and community-based screening programs and to determine if molecular testing alone is adequate in those settings. Clinical data for TSD testing were retrospectively anonymized and subsequently analyzed for 1,036 individuals who participated in these programs. The performance characteristics of the serum and the platelet Hexosaminidase assays were compared, and also correlated with the results of targeted DNA analysis. The serum assay identified 29 carriers and the platelet assay identified 35 carriers for carrier rates of 1/36 and 1/29, respectively. One hundred sixty-nine samples (16.3%) were inconclusive by serum assay in marked contrast to four inconclusive samples (0.4%) by the platelet assay. Molecular analysis alone would have missed four of the 35 carriers detected by the platelet assay, yielding a false negative rate of 11.4% with a sensitivity of 88.6%. Based on the results of this study, platelet assay was superior to serum with a minimal inconclusive rate. Due to changing demographics of the Ashkenazi Jewish population, molecular testing alone in the setting of broad-based population screening programs is not sufficient, and biochemical analysis should be the assay of choice.

  3. How-To-Do-It: Immunological Assays for the Classroom II--Hybridoma Technology: Production of Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Russo, A. J.

    1988-01-01

    Presented is a sample hybridoma assay which can be used in a research or classroom laboratory setting for instructional purposes. Described are experimental methods, materials, and observations made during this activity. (CW)

  4. Evaluation of an in-house dot enzyme-linked immunosorbent assay to detect antibodies against feline panleukopenia virus.

    PubMed

    Mende, Katherina; Stuetzer, Bianca; Truyen, Uwe; Hartmann, Katrin

    2014-10-01

    Measuring antibody titres to determine a cat's immunity to core diseases instead of just administering annual vaccinations has not been established in Germany so far. An in-house test kit for the detection of antibodies against feline panleukopenia virus (FPV), feline herpesvirus-1 and feline calicivirus-- the ImmunoComb Feline VacciCheck--is now available in several European countries. The aim of this study was to assess the quality of the ImmunoComb Feline VacciCheck to determine antibodies by comparing it to a gold standard. The test is aimed for use in practice to assist decision-making when performing an individual health assessment to see whether a cat is potentially unprotected against FPV and requires FPV vaccination. Sera from 347 cats were included in the study. For antibody detection, haemagglutination inhibition (HI) was performed as gold standard. Sensitivity, specificity and positive and negative predictive values of the ImmunoComb Feline VacciCheck were determined for three different HI titre cut-off points (1:20, 1:40, 1:80). In comparison to the HI, the ImmunoComb Feline VacciCheck showed a sensitivity of 79%, 83% and 87%, and a specificity of 89%, 86% and 81%, respectively. Specificity of the ImmunoComb Feline VacciCheck, which was considered the most important parameter, was acceptable in comparison to HI. Especially when considering an antibody titre of 1:20 sufficient for protection (eg, in an adult animal), the ImmunoComb Feline VacciCheck can be recommended for use in veterinary practice.

  5. Prediction of Pneumococcal Conjugate Vaccine Effectiveness against Invasive Pneumococcal Disease Using Opsonophagocytic Activity and Antibody Concentrations Determined by Enzyme-Linked Immunosorbent Assay with 22F Adsorption ▿

    PubMed Central

    Schuerman, L.; Wysocki, J.; Tejedor, J. C.; Knuf, M.; Kim, K.-H.; Poolman, J.

    2011-01-01

    We compared the abilities of two serological readouts, antipolysaccharide IgG antibody concentrations and opsonophagocytic activity (OPA) titers, to predict the clinical effectiveness of the 7-valent pneumococcal conjugate vaccine (7vCRM) against invasive pneumococcal disease (IPD). We also assessed the accuracy of the previously established thresholds for GlaxoSmithKline's enzyme-linked immunosorbent assay with 22F adsorption (22F-ELISA) (≥0.2 μg/ml) and OPA assay (titer, ≥8) in predicting effectiveness. We showed that following a 3-dose 7vCRM primary vaccination, the serological response rates as determined using thresholds of ≥0.2 μg/ml IgG and an OPA titer of ≥8 corresponded well with overall effectiveness against IPD. In addition, the OPA assay seemed to better predict serotype-specific effectiveness than enzyme-linked immunoassay. Finally, when applied to post-dose-2 immune responses, both thresholds also corresponded well with the overall IPD effectiveness following a 2-dose 7vCRM primary vaccination. These results support the importance of the OPA assay in evaluating immune responses to pneumococcal conjugate vaccines. PMID:21994351

  6. Use of Protein AG in an Enzyme-Linked Immunosorbent Assay for Screening for Antibodies against Parapoxvirus in Wild Animals in Japan

    PubMed Central

    Inoshima, Yasuo; Shimizu, Shinya; Minamoto, Nobuyuki; Hirai, Katsuya; Sentsui, Hiroshi

    1999-01-01

    Using protein AG in an enzyme-linked immunosorbent assay (ELISA), we tried to detect antibodies against parapoxvirus in 9 species of wild animals in Japan: the Japanese badger (Meles meles anakuma), Japanese black bear (Ursus thibetanus japonicus), Japanese deer (Cervus nippon centralis), Japanese monkey (Macaca fuscata), Japanese raccoon dog (Nyctereutes procyonoides viverrinus), Japanese serow (Capricornis crispus), Japanese wild boar (Sus scrofa leucomystax), masked palm civet (Paguma larvata), and nutria (Myocastor coypus). A total of 272 serum samples were collected over the period from 1984 to 1995 and were tested by the protein AG-ELISA, the agar gel immunodiffusion test, and an indirect immunofluorescence assay. The protein AG-ELISA was effective in a serological survey for parapoxvirus in wild animals, and antibodies were detected only in Japanese serows. A total of 24 of 66 (36.4%) Japanese serows reacted positively, and they were found in almost all prefectures in all years tested. These results suggest that epizootic cycles of parapoxvirus exist widely in Japanese serows and that they could be reservoirs for the virus in the field in Japan. Moreover, it is probable that they might carry the virus to domestic animals such as cattle, sheep, and goats. PMID:10225841

  7. Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus.

    PubMed

    Huang, Yi; Zhu, Youjie; Yang, Mengshi; Zhang, Zhenqing; Song, Donglin; Yuan, Zhiming

    2014-12-01

    Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

  8. Validation and use of an indirect enzyme-linked immunosorbent assay for detection of antibodies to West Nile virus in American Alligators (Alligator mississippiensis) in Florida.

    PubMed

    Jacobson, Elliott R; Johnson, April J; Hernandez, Jorge A; Tucker, Sylvia J; Dupuis, Alan P; Stevens, Robert; Carbonneau, Dwayne; Stark, Lillian

    2005-01-01

    In October 2002, West Nile virus (WNV) was identified in farmed American alligators (Alligator mississippiensis) in Florida showing clinical signs and having microscopic lesions indicative of central nervous system disease. To perform seroepidemiologic studies, an indirect enzyme-linked immunosorbent assay (ELISA) was developed to determine exposure of captive and wild alligators to WNV. To validate the test, a group of WNV-seropositive and -seronegative alligators were identified at the affected farm using hemagglutination inhibition (HAI) and the plaque reduction neutralization test (PRNT). The indirect ELISA utilized a rabbit anti-alligator immunoglobulins polyclonal antibody as the secondary antibody, and inactivated WNV-infected Vero cells were used as the coating antigen. For all samples (n=58), the results of the ELISA were consistent with the HAI and PRNT findings. Plasma was collected from 669 free-ranging alligators from 21 sites across Florida in April and October 2003. Four samples collected in April and six in October were positive for WNV antibodies using HAI, PRNT, and the indirect ELISA. This indicated that wild alligators in Florida have been exposed to WNV. These findings can be used as a baseline for future surveys.

  9. 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

  10. Evaluation of an indirect enzyme-linked immunosorbent assay for routine screening of Theiler's murine encephalomyelitis virus antibodies in mice colonies.

    PubMed

    Laborde, Juan M; Carbone, Cecilia; Corva, Santiago G; Galosi, Cecilia M

    2008-11-01

    The current study demonstrates the ability of an indirect enzyme-linked immunosorbent assay (iELISA) to detect antibodies against Theiler's murine encephalomyelitis virus in mice colonies. The antigen was produced from infected baby hamster kidney (BHK)-21 cells and treated with 1% Nonidet P40 in saline buffer. Control antigen was prepared following the same procedure using uninfected BHK-21 cells. The optimal antigen and serum dilutions were established. The reaction was revealed using an anti-mouse-horseradish peroxidase conjugate and 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Optimized iELISA was validated by detection of antibodies in known positive and negative serum samples before testing the samples of unknown status. Performance of the iELISA was compared with the indirect fluorescent antibody test, and the cutoff value was determined by receiver operating curve. Indirect ELISA showed 100% sensitivity, 99.38% specificity, and 97.78% predictive positive value. The antigen used is easy to produce, and no special equipment is required. The iELISA developed is simple and provides a rapid and less costly tool for diagnosis and research.

  11. Antibody Response to Plague Vaccination in Humans as Assayed by Staphylococcal Radioimmune Precipitation (St-RIP) Test

    DTIC Science & Technology

    1980-08-22

    Medical Microbiology and Immunology 163, 25-35. Cavanaugh, D. C., Elisberg, B. L., Llewellyn, C. H., Marshall, J. D., Jr., Rust, J. H.,I: Williams J. E...Schaeg, W. (1977). A radioimmunoassay for tetanus antibodies using protein A-containing Staphylococcus aureus. Medical Microbiology and Immunology 163...Putenherpesvirus. Medical Microbiology and Immunology 163, 14l-156. Kessler, S. W. (1975) Rapid isolation of antigens from tells with a staphylococa! protein

  12. An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software

    PubMed Central

    Shah, Pranav; Kerns, Edward; Nguyen, Dac-Trung; Obach, R. Scott; Wang, Amy Q.; Zakharov, Alexey; McKew, John; Simeonov, Anton; Hop, Cornelis E. C. A.

    2016-01-01

    Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t1/2) method] was chosen to determine CLint. The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t1/2 compounds, carbamazepine and antipyrine (t1/2 > 30 minutes); one moderate t1/2 compound, ketoconazole (10 < t1/2 < 30 minutes); and two short t1/2 compounds, loperamide and buspirone (t½ < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (∼12%) for the linear range observed. Using this assay, CYP3A4 CLint and t1/2 values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes. PMID:27417180

  13. Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay.

    PubMed Central

    Cloeckaert, A; de Wergifosse, P; Dubray, G; Limet, J N

    1990-01-01

    A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide O side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development. Images PMID:1701417

  14. Diagnosis and clinical virology of Lassa fever as evaluated by enzyme-linked immunosorbent assay, indirect fluorescent-antibody test, and virus isolation.

    PubMed

    Bausch, D G; Rollin, P E; Demby, A H; Coulibaly, M; Kanu, J; Conteh, A S; Wagoner, K D; McMullan, L K; Bowen, M D; Peters, C J; Ksiazek, T G

    2000-07-01

    The Lassa virus (an arenavirus) is found in West Africa, where it sometimes causes a severe hemorrhagic illness called Lassa fever. Laboratory diagnosis has traditionally been by the indirect fluorescent-antibody (IFA) test. However, enzyme-linked immunosorbent assays (ELISAs) for Lassa virus antigen and immunoglobulin M (IgM) and G (IgG) antibodies have been developed that are thought to be more sensitive and specific. We compared ELISA and IFA testing on sera from 305 suspected cases of Lassa fever by using virus isolation with a positive reverse transcription-PCR (RT-PCR) test as the "gold standard." Virus isolation and RT-PCR were positive on 50 (16%) of the 305 suspected cases. Taken together, Lassa virus antigen and IgM ELISAs were 88% (95% confidence interval [CI], 77 to 95%) sensitive and 90% (95% CI, 88 to 91%) specific for acute infection. Due to the stringent gold standard used, these likely represent underestimates. Diagnosis could often be made on a single serum specimen. Antigen detection was particularly useful in providing early diagnosis as well as prognostic information. Level of antigenemia varied inversely with survival. Detection by ELISA of IgG antibody early in the course of illness helped rule out acute Lassa virus infection. The presence of IFA during both acute and convalescent stages of infection, as well as significant interobserver variation in reading the slides, made interpretation difficult. However, the assay provided useful prognostic information, the presence of IFA early in the course of illness correlating with death. The high sensitivity and specificity, capability for early diagnosis, and prognostic value of the ELISAs make them the diagnostic tests of choice for the detection of Lassa fever.

  15. Comparison and correlation of neisseria meningitidis serogroup B immunologic assay results and human antibody responses following three doses of the Norwegian meningococcal outer membrane vesicle vaccine MenBvac.

    PubMed

    Findlow, Jamie; Taylor, Stephen; Aase, Audun; Horton, Rachel; Heyderman, Robert; Southern, Jo; Andrews, Nick; Barchha, Rita; Harrison, Ewan; Lowe, Ann; Boxer, Emma; Heaton, Charlotte; Balmer, Paul; Kaczmarski, Ed; Oster, Philipp; Gorringe, Andrew; Borrow, Ray; Miller, Elizabeth

    2006-08-01

    The prediction of efficacy of Neisseria meningitidis serogroup B (MenB) vaccines is currently hindered due to the lack of an appropriate correlate of protection. For outer membrane vesicle (OMV) vaccines, immunogenicity has primarily been determined by the serum bactericidal antibody (SBA) assay and OMV enzyme-linked immunosorbent assay (ELISA). However, the opsonophagocytic assay (OPA), surface labeling assay, whole blood assay (WBA), and salivary antibody ELISA have been developed although correlation with protection is presently undetermined. Therefore, the aim of the study was to investigate further the usefulness of, and relationships between, MenB immunologic assays. A phase II trial of the OMV vaccine, MenBvac, with proven efficacy was initiated to compare immunologic assays incorporating the vaccine and six heterologous strains. Correlations were achieved between the SBA assay, OMV ELISA, and OPA using human polymorphonuclear leukocytes and human complement but not between an OPA using HL60 phagocytic cells and baby rabbit complement. Correlations between the surface labeling assay, the SBA assay, and the OMV ELISA were promising, although target strain dependent. Correlations between the salivary antibody ELISA and other assays were poor. Correlations to the WBA were prevented since many samples had results greater than the range of the assay. The study confirmed the immunogenicity and benefit of a third dose of MenBvac against the homologous vaccine strain using a variety of immunologic assays. These results emphasize the need for standardized methodologies that would allow a more robust comparison of assays between laboratories and promote their further evaluation as correlates of protection against MenB disease.

  16. Anti-apical-membrane-antigen-1 antibody is more effective than anti-42-kilodalton-merozoite-surface-protein-1 antibody in inhibiting plasmodium falciparum growth, as determined by the in vitro growth inhibition assay.

    PubMed

    Miura, Kazutoyo; Zhou, Hong; Diouf, Ababacar; Moretz, Samuel E; Fay, Michael P; Miller, Louis H; Martin, Laura B; Pierce, Mark A; Ellis, Ruth D; Mullen, Gregory E D; Long, Carole A

    2009-07-01

    Apical membrane antigen 1 (AMA1) and the 42-kDa merozoite surface protein 1 (MSP1(42)) are leading malaria vaccine candidates. Several preclinical and clinical trials have been conducted, and an in vitro parasite growth inhibition assay has been used to evaluate the biological activities of the resulting antibodies. In a U.S. phase 1 trial with AMA1-C1/Alhydrogel plus CPG 7909, the vaccination elicited anti-AMA1 immunoglobulin G (IgG) which showed up to 96% inhibition. However, antibodies induced by MSP1(42)-C1/Alhydrogel plus CPG 7909 vaccine showed less than 32% inhibition in vitro. To determine whether anti-MSP1(42) IgG had less growth-inhibitory activity than anti-AMA1 IgG in vitro, the amounts of IgG that produced 50% inhibition of parasite growth (Ab(50)) were compared for rabbit and human antibodies. The Ab(50)s of rabbit and human anti-MSP1(42) IgGs were significantly higher (0.21 and 0.62 mg/ml, respectively) than those of anti-AMA1 IgGs (0.07 and 0.10 mg/ml, respectively) against 3D7 parasites. Ab(50) data against FVO parasites also demonstrated significant differences. We further investigated the Ab(50)s of mouse and monkey anti-AMA1 IgGs and showed that there were significant differences between the species (mouse, 0.28 mg/ml, and monkey, 0.14 mg/ml, against 3D7 parasites). Although it is unknown whether growth-inhibitory activity in vitro reflects protective immunity in vivo, this study showed that the Ab(50) varies with both antigen and species. Our data provide a benchmark for antibody levels for future AMA1- or MSP1(42)-based vaccine development efforts in preclinical and clinical trials.

  17. Enzyme-linked immunosorbent assay for human insulin-like growth factor-I using monoclonal and polyclonal antibodies with defined epitope recognition.

    PubMed

    Tamura, K; Kobayashi, M; Suzuki, S; Ishii, Y; Koyama, S; Yamada, H; Hashimoto, K; Niwa, M; Shibayama, F

    1990-05-01

    Monoclonal antibodies (McAb) and polyclonal antibodies (PcAb) against human insulin-like growth factor-I (somatomedin C; hIGF-I) were produced. Using these two antibodies, an enzyme-linked immunosorbent assay (ELISA) system for hIGF-I was established. The ELISA system was able to detect hIGF-I at a range of 1-25 micrograms/l, compared with the range of 1-50 micrograms/l detected by radioimmunoassay (RIA). Human IGF-II and human insulin could not be recognized in this system. The plasma concentrations of IGF-I found using the ELISA agreed well with those found using RIA after conventional Sep-Pak C18 cartridge pretreatment. Epitopes of hIGF-I to McAb and PcAb were investigated by enzymatic digestion of hIGF-I followed by comparing the affinity of the antibodies to the peptides obtained proteolytically. The epitope to McAb was found to be a peptide containing Leu10-Val11-Asp12 (epitope 2). Five epitopes to PcAb containing the following key fragments were identified: a conformational structure formed by the disulphide bonds between Cys6 and Cys48, and between Cys47 and Cys52 (epitope 1), Leu10-Val11-Asp12 (epitope 2), Val17-Cys18-Gly19-Asp20 (epitope 3), Arg21-Gly22-Phe23-Tyr24 (epitope 4) and Lys68-Ser69-Ala70 (epitope 5). Of these, the peptide containing epitope 5 showed the highest affinity to PcAb. The results indicated that our ELISA system combined recognition by epitope 2 of McAb and recognition by epitope 5 of PcAb to obtain its good specificity.

  18. Antithyroglobulin antibody

    MedlinePlus

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  19. Antibody levels correlate with detection of Trypanosoma cruzi DNA by sensitive PCR assays in seropositive blood donors and possible resolution of infection over time

    PubMed Central

    Sabino, E.C.; Lee, T.H.; Montalvo, L.; Nguyen, M.L.; Leiby, D.A.; Carrick, D.M.; Otani, M.M.; Vinelli, E.; Wright, D.; Stramer, S.L.; Busch, M.

    2013-01-01

    Background The clinical significance of anti-T. cruzi low-level reactive samples is incompletely understood. PCR-positive rates and antibody levels among seropositive blood donors in three countries are described. Methods Follow-up whole blood and plasma samples were collected from T. cruzi-seropositive donors from 2008-2010 in the US (n=195) and Honduras (n=58). Also 143 samples from Brazil in 1996-2002, originally positive by three serological assays, were available and paired with contemporary follow-up samples from these donors. All samples were retested with the FDA-approved Ortho ELISA. PCR assays were performed on coded sample panels by two laboratories (BSRI and ARC) that amplified kinetoplast minicircle DNA sequences of T. cruzi. Results PCR testing at BSRI yielded slightly higher overall sensitivity and specificity (33% and 98%) compared with the ARC lab (28% and 94%). Among seropositive donors, PCR-positive rates varied by country (p<0.0001) for the BSRI laboratory: Brazil (57%), Honduras (32%) and the US (14%). ELISA signal/cutoff (S/CO) ratios were significantly higher for PCR-positive compared to PCR-negative donors (p<0.05 for all comparisons). Additionally, PCR-negative Brazilian donors exhibited greater frequencies of antibody decline over time versus PCR-positive donors (p=0.003). Conclusion For all three countries, persistent DNA positivity correlated with higher ELISA S/CO values, suggesting that high-level seroreactivity reflects chronic parasitemia. The higher rate of PCR positivity for Brazilian donors was likely attributable to required reactivity on three assays available a decade ago. Significant S/CO declines in 10% of the PCR-negative Brazilian donors may indicate seroreversion following parasite clearance in the absence of treatment. PMID:23002996

  20. Detection of Salmonella enterica serovar Enteritidis (SE) Antibodies in Serum Using A Polystyrene Bead/SE Flagella Agglutination Assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serologic screening of flocks can be an important method to detect Salmonella enteritidis (SE) infections but can be labor intensive or lack specificity. Our goal was to develop a rapid agglutination assay using SE flagella adsorbed to polystyrene beads as a simple, relatively specific test to dete...

  1. Detection of Lassa virus antinucleoprotein immunoglobulin G (IgG) and IgM antibodies by a simple recombinant immunoblot assay for field use.

    PubMed

    Ter Meulen, J; Koulemou, K; Wittekindt, T; Windisch, K; Strigl, S; Conde, S; Schmitz, H

    1998-11-01

    The nucleoprotein of Lassa virus, strain Josiah, was expressed in Escherichia coli as an N-terminally truncated, histidine-tagged recombinant protein. Following affinity purification the protein was completely denatured and spotted onto nitrocellulose membrane. A total of 1 microgram of protein was applied for detection of Lassa virus antibodies (LVA) in a simple immunoblot assay. Specific anti-Lassa immunoglobulin M (IgM) antibodies could be detected by increasing the amount of protein to 5 microgram. A panel of 913 serum specimens from regions in which Lassa virus was endemic and from regions in which Lassa virus was not endemic was used for evaluating the sensitivity and specificity of the LVA immunoblot in comparison to those of an indirect immunofluorescence (IIF) assay. The sera originated from field studies conducted in the Republic of Guinea (570 serum samples) and Liberia (99 serum samples), from inpatients of the clinical department of the Bernhard-Nocht-Institute, Hamburg, Germany (94 serum samples), and from healthy German blood donors (150 serum samples). In comparison to the IIF assay the LVA immunoblot assay had a specificity of 90.0 to 99.3%, depending on the origin of the specimens. The sensitivity was found to be highest for the Guinean samples (90.7%) and was lower for the Liberian samples (75%). Acute Lassa fever was diagnosed by PCR in 12 of 59 (20.3%) patients with fever of unknown origin (FUO) from the Republic of Guinea. On admission to the hospital, nine Lassa fever patients (75%) were reactive by the IgM immunoblot assay. One of the patients was infected with a new Lassa variant, which showed 10.4% variation on the amino acid level in comparison to the prototype strain of Lassa virus, Josiah. Seven PCR-negative patients were reactive by immunoblotting. The positive and negative predictive values of a single IgM immunoblot result for acute, PCR-confirmed Lassa fever were therefore 53.6 and 93.0%, respectively. Because of its high negative

  2. Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins.

    PubMed

    Hu, Jenny; Wala, Iwona; Han, Hong; Nagatani, Janice; Barger, Troy; Civoli, Francesca; Kaliyaperumal, Arunan; Zhuang, Yao; Gupta, Shalini

    2015-04-01

    Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs.

  3. Animal models and antibody assays for evaluating candidate SARS vaccines: summary of a technical meeting 25-26 August 2005, London, UK.

    PubMed

    Roberts, Anjeanette; Wood, John; Subbarao, Kanta; Ferguson, Morag; Wood, David; Cherian, Thomas

    2006-11-30

    Severe acute respiratory syndrome (SARS) emerged in the Guangdong province of China in late 2002 and spread to 29 countries. By the end of the outbreak in July 2003, the CDC and WHO reported 8437 cases with a 9.6% case fatality rate. The disease was caused by a previously unrecognized coronavirus, SARS-CoV. Drawing on experience with animal coronavirus vaccines, several vaccine candidates have been developed and evaluated in pre-clinical trials. Available data suggest that vaccines should be based on the the 180kDa viral spike protein, S, the only significant neutralization antigen capable of inducing protective immune responses in animals. In the absence of clinical cases of SARS, candidate vaccines should be evaluated for efficacy in animal models, and although it is uncertain whether the United States Food and Drug Administration's "animal rule" would apply to licensure of a SARS vaccine, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. This report summarizes the recommendations from a WHO Technical Meeting on Animal Models and Antibody Assays for Evaluating Candidate SARS Vaccines held on 25-26 August 2005 in South Mimms, UK, provides guidance on the use of animal models, and outlines the steps to develop standard reagents and assays for immunological evaluation of candidate SARS vaccines.

  4. Detection of a low-molecular-weight antigen on melanoma cells by a human antiserum in leukocyte-dependent antibody assays.

    PubMed

    Hersey, P; Murray, E; Werkmeister, J; McCarthy, W H

    1979-10-01

    Biochemical characterization of serologically detected human melanoma antigens was undertaken for the development of immunodiagnostic assays in melanoma. An antiserum from a human melanoma patient, which detected melanoma antigens expressed on a large proportion of different melanoma cells, was used in leucocyte-dependent cytotoxic antibody (LDA) 51Cr-release assays to monitor the purification of melanoma antigens in urea/acetate extracts of lactoperoxidase 125I-labelled melanoma cell membranes. The separation procedures included affinity chromatography on Concanavalin A, gel filtration on porous polyacrylamide beads and preparative isoelectric focusing. The fractions were also monitored by polyacrylamide electrophoresis in sodium dodecyl sulphate and by measurement of beta 2 microglobulin and carcinoembryonic antigen content. The antigens detected by this antiserum appeared to be acidic (pI 3.5) low-mol.-wt glycoproteins of approximately 15,000 daltons which were resistant to heating at 56 degrees C and digestion with neuraminidase, but susceptible to repeated freeze-thawing and trypsin digestion. They did not appear to be related to HLA antigens, beta 2 microglobulin or known foetal antigens. The nature of the antigens detected in these studies is as yet unknown, but they appear similar to those described in the sera and urine of melanoma patients in previous reports. Thes combined results and the frequent expression of these antigens on melanoma cells from different patients suggest that assays to detect this antigen may provide a valuable immunodiagnostic aid in the management of melanoma.

  5. Driving efficiency in a high-throughput metabolic stability assay through a generic high-resolution accurate mass method and automated data mining.

    PubMed

    Shui, Wenqing; Lin, Song; Zhang, Allen; Chen, Yan; Huang, Yingying; Sanders, Mark

    2011-08-01

    Improving analytical throughput is the focus of many quantitative workflows being developed for early drug discovery. For drug candidate screening, it is common practice to use ultra-high performance liquid chromatography (U-HPLC) coupled with triple quadrupole mass spectrometry. This approach certainly results in short analytical run time; however, in assessing the true throughput, all aspects of the workflow needs to be considered, including instrument optimization and the necessity to re-run samples when information is missed. Here we describe a high-throughput metabolic stability assay with a simplified instrument set-up which significantly improves the overall assay efficiency. In addition, as the data is acquired in a non-biased manner, high information content of both the parent compound and metabolites is gathered at the same time to facilitate the decision of which compounds to proceed through the drug discovery pipeline.

  6. Enzyme-linked immunosorbent assay for the detection of canine Leptospira antibodies using recombinant OmpL1 protein.

    PubMed

    Okuda, Masaru; Sakai, Yoshiko; Matsuuchi, Megumi; Oikawa, Tatsuo; Watanabe, Malaika; Itamoto, Kazuhito; Iwata, Hiroyuki; Kano, Rui; Hasegawa, Atsuhiko; Onishi, Takafumi; Inokuma, Hisashi

    2005-03-01

    OmpL1 is a 31-kDa outer membrane protein characterized in 1993 and known to be expressed only in pathogenic Leptospira spp. Recombinant OmpL1 (GST-rOmpL1) was expressed for use as an ELISA antigen for the detection of anti-Leptospira antibodies. In immunoblot analysis, the protein reacted with sera of dogs infected with three different serotypes of Leptospira interrogans, while did not react with sera of dogs both uninfected negative controls and infected with Borrelia burgdorferi, which is closely related to Leptospira spp. Moreover, in ELISA using GST-rOmpL1, the optical density (O.D.) values from the positive controls were very high (1.125 +/- 0.549). In contrast, the O.D. values from clinically healthy dogs and dogs with diseases other than leptospirosis were very low (0.109 +/- 0.046 and 0.089 +/- 0.046, respectively). These data suggest that the detection of anti-Leptospira antibodies by ELISA using the GST-rOmpL1 protein can be applied for diagnosis of canine leptospirosis.

  7. An antibody induction method in mice for the potency assays of diphtheria and tetanus components in combined vaccines.

    PubMed

    Maheshwari, S C; Sharma, S B; Kumar, A; Sokhey, J

    1998-09-01

    Eleven batches of Adsorbed Diphtheria-Tetanus (DT) vaccines and thirteen batches of Adsorbed Diphtheria-Pertussis-Tetanus (DTP) vaccines were tested for the potency of diphtheria and tetanus components by an Antibody Induction Method (AIM) developed in mice. The potency results obtained were found comparable and did not show any statistically significant difference with those obtained by WHO recommended lethal challenge tests for diphtheria in guinea pigs and for tetanus in mice. AIM in mice is more economical as both diphtheria and tetanus components of combined vaccine can be tested in the same experiment and the procedure also eliminates the use of guinea pigs required in the lethal challenge/conventional tests. The data obtained while testing tetanus component by the conventional antibody induction (IP) method in guinea pigs suggests that minimum requirements laid down in i.p. is too low which may be fixed as at least 3 out of 9 guinea pig sera and should contain > or = 4 units of tetanus antitoxin per ml.

  8. Sensitive and accurate identification of protein–DNA binding events in ChIP-chip assays using higher order derivative analysis

    PubMed Central

    Barrett, Christian L.; Cho, Byung-Kwan

    2011-01-01

    Immuno-precipitation of protein–DNA complexes followed by microarray hybridization is a powerful and cost-effective technology for discovering protein–DNA binding events at the genome scale. It is still an unresolved challenge to comprehensively, accurately and sensitively extract binding event information from the produced data. We have developed a novel strategy composed of an information-preserving signal-smoothing procedure, higher order derivative analysis and application of the principle of maximum entropy to address this challenge. Importantly, our method does not require any input parameters to be specified by the user. Using genome-scale binding data of two Escherichia coli global transcription regulators for which a relatively large number of experimentally supported sites are known, we show that ∼90% of known sites were resolved to within four probes, or ∼88 bp. Over half of the sites were resolved to within two probes, or ∼38 bp. Furthermore, we demonstrate that our strategy delivers significant quantitative and qualitative performance gains over available methods. Such accurate and sensitive binding site resolution has important consequences for accurately reconstructing transcriptional regulatory networks, for motif discovery, for furthering our understanding of local and non-local factors in protein–DNA interactions and for extending the usefulness horizon of the ChIP-chip platform. PMID:21051353

  9. Seroprevalence of antibodies to Salmonella spp in semidomesticated reindeer in Norway, determined by enzyme-linked immunosorbent assay.

    PubMed

    Aschfalk, Ansgar; Laude, Sabine; Denzin, Nicolai

    2002-01-01

    An indirect ELISA was developed as a possible tool to detect the seroprevalence of antibodies to Salmonella spp in semidomesticated reindeer. To cover a broad spectrum of serogroups a lipopolysaccharide mix of S. typhimurium and S. choleraesuis was used as antigen in this pilot study. Sera from 31 culture-negative reindeer with no clinical or historical evidence of salmonellosis were used as negative serum control. After immunisation with an inactivated S. typhimurium vaccine, pooled sera from 6 reindeer were used as positive serum control as no serum from naturally infected animals was available. A seroprevalence of 0.6% in 2000 clinically healthy, slaughter-reindeer from Norway was determined by using this ELISA. No more information on Salmonella in reindeer in Norway is known to the authors. This is the first ELISA established for indirect detection of Salmonella in reindeer.

  10. Evaluation of modified Ziehl-Neelsen, direct fluorescent-antibody and PCR assay for detection of Cryptosporidium spp. in children faecal specimens.

    PubMed

    Aghamolaie, S; Rostami, A; Fallahi, Sh; Tahvildar Biderouni, F; Haghighi, A; Salehi, N

    2016-09-01

    To determine the sensitivity and specificity of routine screening methods for cryptosporidiosis, three methods including conventional modified Ziehl-Neelsen (MZN), direct fluorescent-antibody (DFA) and Nested-PCR assay compared together. To this end, their ability to identify the low concentrations of Cryptosporidium spp. oocysts in children fecal samples was evaluated. The sample population of this study was children under 12 years old who had diarrhea and referred to pediatric hospitals in Tehran, Iran. 2,510 stool specimens from patients with diarrhea were screened for Cryptosporidium oocysts by concentration method and MZN. To determine sensitivity and specificity, Nested-PCR and DFA were performed on 30 positive and 114 negative samples which previously had been proved by MZN. By using the microscopic method, DFA assay and PCR analysis, a total of 30 (1.2 %), 28 (1.1 %) and 32 (1.27 %) positive samples were detected respectively. According to the results, the sensitivity, specificity, and positive and negative predictive values of the Nested-PCR assay were 100 %, compared to 94, 100, 100, and 98 %, respectively, for MZN and 87.5, 100, 100, and 96 %, respectively, for DFA. Results of the present study showed that the Nested-PCR assay was more sensitive than the other two methods and laboratories can use the Nested-PCR method for precise diagnosis of Cryptosporidium spp. However, regarding the costs of Nested-PCR and its unavailability in all laboratories and hospitals, MZN staining on smears has also enough accuracy for Cryptosporidium diagnosis.

  11. Rapid determination of members of the family Enterobacteriaceae in drinking water by an immunological assay using a monoclonal antibody against enterobacterial common antigen.

    PubMed Central

    Hübner, I; Steinmetz, I; Obst, U; Giebel, D; Bitter-Suermann, D

    1992-01-01

    An immunological method for the detection of members of the family Enterobacteriaceae in drinking water was developed. The method was based on a sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody immunoglobulin G2a 898 against enterobacterial common antigen. The enterobacterial common antigen sandwich ELISA combined with selective preenrichment culture could be performed in only 24 h. Six hundred sixty-eight water samples from a variety of German public water supplies were screened to verify the effectiveness of the new method. Ninety-eight percent of the results obtained by the immunological method could be confirmed by conventional microbiological methods. The immunological method proved to be considerably faster and more specific and sensitive than the standard method specified by the German drinking water regulations. PMID:1444435

  12. Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.

    PubMed

    Pierce, Lindsey R; Willey, James C; Palsule, Vrushalee V; Yeo, Jiyoun; Shepherd, Brian S; Crawford, Erin L; Stepien, Carol A

    2013-01-01

    Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R(2) = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/10(6) actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.

  13. Accurate Detection and Quantification of the Fish Viral Hemorrhagic Septicemia virus (VHSv) with a Two-Color Fluorometric Real-Time PCR Assay

    PubMed Central

    Palsule, Vrushalee V.; Yeo, Jiyoun; Shepherd, Brian S.; Crawford, Erin L.; Stepien, Carol A.

    2013-01-01

    Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain – IVb – appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R2 = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/106 actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics. PMID:23977162

  14. Epidemiological, biological and histological characterization of patients with indeterminate third-generation recombinant immunoblot assay antibody results for hepatitis C virus.

    PubMed

    Ríos, M; Diago, M; Rivera, P; Tuset, C; Cors, R; García, V; Carbonel, P; Gonzalez, C

    2006-03-01

    We studied the epidemiological, laboratory and histological characteristics of a group of patients with positive antibodies against hepatitis C virus (HCV) as determined by third-generation enzyme-linked immunosorbent assay (ELISA), and with indeterminate HCV antibody positivity as established by third-generation recombinant immunoblot assay (RIBA-3). The results obtained were compared with those recorded in a group of RIBA-3-positive patients. Both groups correspond to blood donors in whom the prevalence of hepatitis C is low. There were no statistically significant intergroup differences in mean age, or in the presence of infection risk factors. RNA positivity was much more frequent in the RIBA-positive group (71%vs 10%; P < 0.05), as was transaminase elevation during the 3 years of follow-up (54%vs 13%; P < 0.05). In 46% of the RIBA-indeterminate patients the liver biopsy proved normal, or only liver steatosis or minimal changes were detected, while 33% had persistent chronic hepatitis, and 21% showed active chronic hepatitis. A mean Knodell index score of 2.28 was recorded; 50% of the subjects showed no fibrosis, 46% grade 1 fibrosis (fibrous portal expansion), 4% grade 2 fibrosis (bridging fibrosis), and none grade 3 fibrosis (liver cirrhosis). In the RIBA-positive group, a greater percentage of patients had active chronic hepatitis, a greater Knodell index, and increased-grade fibrosis. It can be concluded that the RIBA-3-indeterminate group is epidemiologically similar to the RIBA-3-positive series, although with a lesser prevalence of laboratory test alterations, a lower viral replication index, and more likely to have benign disease - particularly in subjects without viral replication.

  15. Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

    PubMed Central

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-01-01

    Background Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. Methods In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. Results The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). Conclusions These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:26731402

  16. Qualification and application of a surface plasmon resonance-based assay for monitoring potential HAHA responses induced after passive administration of a humanized anti Lewis-Y antibody.

    PubMed

    Szolar, O H J; Stranner, S; Zinoecker, I; Mudde, G C; Himmler, G; Waxenecker, G; Nechansky, A

    2006-06-16

    A sensitive, surface plasmon resonance (SPR)-based assay monitoring potential human-anti-human antibody (HAHA) reactions against the monoclonal antibody (mAb) IGN311 is presented. The latter is a fully humanized Lewis-Y carbohydrate specific mAb that is currently tested in a passive immune therapy approach in a clinical phase I trial. For the SPR experiments a BIACORE 3000 analyzer was used. The ligand IGN311 was covalently coupled to the carboxy-methylated dextran matrix of a CM5 research grade chip (BIACORE). In the course of a fully nested experimental design, a four parameter logistic equation was identified as appropriate calibration model ranging from 0.3 microg/mL (lower limit of quantitation, LLOQ) to 200 microg/mL (upper limit of quantitation, ULOQ) using an anti-idiotypic mAb ('HAHA mimic') as calibrator. The bias ranged from -2.4% to 5.5% and the intermediate precision expressed as 95% CI revealed values from 5.6% to 8.3%. Specificity was evaluated using six human serum matrices from healthy donors spiked with calibrator at the limit of quantitation (LOQ) with >80% of values being recovered with less than 25% relative error. The qualified assay was applied to monitor potentially induced HAHA reactivity in 11 patients from a clinical phase I trial with passively administered IGN311. Of the 11 patients, one high HAHA responder and several low responders were identified. Protein-G depletion experiments with human serum samples revealed that the observed response is predominantly caused by IgG binding to the ligand. The characteristics of these HAHA responses were all of the so-called 'Type I' which is defined by a peak response around day 15 that decreases from this point steadily suggesting that some kind of tolerance is established. Therefore, this type of HAHA response is regarded as non critical for the patient's safety.

  17. Development and validation of an indirect Enzyme-linked immunosorbent assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants.

    PubMed

    van der Heijden, H M J F; Bouwstra, R J; Mars, M H; van der Poel, W H M; Wellenberg, G J; van Maanen, C

    2013-10-01

    To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood samples was developed. Schmallenberg virus antigen was produced by propagation on Vero cells, partly purified and coated onto ELISA plates. The indirect ELISA procedure included the subsequent incubation of diluted samples, protein-G-HRP conjugate and TMB substrate solution. Net Optical Densities (OD) values were calculated and expressed as a sample to positive percentage (S/P%) by comparison of the average net OD with the OD of the positive control. Validation of this assay was performed using 633 samples from SBV-free sheep, goats and cattle, and 141 samples from SBV suspect ruminants. The diagnostic specificity was 98.8%. Test results of 86 ruminant serum samples using both the SBV-ELISA and an SBV virus neutralization test (VNT), designated as the gold standard serological test for SBV, showed good correlation: at an S/P cut-off of 15% only one VNT positive sample tested negative in the SBV ELISA. The diagnostic sensitivity of the ELISA, relative to the VNT, was 98.8% (95% CI: 93.3-100.0%). The ELISA showed a high repeatability (cv=6.5%) and reproducibility (100% agreement). It was concluded that this ELISA is a suitable test method for the detection of SBV antibodies in sera from cows, sheep and, possibly, goats.

  18. Analysis and solution of false-positives when testing CVA16 sera using an antibody assay against the EV71 virus.

    PubMed

    Wang, Changbing; You, Aiping; Tian, Xingui; Zhao, Mingqi; Chen, Yi; Lin, Tao; Zheng, Jianbin; Xiao, Misi; Zhang, Yingying; Kuang, Lu; Zhou, Zhenwen; Zhu, Bing

    2013-09-01

    Hand, foot and mouth disease (HFMD) in humans is caused mainly by Enterovirus 71(EV71) and Coxsackievirus A16 (CVA16). EV71 is associated with severe HFMD cases but not CVA16. Use of IgM-capture enzyme-linked immunosorbent assay (ELISA) is important for the early diagnosis of EV71 infection, but cross-reactivity of the anti-CVA16 IgM antibody with EV71 produces false-positive results. In this report, we designed a new EV71 IgM-capture ELISA method using the EV71 VP1 peptide instead of the EV71 virion as the detectable antigen, and tested sera from patients infected with EV71 or CVA16. The results showed that acute sera from 76 EV71-infected patients had similar sensitivity for virus detection (98.68%) or VP1 detection (97.37%). When acute sera from patients infected with CVA16 were used, significant differences between the two methods were observed. The cross-reactivity rate of the virus detection method was 29.4% (5/17), but no cross-reactivity was observed using the VP1 detection method. Western immunoblotting demonstrated that EV71 VP3 cross-reacted with part of the CVA16 IgM antibody. The results demonstrate that EV71 VP3 is the cross-reactive antigen in the EV71 IgM-capture ELISA when testing CVA16 sera using the virus-antibody detection method. The problem of false-positive results was resolved by using the VP1 peptide as the detectable antigen.

  19. Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red.

    PubMed

    Ju, Chunmei; Tang, Yong; Fan, Huiying; Chen, Jinding

    2008-07-28

    To set up an immunoassay-based method to detect Sudan dyes and Para red, we generated a monoclonal antibody (Mab) using a specially designed carboxyl derivative of Sudan I (CSD I) as the immunogen. CSD I was synthesized by azocoupling reaction using 2-naphthol and diazotised 4-aminobenzoic acid. The antibody was obtained from a hybridoma, which was derived from the fusion of the mouse myeloma SP2/0 cells and the splenocytes from the mice immunized with the CSD I-bovine serum albumin (BSA) conjugate. In addition, we showed that the Mab was highly specific for Sudan I, III and Para red. The limit of detection was approximately 0.01ngmL(-1) in phosphate-buffered saline (PBS) buffer and 0.5ngg(-1) in chilli tomato sauce. The recoveries of Sudan I, III and Para red for the chilli tomato sauce were from 84% to 99% and coefficients of variation were from 14.9% to 33.3%. Thus, the enzyme-linked immunosorbent assay (ELISA) method is a rapid and high throughput screening tool to detect Sudan dyes and Para red in food products.

  20. Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay.

    PubMed

    Malgor, R; Nonaka, N; Basmadjian, I; Sakai, H; Carámbula, B; Oku, Y; Carmona, C; Kamiya, M

    1997-12-01

    A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotinylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67,700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, showing a sensitivity of 100% and a specificity of 96%.

  1. Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column.

    PubMed Central

    Park, E M; Shigenaga, M K; Degan, P; Korn, T S; Kitzler, J W; Wehr, C M; Kolachana, P; Ames, B N

    1992-01-01

    An immunoaffinity column is described that facilitates the analysis of oxidative damage products of DNA and RNA in urine, blood plasma, and medium isolated from cultures of Escherichia coli. In intact animals, lesions (adducts) excised from DNA are transported from the cell through the circulation and excreted in urine. In bacteria, DNA adducts are excreted directly into the medium. In either case, the adducts can be assayed as a measure of oxidative damage to DNA. A monoclonal antibody that recognizes 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG;8-hydroxy-2'-deoxyguanosine), a bio-marker of oxidative damage to DNA, has been isolated, and its substrate binding properties have been characterized. The relative binding affinities of this monoclonal antibody for oxo8dG, unmodified nucleosides, or derivatives of Gua made it suitable for the preparation of immunoaffinity columns that greatly facilitate the isolation of oxo8dG, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroguanosine from various biological fluids. Quantitative analysis of these adducts in urine of rats fed a nucleic acid-free diet and in the medium from cultures of E. coli suggests that oxo8-7,8-dihydroguanine is the principal repair product from oxo8-dG in DNA of both eukaryotes and prokaryotes. The results support our previous estimate of about 10(5) oxidative lesions to DNA being formed and excised in an average rat cell per day. PMID:1565629

  2. Coxsackievirus B1-based antibody-capture enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG), IgM, and IgA with broad specificity for enteroviruses.

    PubMed Central

    Swanink, C M; Veenstra, L; Poort, Y A; Kaan, J A; Galama, J M

    1993-01-01

    An antibody-capture enzyme-linked immunosorbent assay (ELISA) with coxsackievirus B1 as the antigen was evaluated for detection of immunoglobulin G (IgG), IgM, and IgA antibodies and showed broad specificity for enteroviruses. In total, 116 serum or cerebrospinal fluid samples from 62 patients were tested by ELISA and the complement fixation test (CFT). Additionally, 15 serum samples that contained poliovirus-specific IgM antibody were tested. Serum samples from 200 healthy blood donors were used for standardization of the assays. The sensitivity of the ELISA varied with time of serum sampling, with a relatively low sensitivity when serum was collected within 3 days after the onset of symptoms (23%; 5 of 22) but good sensitivity when serum was collected later (83%; 20 of 24). The sensitivity was better than that of the CFT. The ELISAs were broadly reactive as concluded from typing of virus isolates that were simultaneously obtained. The assay did, furthermore, detect antibody against poliovirus type 3. Sera that contained rheumatoid factor, antinuclear antibody, or cardiolipin antibody (by the Venereal Disease Research Laboratory test) did not react in this ELISA. Nonspecific reactivity did occur, however, in cases of infectious mononucleosis and in Mycoplasma pneumoniae infection. The enterovirus-specific ELISA is found to be simple to perform, more sensitive than the CFT, and far less laborious than the neutralization test. PMID:8308117

  3. Identification of specific antinuclear antibodies in dogs using a line immunoassay and enzyme-linked immunosorbent assay.

    PubMed

    Bremer, Hanna D; Lattwein, Erik; Renneker, Stefanie; Lilliehöök, Inger; Rönnelid, Johan; Hansson-Hamlin, Helene

    2015-12-15

    Circulating antinuclear antibodies (ANA) are commonly present in the systemic autoimmune disease Systemic Lupus Erythematosus (SLE) and in other systemic rheumatic diseases, in humans as well as in dogs. The indirect immunofluorescence (IIF)-ANA test is the standard method for detecting ANA. Further testing for specific ANA with immunoblot techniques or ELISAs is routinely performed in humans to aid in the diagnosis and monitoring of disease. Several specific ANA identified in humans have been identified also in suspected canine SLE but, in contrast to humans, investigation of autoantibodies in canine SLE is mainly restricted to the IIF-ANA test. Our aim was to identify both known and novel specific ANA in dogs and to investigate if different IIF-ANA patterns are associated with different specific ANA in dogs. Sera from 240 dogs with suspicion of autoimmune disease (210 IIF-ANA positive (ANA(pos)) and 30 IIF-ANA negative (ANA(neg))) as well as sera from 27 healthy controls were included. The samples were analysed with a line immunoassay, LIA (Euroline ANA Profile 5, Euroimmun, Lübeck, Germany) and four different ELISAs (Euroimmun). The ANA(pos) dogs were divided in two groups depending on the type of IIF-ANA pattern. Of the 210 ANA(pos) samples 68 were classified as ANA homogenous (ANA(H)) and 141 as ANA speckled (ANA(S)), one sample was not possible to classify. Dogs in the ANA(H) group had, compared to the other groups, most frequently high levels of anti-double stranded deoxyribonucleic acid (dsDNA) and anti-nucleosome ANA. Anti-dsDNA antibodies were confirmed in some dogs with the Crithidia luciliae indirect immunofluorescence test (CLIFT). The frequency of ANA(H) dogs with values above those observed in the healthy group was significantly higher compared to ANA(S) dogs for anti-dsDNA, anti-nucleosome, and anti-histone reactivity. Dogs in the ANA(S) group had, compared to the other groups, most frequently high levels of anti-ribonucleoproteins (RNP) and

  4. Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay.

    PubMed

    Zhang, Xian; Sun, Mengjiao; Kang, Yue; Xie, Hui; Wang, Xin; Song, Houhui; Li, Xiaoliang; Fang, Weihuan

    2015-11-01

    Ochratoxin A (OTA) is one of the most commonly occurring mycotoxins produced by some species of Aspergillus and can contaminate cereal and cereal products. A high-affinity anti-OTA monoclonal antibody (mAb) was generated from a hybridoma cell line 2D8 using splenocytes from a BALB/c mouse immunized with synthesized OTA-bovine serum albumin conjugate. The mAb 2D8 is specific with high affinity (3.75 × 10(9) L/M). An indirect competitive ELISA (ic-ELISA) was then developed using this mAb for quantitative determination of OTA in corn and feed samples. Using the optimized conditions, there was good linearity between OTA concentration and competitive inhibition (y = -0.6076x + 0.2441, R(2) = 0.9923) with the working range from 2.4 to 23.6 μg/kg, IC50 at 7.6 μg/kg and lower limit of detection at 1.4 μg/kg. The recovery rates in spiked samples were 91.2-110.3%. Of the 56 corn and feed samples, this ic-ELISA and a commercial kit both found the same 13 samples positive for OTA with good linear correlation between the two methods in OTA quantification (R(2) = 0.9706). We conclude that this ic-ELISA can be used for rapid and quantitative screening of corn and feed samples for the presence of OTA.

  5. An inhibition enzyme immuno assay exploring recombinant invariant surface glycoprotein and monoclonal antibodies for surveillance of surra in animals.

    PubMed

    Rudramurthy, G R; Sengupta, P P; Ligi, M; Rahman, H

    2017-02-20

    The present study is aimed at the development of inhibition ELISA (I-ELISA) exploring monoclonal antibodies (MAbs) and recombinant invariant surface glycoprotein. The extracellular domain (ED) of invariant surface glycoprotein (ISG-75) from Trypanosoma evasni has been heterologously expressed in Pichia pastoris (X-33). The recombinant ISG-75 (rISG-75ED) was characterized by immunoblot and ELISA, followed by the production of MAbs against rISG-75ED. The MAbs were characterized by immunoblot and then explored in the development of I-ELISA for the detection of surra. The diagnostic potential of the developed test has been evaluated using 1192 field sera sample including cattle, buffalo, donkey, horse and camel. The statistical analysis of the data showed optimum combination of diagnostic sensitivity and specificity at 98.8% and 99.2% respectively, with cut-off percentage inhibition (PI) value of >45. The Cohen's kappa coefficient of agreement was found to be 0.98. Hence, the diagnostic test developed in the present study can be exploited as a potential and reliable tool in the serodiagnosis and surveillance of surra in animals.

  6. Highly sensitive and reproducible immunoradiometric assay for total human renin using monoclonal antibodies, iodogen labelling and polystyrene star tubes

    SciTech Connect

    Nielsen, M.D.; Rasumussen, P.H.; Giese, J.

    1987-01-01

    A two-site immunoradiometric assay for total renin concentration in human plasma is described. A new type of polystyrene tubes with a special bottom geometry, so-called Star Tubes were used. The lower limit of detection was 2 microIU per ml and the working range from 2 to 2000 microIU per ml. The variation for duplicate determinations on standards and plasma samples was 2.7% and the day to day variation was 4.8%. No significant interferences or cross reactivities were identified. EDTA-plasma was analyzed after dilution with a phosphate buffer. Plasma samples from different patient categories were analyzed. The results were compared with those obtained by our substrate enrichment method after acid activation of the samples had taken place. A linear correlation (r = 0.990) between the results obtained with the two methods was found.

  7. Single-Cell Analysis of B Cell/Antibody Cross-Reactivity Using a Novel Multicolor FluoroSpot Assay.

    PubMed

    Hadjilaou, Alexandros; Green, Angela M; Coloma, Josefina; Harris, Eva

    2015-10-01

    Dengue is a major public health problem globally. It is caused by four antigenically distinct serotypes of dengue virus (DENV1-4), and although serotype-specific and strongly neutralizing cross-reactive immune responses against the four DENV serotypes are thought to be protective, subneutralizing Abs can contribute to increased disease severity upon secondary infection with a different DENV serotype. Understanding the breadth of the immune response in natural DENV infections and in vaccinees is crucial for determining the correlates of protection or disease severity. Transformation of B cell populations to generate mAbs and ELISPOT assays have been used to determine B cell and Ab specificity to DENV; however, both methods have technical limitations. We therefore modified the conventional ELISPOT to develop a Quad-Color FluoroSpot to provide a means of examining B cell/Ab serotype specificity and cross-reactivity on a single-cell basis. Abs secreted by B cells are captured by an Fc-specific Ab on a filter plate. Subsequently, standardized concentrations of all four DENV serotypes are added to allow equal stoichiometry for Ag binding. After washing, the spots, representing individual B cells, are visualized using four fluorescently labeled DENV serotype-specific detection mAbs. This method can be used to better understand the breadth and magnitude of B cell responses following primary and secondary DENV infection or vaccination and their role as immune correlates of protection from subsequent DENV infections. Furthermore, the Quad-Color FluoroSpot assay can be applied to other diseases caused by multiple pathogen serotypes in which determining the serotype or subtype-specific B cell response is important.

  8. TSH receptor antibody titers measured with a third-generation assay did not reflect the activity of Graves' ophthalmopathy in untreated Japanese Graves' disease patients.

    PubMed

    Mukasa, Koji; Yoshimura Noh, Jaeduk; Kouzaki, Ai; Ohye, Hidemi; Kunii, Yo; Watanabe, Natsuko; Yoshihara, Ai; Matsumoto, Masako; Suzuki, Miho; Ito, Koichi

    2016-01-01

    TSH receptor antibody (TRAb) titer has been reported to be correlated with Graves' ophthalmopathy (GO). However, the correlation between GO activity and TRAb titer assessed with a third-generation assay has not been reported. We enrolled 238 untreated Graves' disease patients who came to the outpatient clinic of Ito Hospital and 28 patients who were euthyroid. All of the patients were assessed for GO by an ophthalmologist within 3 months of their initial visit to Ito Hospital. Clinical activity score (CAS), short inversion time inversion recovery (STIR), and sum of the maximum external orbital muscle areas (SEOMA) on a frontal sectional magnetic resonance imaging (MRI). The TRAb titer was significantly higher in patients with inactive ophthalmopathy (the inactive-GO group) than in patients with active ophthalmopathy (the active-GO group) (17.7 ± 13.5 IU/L vs. 13.0 ± 13.1 IU/L, p=0.0082). The SEOMA values were not correlated with TRAb titer. The prevalence of active-GO was higher in euthyroid patients than in hyperthyroid patients although the difference was not significant. In conclusion, TRAb titer measured with a third-generation assay dose not correlate with GO activity based on MRI findings in untreated Graves' disease patients, and the prevalence of active-GO is higher in euthyroid patients with lower TRAb titers than in hyperthyroid patients.

  9. Comparative assessment of the leprosy antibody absorption test, Mycobacterium leprae extract enzyme-linked immunosorbent assay, and gelatin particle agglutination test for serodiagnosis of lepromatous leprosy.

    PubMed Central

    Escobar-Gutiérrez, A; Amezcua, M E; Pastén, S; Pallares, F; Cázares, J V; Pulido, R M; Flores, O; Castro, E; Rodríguez, O

    1993-01-01

    A comparative assessment of three serological methods for leprosy diagnosis (the fluorescent leprosy antibody absorption [FLA-ABS] test, the Mycobacterium leprae soluble-extract enzyme-linked immunosorbent assay [ELISA], and the M. leprae particle agglutination [MLPA] test) was carried out. The objective was to identify their performance in clinical and epidemiological diagnosis of leprosy. The study group included 45 lepromatous leprosy patients under treatment. Specificity was > 95% for all three assays, and sensitivity was 95, 58, and 74% for the FLA-ABS test, the MLPA test, and the ELISA, respectively. The only cross-reactivity for M. tuberculosis-infected patients was with the soluble-extract ELISA. Although the FLA-ABS test displayed the highest specificity and sensitivity values, it can only be used in well-developed laboratories, and the patient's clinical and epidemiological background must be considered when results are interpreted because the test remains positive after therapeutic success and could be positive for some household contacts. The MLPA test is easier to perform and interpret, and it is adequate for small laboratories and epidemiological studies intended to detect active untreated or irregularly treated leprosy cases. Therefore, the FLA-ABS and MLPA tests are complementary, and both should be used for serodiagnosis of leprosy. PMID:8501238

  10. Re-assessment of direct fluorescent antibody negative brain tissues with a real-time PCR assay to detect the presence of raccoon rabies virus RNA.

    PubMed

    Szanto, Annamaria G; Nadin-Davis, Susan A; Rosatte, Richard C; White, Bradley N

    2011-06-01

    The first report of the raccoon variant of rabies virus was in Ontario, Canada in 1999. As part of the control of this outbreak a Point Infection Control (PIC) strategy of trapping and euthanizing vector species was implemented. To evaluate whether this strategy was indeed removing diseased animals, rabies diagnosis was performed on these specimens. During a PIC program conducted in 2003, 721 animals (raccoons, striped skunks and red foxes) were collected and euthanized and brain material from each specimen was divided into two halves; one half was submitted for rabies diagnosis by a direct fluorescent antibody (DFA) test while the other was tested using a sensitive real-time reverse-transcriptase polymerase chain reaction (RT-qPCR), to detect raccoon rabies virus (RRV) RNA. This latter assay can detect less than ten viral copies in 200ng of total cellular RNA. All 721 PIC brain samples were negative by the DFA test but ten of them (5 raccoons, 5 skunks) tested positive for raccoon rabies virus by the RT-qPCR assay albeit at low levels. Three of these samples were confirmed by sequencing of the PCR products. Little correlation was observed between clinical rabies DFA positive scoring categories and viral copy number as determined by RT-qPCR.

  11. Diagnosis of amebic dysentery by detection of Entamoeba histolytica fecal antigen by an invasive strain-specific, monoclonal antibody-based enzyme-linked immunosorbent assay.

    PubMed Central

    Gonzalez-Ruiz, A; Haque, R; Rehman, T; Aguirre, A; Hall, A; Guhl, F; Warhurst, D C; Miles, M A

    1994-01-01

    An invasive strain-specific monoclonal antibody against Entamoeba histolytica has been used in a capture enzyme-linked immunosorbent assay (ELISA) for the detection of invasive E. histolytica fecal antigen in clinical specimens and for the diagnosis of amebic dysentery in patients from Bangladesh. The fecal antigen capture ELISA (FAC-ELISA) did not cross-react with other parasite species in the clinical specimens or with noninvasive E. histolytica present in those specimens and in experimentally seeded stools. The limit of detection of the assay for invasive E. histolytica crude antigen diluted in phosphate-buffered saline or in stools was 0.58 and 3.9 micrograms/ml, respectively, which is the equivalent of approximately 72 and 487 E. histolytica trophozoites per well, respectively. The sensitivity, specificity, and efficiency of the FAC-ELISA were 87, 100, and 98%, respectively, for the detection of invasive E. histolytica antigens and 100, 100, and 100%, respectively, for the diagnosis of amebic dysentery. The FAC-ELISA is a potential alternative for the field diagnosis of amebic dysentery and for epidemiological studies to define the distribution of invasive E. histolytica. PMID:8027351

  12. Detection of antibodies to hepatitis C virus (HCV) structural proteins in anti-HCV-positive sera by an enzyme-linked immunosorbent assay using synthetic peptides as antigens.

    PubMed Central

    Ishida, C; Matsumoto, K; Fukada, K; Matsushita, K; Shiraki, H; Maeda, Y

    1993-01-01

    We have defined 10 linear immunogenic regions encoded by the putative hepatitis C virus (HCV) structural proteins (core and envelope) by employing an enzyme-linked immunosorbent assay (ELISA) and by using 17 sequential synthetic peptides covering the N-terminal 330 amino acids of the structural polyproteins as antigens. These peptides correspond to amino acids 1 to 24, 21 to 44, 42 to 68, 64 to 91, and 100 to 120 of the putative core protein and amino acids 192 to 212, 223 to 238, 236 to 258, 250 to 266, and 307 to 330 of the putative envelope protein. In particular, the peptide covering amino acids 21 to 44 of the core protein was reactive with all but one (40 of 41) of the serum samples giving a positive signal in the passive hemagglutination assay (PHA) using the core and nonstructural proteins (NS 3/4) of the virus as antigens. We detected the HCV genome in 25 (61%) of 41 PHA-positive serum samples by the polymerase chain reaction (PCR) test. Of 25 PCR-positive serum samples, 17 serum samples had reactivity to the peptides derived from the envelope protein. On the other hand, only 1 of the 16 PCR-negative serum samples had reactivity to the peptides derived from the envelope protein. Interestingly, we often observed high serum alanine aminotransferase levels in PCR-positive individuals bearing antibodies to the envelope protein. PMID:7681849

  13. Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

    PubMed

    Halling, V W; Jones, M F; Bestrom, J E; Wold, A D; Rosenblatt, J E; Smith, T F; Cockerill, F R

    1999-10-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a

  14. Monoclonal antibody-based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors.

    PubMed

    Wu, Jianxiang; Ni, Yuequn; Liu, Huan; Ding, Ming; Zhou, Xueping

    2014-01-01

    Rice dwarf virus (RDV) causes Rice dwarf disease, which leads to considerable losses in rice production in Asia. Purified RDV virions were used as the immunogen to prepare monoclonal antibodies (mAbs). Three murine mAbs against RDV were prepared. Plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and immunocapture-RT-PCR (IC-RT-PCR) were then developed for sensitive, specific, and rapid detection of RDV in rice and leafhopper samples obtained in the field using the mAbs. The PTA-ELISA, dot-ELISA and IC-RT-PCR detected the virus in infected tissue crude extracts diluted at 1:81,920, 1:10,240 and 1:655,360 (w/v, g mL(-1)), in individual viruliferous rice green leafhopper crude extracts diluted at 1:25,600, 1:6400 and 1:3,276,800 (individual leafhopper/μL), respectively. 878 rice field samples and 531 leafhopper field samples from ten provinces of China were screened for the presence of RDV using the two serological assays and the IC-RT-PCR and the results indicated that 37 of the 878 rice samples and 22 of the 531 leafhopper samples were infected by RDV. All positive samples were from Yunnan Province, indicating that RDV is prevalent in this province, but not in the other nine provinces. The dot-ELISA is suitable for routine detection of large-scale rice and leafhopper samples in field surveys.

  15. Development and Evaluation of a Multiplex Microsphere Assay for Quantitation of IgG and IgA Antibodies against Neisseria meningitidis Serogroup A, C, W, and Y Polysaccharides

    PubMed Central

    Bårnes, Guro K.; Kristiansen, Paul A.; Caugant, Dominique A.

    2015-01-01

    We developed and evaluated a rapid and simple multiplex microsphere assay for the quantification of specific IgG and IgA antibodies against meningococcal serogroup A, C, W, and Y capsular polysaccharides in serum and saliva. Meningococcal polysaccharides were conjugated to distinct magnetic carboxylated microspheres, and the performance of the assay was assessed using the CDC1992 standard meningococcal reference serum and a panel of serum and saliva samples. The standard curve was linear over an eight 3-fold dilution range in the IgG assay and a seven 3-fold dilution range in the IgA assay. No cross-reactivity was discovered, and the assay showed high specificity with ≥91% homologous inhibition and ≤11% heterologous inhibition for all serogroups and immunoglobulin classes. Lower limits of detections were ≤280 pg/ml for IgG and ≤920 pg/ml for IgA antibodies. The assay was reproducible, with a mean coefficient of variation of ≤5% for intra-assay duplicates, a mean coefficient of variation of ≤20% for interassay repeated analysis with different conjugations of microspheres, and a mean coefficient of variation within 25.8% for interoperator variation. The assay showed good correlation to the standard meningococcal polysaccharide enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies. This multiplex assay is robust and reliable and requires less sample volume, and less time and workload are needed than for ELISA, making this method highly relevant for serological and salivary investigations on the effect of meningococcal vaccines and for immunosurveillance studies. PMID:25924767

  16. Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark®) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens

    PubMed Central

    Larson, Jeffrey S.; Goodman, Laurie J.; Tan, Yuping; Defazio-Eli, Lisa; Paquet, Agnes C.; Cook, Jennifer W.; Rivera, Amber; Frankson, Kristi; Bose, Jolly; Chen, Lili; Cheung, Judy; Shi, Yining; Irwin, Sarah; Kiss, Linda D. B.; Huang, Weidong; Utter, Shannon; Sherwood, Thomas; Bates, Michael; Weidler, Jodi; Parry, Gordon; Winslow, John; Petropoulos, Christos J.; Whitcomb, Jeannette M.

    2010-01-01

    We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7–10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH). PMID:21151530

  17. Influence of anti-FloR antibody on florfenicol accumulation in florfenicol-resistant Escherichia coli and enzyme-linked immunosorbent assay for detection of florfenicol-resistant E. coli isolates.

    PubMed

    Wu, Beibei; Xia, Chun; Du, Xiangdang; Cao, Xingyuan; Shen, Jianzhong

    2006-02-01

    To detect florfenicol-resistant Escherichia coli isolates by enzyme-linked immunosorbent assay (ELISA), anti-FloR1 antibodies were produced in mice using a recombinant glutathione S-transferase (GST)-FloR1 protein, which was expressed in a prokaryote expression system, as the antigen. The specificity of the murine anti-GST-FloR1 antibody and its influence on florfenicol accumulation in florfenicol-resistant isolates were investigated using Western blotting and high-performance liquid chromatography, respectively. Western blotting using the anti-FloR1 antibody showed specific binding of the antibody to the florfenicol-resistant FloR protein. Preincubation of florfenicol-resistant strains with the antibody significantly increased the intracellular accumulation of florfenicol and enhanced the bacterial susceptibility to florfenicol, suggesting that antibody binding to the FloR protein inhibited the activity of the efflux protein conferred by the floR gene. Analyses of florfenicol-resistant and -sensitive isolates by ELISA using the anti-FloR1 antibody showed good correlation between FloR protein expression and the floR genotype. The anti-FloR1 antibody-based ELISA is a useful tool for the detection of florfenicol-resistant bacteria harboring the floR gene.

  18. Prevalence estimation of tick-borne encephalitis virus (TBEV) antibodies in dogs from Finland using novel dog anti-TBEV IgG MAb-capture and IgG immunofluorescence assays based on recombinant TBEV subviral particles.

    PubMed

    Levanov, Lev; Vera, Cristina Pérez; Vapalahti, Olli

    2016-07-01

    Tick-borne encephalitis (TBE) is one of the most dangerous human neurological infections occurring in Europe and Northern parts of Asia with thousands of cases and millions vaccinated against it. The risk of TBE might be assessed through analyses of the samples taken from wildlife or from animals which are in close contact with humans. Dogs have been shown to be a good sentinel species for these studies. Serological assays for diagnosis of TBE in dogs are mainly based on purified and inactivated TBEV antigens. Here we describe novel dog anti-TBEV IgG monoclonal antibody (MAb)-capture assay which is based on TBEV prME subviral particles expressed in mammalian cells from Semliki Forest virus (SFV) replicon as well as IgG immunofluorescence assay (IFA) which is based on Vero E6 cells transfected with the same SFV replicon. We further demonstrate their use in a small-scale TBEV seroprevalence study of dogs representing different regions of Finland. Altogether, 148 dog serum samples were tested by novel assays and results were compared to those obtained with a commercial IgG enzyme immunoassay (EIA), hemagglutination inhibition test and IgG IFA with TBEV infected cells. Compared to reference tests, the sensitivities of the developed assays were 90-100% and the specificities of the two assays were 100%. Analysis of the dog serum samples showed a seroprevalence of 40% on Åland Islands and 6% on Southwestern archipelago of Finland. In conclusion, a specific and sensitive EIA and IFA for the detection of IgG antibodies in canine sera were developed. Based on these assays the seroprevalence of IgG antibodies in dogs from different regions of Finland was assessed and was shown to parallel the known human disease burden as the Southwestern archipelago and Åland Islands in particular had considerable dog TBEV antibody prevalence and represent areas with high risk of TBE for humans.

  19. Evaluation of a commercial enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody in diagnosis of human leptospiral infection.

    PubMed Central

    Winslow, W E; Merry, D J; Pirc, M L; Devine, P L

    1997-01-01

    The PanBio Leptospira immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) is a commercially available screening test for the diagnosis of acute leptospiral infection. The ability of the test to diagnose early or recent Leptospira interrogans infection was assessed by testing sera with known microagglutination test (MAT) titers to serovars pomona, hardjo, copenhageni, and australis. The IgM ELISA detected all 41 cases of early or recent leptospiral infection (sensitivity, 100%), with a positive ELISA result seen in many cases before MAT antibody titers reached 1:50. Thirty-eight of 41 patients showed seroconversion (fourfold or greater increase in titer by MAT, 2 of 41 patients had a single sample with elevated titer, and 1 patient from whom leptospires were isolated from a blood sample failed to show MAT titers, despite a seroconversion (negative to positive result) in the ELISA. Follow-up sera obtained from 8 of 12 patients (67%) for 3 to 48 months after the acute stage of illness showed persisting IgM antibody. However, the range of levels detected in these samples (maximum ELISA ratio, 2.0) was lower than the range seen when infection was recent. Reactivity in the IgM ELISA was observed for only 1 of 59 serum samples from asymptomatic donors (specificity, 98%) and 16 of 233 serum samples from patients with Ross River virus, brucella, Epstein-Barr virus, cytomegalovirus, mycoplasma, Q-fever, toxoplasma, hepatitis A virus, Treponema pallidum, or Borrelia burgdorferi infection (specificity, 93%), with the majority of these patients showing lower levels of IgM in comparison to those in patients with leptospiral infection. We conclude that this ELISA is sufficiently sensitive for use as an initial screen for leptospiral infections, with subsequent confirmation of positive test results by MAT. PMID:9230359

  20. Acetone in drinking water does not modulate humoral immunity in mice as measured by the antibody, plaque-forming cell assay.

    PubMed

    Woolhiser, Michael R; Houtman, Carrie E; Waechter, John M

    2006-01-01

    It has been reported that the repeated topical, nonoccluded application of acetone may modulate antibody production in mice, thus producing humoral immunosuppression. However, the evaporative loss expected following nonoccluded dermal application of acetone makes the systemic effect seem unlikely. This study was designed to investigate the immunotoxicity potential of acetone in mice following a more direct systemic route of dosing via drinking water for 28 days. CD-1 male mice consumed average daily acetone doses of 121, 621 or 1144 mg/kg/day. The antibody, plaque-forming cell (AFC) assay was performed to measure the T cell-dependent, anti-sheep red blood cell immunoglobulin M (IgM) response, and hematology and thymus weights were evaluated to provide additional insight into the potential effects to the immune system. Body weights, white blood cell (WBC), numbers, red blood cell (RBC) counts, and hemoglobin and hematocrit levels showed no treatment-related effects at any dose of acetone. Eosinophil percentages were variable but also showed no dose-related trends. Spleen and thymus weights were not statistically different from controls and there were no effects on spleen cellularity or AFC response as a result of acetone administration. The AFC responses ranged from 1088 to 1401 AFCs/10(6) splenocytes and were not statistically different from controls (1277 AFCs/10(6) cells). Mice treated with cyclophosphamide (20 mg/kg) on days 25 to 28 demonstrated a 94% reduction in AFC/10(6) cells. Thus, the direct systemic administration of acetone did not produce evidence for immunotoxicity in CD-1 mice and the no observed adverse effect level (NOAEL) in this study was determined to be 1144 mg/kg/day.

  1. Development of a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for nitroimidazoles in edible animal tissues and feeds.

    PubMed

    Han, Wei; Pan, Yuanhu; Wang, Yulian; Chen, Dongmei; Liu, Zhenli; Zhou, Qi; Feng, Liang; Peng, Dapeng; Yuan, Zonghui

    2016-02-20

    The misuse of nitroimidazoles (NDZs) can lead to NDZs residues in edible animal tissues, which would be harmful to consumer health. To quickly monitor NDZs residues in edible animal tissues and feed, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with a simple sample preparation method and clean-up was developed in the present study. At first, a broad-specificity monoclonal antibody, 1D5, against NDZs has been produced, which the IC50 values of the NDZs, dimetridazole, ipronidazole, ronidazole hydroxydimetridazole, and hydroxyipronidazole, were 4.79μgL(-1), 0.47μgL(-1), 5.97μgL(-1), 23.48μgL(-1), and 15.03μgL(-1), respectively. The limit of detection of the method for the NDZ matrix calibration ranged from 4.2μgkg(-1) to 50.3μgkg(-1) in the feed matrices and from 0.11μgkg(-1) to 4.11μgkg(-1) in the edible animal tissues matrices. The recoveries of the NDZs were in the range of 75.5-111.8%. The CVs were less than 14.4%. A good correlation (r=0.9905) between the ELISA and HPLC-MS results of the tissues demonstrated the reliability of the developed ic-ELISA, which makes it a useful tool for screening of NDZs in animal edible tissue and feed.

  2. Localization and Distribution of 'Candidatus Liberibacter asiaticus’ in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody

    PubMed Central

    Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H.; Hartung, John S.

    2015-01-01

    ‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant. PMID:25946013

  3. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    PubMed

    Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H; Hartung, John S

    2015-01-01

    'Candidatus Liberibacter asiaticus' (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.

  4. Application of the fluorescence polarization assay for detection of caprine antibodies to Brucella melitensis in areas of high prevalence and widespread vaccination.

    PubMed

    Ramírez-Pfeiffer, C; Nielsen, K; Smith, P; Marín-Ricalde, F; Rodríguez-Padilla, C; Gomez-Flores, R

    2007-03-01

    The screening Rose Bengal test (RBT), the buffered plate agglutination test (BPAT), and the confirmatory complement fixation test (CFT) are currently approved by the World Organization for Animal Health (OIE) for diagnosis of goat brucellosis. However, RBT (at 3% or 8% cell concentration) is known to be affected by vaccinal antibodies. In the present study, Mexican and Canadian OIE tests were compared with the fluorescence polarization assay (FPA), alone or in combination, using indirect and competitive enzyme-linked immunosorbent assays as classification variables for goat sera obtained from an area of high prevalence and widespread vaccination. The relative sensitivities and specificities were, respectively, 99.7% and 32.5% for RBT3, 92.8% and 68.8% for RBT8, 98.4% and 84.8% for Canadian CFT, 83.7% and 65.5% for Mexican CFT, and 78.1% and 89.3% for FPA. The use of FPA as the confirmatory test in combination with other tests significantly increased the final specificities of the screening tests alone; BPAT, RBT3, and RBT8 plus FPA resulted in final specificities of 90%, 91.2%, and 91.3%, respectively, whereas for the combinations RBT3 plus Mexican CFT, RBT8 plus Mexican CFT, and BPAT plus Canadian CFT, specificities were 65.5%, 63.2%, and 91.7%, respectively. We suggest that FPA may be routinely applied as an adaptable screening test for diagnosis of goat brucellosis and as a confirmatory test for screening test series. Some advantages of FPA are that its cutoff can be adjusted to improve its sensitivity or specificity, it is a low-cost and easy-to-perform test of choice when specificity is relevant or when an alternative confirmatory test is not available, and it is not affected by vaccination, thus reducing the number of misdiagnosed and killed goats.

  5. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Adungo, Ferdinard; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu

    2016-01-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

  6. Immunoglobulin detection in wild birds: Effectiveness of three secondary anti-avian IgY antibodies in direct ELISAs in 41 avian species

    USGS Publications Warehouse

    Fassbinder-Orth, Carol A.; Wilcoxen, Travis E.; Tran, Tiffany; Boughton, Raoul K.; Fair, Jeanne M.; Hofmeister, Erik K.; Grindstaff, Jennifer L.; Owen, Jen C.

    2016-01-01

    4.This study indicates that the anti-passerine IgY secondary antibody can effectively be used in immunological assays to detect passerine IgY for species in most passerine families and is preferred over anti-chicken and anti-bird secondary antibodies for the majority of passerine species. This anti-passerine antibody will allow for more accurate detection and quantification of IgY in more wild bird species than was possible with previously available secondary antibodies.

  7. Development and validation of a sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for the determination of the aflatoxin M1 levels in milk.

    PubMed

    Peng, Dapeng; Yang, Bijia; Pan, Yuanhu; Wang, Yulian; Chen, Dongmei; Liu, Zhenli; Yang, Wenxiang; Tao, Yanfei; Yuan, Zonghui

    2016-04-01

    A sensitive monoclonal antibody (mAb) against aflatoxin M1 (AFM1) was generated to quickly monitor the AFM1 residues in milk. Then, a mAb-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established that utilizes simple sample preparation and clean-up methods. The obtained 3D8 mAb, which is an IgG1 isotype mAb, displayed an IC50 value of 64.75 ng L(-1) for AFM1 and did not exhibit measurable cross-reactivity with other aflatoxins and antibiotics. The decision limit (CCα, α = 1%), detection capability (CCβ, β = 5%), and LOQ value for the AFM1 matrix calibration method were 24 ng L(-1), 27.5 ng L(-1), and 35 ng L(-1) in the milk matrices, respectively. The AFM1 recovery ranged from 85.3% to 107.6%. The CVs were less than 13.8%. A positive correlation (r > 0.99) was observed between the ic-ELISA and HPLC-MS/MS results. This ic-ELISA would be a useful tool for screening the AFM1 residues in milk.

  8. Utilizing three monoclonal antibodies in the development of an immunochromatographic assay for simultaneous detection of sulfamethazine, sulfadiazine, and sulfaquinoxaline residues in egg and chicken muscle.

    PubMed

    Guo, Yancheng; Ngom, Babacar; Le, Tao; Jin, Xiue; Wang, Liping; Shi, Deshi; Wang, Xiliang; Bi, Dingren

    2010-09-15

    A rapid and sensitive immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneous detection of sulfamethazine (SM(2)), sulfadiazine (SDZ), and sulfaquinoxaline (SQX) in chicken breast muscle and egg samples. For this purpose, three monoclonal antibodies raised against those three sulfonamides were conjugated to colloidal gold particles and applied to the conjugate pads of the test strip. The competitors of the sulfonamides (SM(2)/SDZ/SQX-bovine serum albumin conjugates) were immobilized onto a nitrocellulose membrane at three detection zones to form T(1), T(2), and T(3), respectively. With this method, the cutoff values for the three test lines were achieved at 80 μg/kg, which is lower than the maximum residue levels (MRLs) established for sulfonamides. The recoveries in negative samples spiked at concentrations of 10, 50, and 100 μg/kg ranged from 75% to 82% for egg samples and from 78% to 81% for chicken samples. The method was compared with the HPLC method by testing 180 eggs and chicken breast samples from local markets, and an agreement rate of 99.7% was obtained between the two methods.

  9. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

    USGS Publications Warehouse

    Pascho, Ronald J.; Chase, Dorothy M.; McKibben, Constance L.

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 × 109cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 × 104cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

  10. Hep-2 cell based indirect immunofluorescence assay for antinuclear antibodies as a potential diagnosis of drug-induced autoimmunity in nonclinical toxicity testing.

    PubMed

    Hong, Min; Ma, Ben; Lin, Zhi; Zhou, Xiaobing; Geng, Xingchao; Shen, Lianzhong; Li, Bo

    2015-03-01

    Antinuclear antibodies (ANAs) are important biomarkers in the diagnosis of autoimmune diseases in humans; however, the diagnostic performance of ANA in nonclinical safety studies are not well understood. Here, we studied the use of ANAs as potential nonclinical biomarkers for drug-induced autoimmunity (DIA) using a Hep-2 based indirect immunofluorescence assay (IFA). Initially, MRL-fas(lpr)/J mice and HgCl₂-treated rats were used as SLE-positive models. Serum samples obtained from 94 normal mice or 204 normal rats aged one to four months served as the negative control. The IFA effectively distinguished ANAs-positive samples in both species with a cut-off titer of 1:100. Brown Norway rats were treated with 450 mg/kg D-penicillamine for 30 consecutive days. ANAs were generated and corresponded with DIA development. Human Hep-2 cells, mice Neuro 2A cells, and Chinese Hamster Lung cells served as antigen from different species, which were found cross-reactive with ANA-positive serum samples from mice, rats, and humans without any differences in diagnosis. This methodology showed no species-specificity for ANA detection. Furthermore, we found approximately 20 percentage of the mice aged seven to eight months demonstrated age-related ANAs, which was consistent with humans. Overall, our findings demonstrated the use of ANA detection using IFA in the nonclinical diagnosis of murine drug-induced autoimmunity, and age-related ANAs should be considered when aged animals are used.

  11. Qualitative and quantitative analyses of Epstein-Barr virus early antigen diffuse component by western blotting enzyme-linked immunosorbent assay with a monoclonal antibody.

    PubMed Central

    Lin, J C; Choi, E I; Pagano, J S

    1985-01-01

    We report the use of monoclonal antibody against the early antigen diffuse component (anti-EA-D) of Epstein-Barr virus (EBV) to analyze, both qualitatively and quantitatively, the expression of EA-D in various human lymphoblastoid cell lines activated by chemical inducers. The kinetics of synthesis of EA-D in P3HR-1, B95-8, and Ramos/AW cells were similar in that they all reached the peak of synthesis on day 5 after induction. Surprisingly, no expression of EA-D was found in induced BJAB/GC, an EBV-genome-containing cell line. EBV-negative cell lines, BJAB and Ramos, were negative for EA-D. Raji cells had no detectable EA-D but responded rapidly to induction, reaching a peak on day 3. Superinfection of Raji cells also resulted in marked induction of EA-D, which reached a plateau between 8 to 12 h postinfection. Western blotting coupled with the enzyme-linked immunosorbent assay was employed to identify polypeptides representing EA-D. A family of four polypeptides with molecular weights of 46,000 (46K protein), 49,000, 52,000, and 55,000 were identified to be reactive with monoclonal anti-EA-D antiserum. The pattern of EA-D polypeptides expressed in each cell line was different. Of particular interest was the expression of a large quantity of 46K protein both in induced Raji and P3HR-1 cells, but not in superinfected Raji cells. A 49K doublet was expressed in activated p3HR-1, B95-8, and Ramos/AW cells and in superinfected Raji cells. In addition, two distinct 52K and 55K polypeptides were expressed in induced Ramos/AW and superinfected Raji cells. However, none of these EA-D polypeptides was detectable in BJAB/GC, BJAB, Ramos, and mock-infected Raji cells. To approximate relative concentrations of EA-D in cell extracts, we employed the enzyme-linked immunosorbent assay and immunoblot dot methods by using one of the purified EA-D components to construct a standard curve. Depending upon the cell lines, it was estimated that ca. 1 to 3% (determined by the enzyme

  12. Evaluation of enzyme-linked immunosorbent assays and a haemagglutination inhibition tests for the detection of antibodies to Newcastle disease virus in village chickens using a Bayesian approach.

    PubMed

    Chaka, H; Thompson, P N; Goutard, F; Grosbois, V

    2015-04-01

    Newcastle disease (ND) is an endemic disease in village chickens in Ethiopia with substantial economic importance. The sensitivity (Se) and specificity (Sp) of a blocking enzyme-linked immunosorbent assay (bELISA, Svanova Biotech), indirect ELISA (iELISA, Laboratoire Service International) and haemagglutination inhibition (HI) test for ND virus (NDV) antibody detection were evaluated in a Bayesian framework in the absence of a gold standard test, on sera collected from unvaccinated chickens kept under the village production system in household flocks and at markets in two woredas (i.e. districts) of the Eastern Shewa zone, Ethiopia. The outcomes of the iELISA test differed dramatically from those of the two other tests with 92% of the samples testing positive as compared with less than 15% for bELISA and HI. iELISA results were also inconsistent with previous estimations of Newcastle serological prevalence. The information provided by the iELISA test was thus considered as highly unreliable, probably due to an extremely low specificity, and thus not considered in the Bayesian models aiming at estimating serological prevalence and test performance parameters. Bayesian modelling of HI and bELISA test results suggested that bELISA had both the highest Se (86.6%; 95% posterior credible interval (PCI): 61.8%; 98.5%), and the highest Sp (98.3%; 95% PCI: 97.2%; 99.5%), while HI had a Se of 80.2% (95% PCI: 59.1%; 94.3%), and a Sp of 96.1% (95% PCI: 95.1%; 97.4%). Model selection and the range of the posterior distribution of the correlation between bELISA and HI test outcomes for truly seropositive animals (median at 0.461; PCI: -0.055; 0.894) suggested a tendency for bELISA and HI to detect the same truly positive animals and to fail to detect the same truly positive animals. The use of bELISA in screening and surveillance for NDV antibodies is indicated given its high Se and Sp, in addition to its ease of automation to handle large numbers of samples compared to HI. The

  13. Infectious hematopoietic necrosis (IHN) and viral hemorrhagic septicemia (VHS): Detection of the trout antibodies to the causative viruses by means of plaque neutralization, immunofluorescence, and enzyme-linked immunosorbent assay

    USGS Publications Warehouse

    Vestergard Jorgensen, P. E.; Olesen, N.J.; Lorenzen, N.; Winton, J.R.; Ristow, S.S.

    1991-01-01

    Sera collected from cultured rainbow trout Oncorhynchus mykiss surviving outbreaks of infectious hematopoietic necrosis (IHN) or viral hemorrhagic septicemia (VHS) were examined for the presence of antibodies to both of the causative viruses, infectious hematopoietic necrosis virus (IHNV) and Egtved virus (viral hemorrhagic septicemia virus: VHSV). Sera were screened with three serological tests: 50% plaque neutralization test (PNT), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA). In sera from 20 rainbow trout surviving IHN, antibodies to IHNV were detected in 9 fish by PNT, in 12 fish by IF, and in 9 fish by ELISA. In these sera, antibodies cross-reacting with VHSV were rare (detected in 0 fish by PNT, in 1 by IF, and in 1 by ELISA). In sera from 20 rainbow trout surviving VHS, antibodies to VHSV were detected in 9 fish by PNT, in 16 fish by IF, and in 18 fish by ELISA. A considerable percentage of the VHS-survivor sera contained antibodies that cross-reacted with IHNV, as detected by ELISA (16 fish) and 1F (7 fish) but not by PNT (0 fish). The three serological tests appear to be useful tools for IHNV and VHSV epidemiology; however, the presence of cross-reacting antibodies in some sera suggests caution when farms require specific pathogen-free certification for one of the viruses in the presence of the other.

  14. Identification of an immunodominant epitope in glycoproteins B and G of herpes simplex viruses (HSVs) using synthetic peptides as antigens in assay of antibodies to HSV in herpes simplex encephalitis patients.

    PubMed

    Bhullar, S S; Chandak, N H; Baheti, N N; Purohit, H J; Taori, G M; Daginawala, H F; Kashyap, R S

    2014-01-01

    Herpes simplex encephalitis (HSE) is a severe viral infection of the central nervous system (CNS). Assay of antibody response is widely used in diagnostics of HSE. The aim of this study was to identify an immunodominant epitope determining the antibody response to herpes simplex viruses (HSVs) in cerebrospinal fluid (CSF) of HSE patients. The synthetic peptides that resembled type-common as well as type-specific domains of glycoproteins B (gB) and G (gG) of these viruses were evaluated for binding with IgM and IgG antibodies in CSF samples from HSE and non-HSE patients in ELISA. The QLHDLRF peptide, derived from gB of HSV was found to be an immunodominant epitope in the IgM and IgG antibody response. The patients with confirmed and suspected HSE showed in ELISA against this peptide 26% and 23% positivities for IgM, 43% and 37% positivities for IgG and 17% and 15% for both IgM and IgG antibodies, respectively. The total positivities of 86% and 75% for both IgM and IgG antibodies were obtained in the patients with confirmed and suspected HSE, respectively. These results demonstrate that a synthetic peptide-based diagnostics of HSE can be an efficient and easily accessible alternative. This is the first report describing the use of synthetic peptides derived from HSVs in diagnostics of HSE using patientsʹ CSF samples.

  15. Immunofluorescence assay reactivity patterns of serum samples presenting indeterminate Western blot results for antibodies to HIV-1 and HTLV-I/II in Cordoba, Argentina.

    PubMed

    Gastaldello, R; Gallego, S; Isa, M B; Maturano, E; Sileoni, S; Nates, S; Medeot, S

    2001-01-01

    Serum samples (n: 110) from blood donors and high risk individuals from Cordoba, Argentina with indeterminate HIV-1 and HTLV-I/II Wb profiles were studied for specific antibodies to HTLV-I/II and HIV-1 by indirect immunofluorescence assay (IFA) and for the presence or absence of HIV-1 and HTLV-I/II specific bands by Wb. This study was carried out in order to characterize their putative reactions with HIV-1 and HTLV-I/II proteins and to resolve the retrovirus infection status of these individuals. Results indicated that blood donors sera displaying indeterminate HIV-1 or HTLV-I/II Wb patterns were not immunoreactive to HTLV-I/II and HIV-1 on IFA. However, a high rate of indeterminate HIV-1 and HTLV-I/II Wb samples from high risk individuals had positive HTLV-I/II and HIV-1 IFA results respectively. Our study supports the growing evidence that HTLV-HIV indeterminate seroreactivity in low risk population is due to a cross reaction against nonviral antigens, and in high risk populations the indeterminate samples show serological cross-recognition between HIV-1 proteins and HTLV-I/II proteins on Wb. These results point out the necessity to investigate the HTLV-I/II reactivity in indeterminate HIV-1 samples and vice versa in order to confirm the diagnosis. Finally, this study shows the potential usefulness of IFA in elucidating the status of HIV-1 and HTLV-I/II infection of individuals with indeterminate Wb profiles, thus enabling resolution of retrovirus infection status.

  16. Enzyme-linked immunosorbent assay using recombinant SAG1 antigen to detect Toxoplasma gondii-specific immunoglobulin G antibodies in human sera and saliva.

    PubMed

    Chahed Bel-Ochi, Nouha; Bouratbine, Aïda; Mousli, Mohamed

    2013-04-01

    Serologic detection of Toxoplasma gondii IgG antibodies is widely accepted as a means to determine immune status and susceptibility to Toxoplasma infection during pregnancy. However, current commercial kits present some drawbacks, such as a requirement for whole-parasite antigen preparation or interassay variability. To address these problems, the purpose of this study was to produce a whole sequence of the recombinant T. gondii SAG1 antigen (rSAG1) to assess its diagnostic performance in Toxoplasma IgG screening and to explore a saliva-based method as a noninvasive alternative to serum-based testing. rSAG1 was expressed in recombinant bacteria as inclusion bodies, purified through one-step affinity chromatography, and refolded in native form by dialysis. A large amount was obtained, and the specific antigen immunoreactivity was confirmed by immunoblotting. Two rSAG1-based enzyme-linked immunosorbent assays (ELISAs) applied to paired serum and saliva samples were designed. The rSAG1-based ELISA evaluation consisted of testing intrinsic sensitivity and specificity of 49 serum samples from patients immune to toxoplasmosis and 42 serum samples from nonimmune controls identified by routinely used kits. To assess agreement between serum-based and saliva-based tests, the positive percent agreement (PPA) and negative percent agreement (NPA) between the 2 tests were estimated. The rSAG1 serum-based ELISA detected specific IgG with 100% sensitivity and specificity. The PPA and NPA between the serum-based and saliva-based tests varied according to the selected optical density threshold in saliva. Thus, for a selected cutoff of 0.14, the PPA was 100% and the NPA was 88.1%, whereas for a selected cutoff of 0.29, the PPA was 67.3% and the NPA was 100%.

  17. The use of a microagglutination assay for the detection of antibodies to Corynebacterium pseudotuberculosis in naturally infected sheep and goat flocks.

    PubMed Central

    Menzies, P I; Muckle, C A

    1989-01-01

    Two goat flocks comprising 326 animals and four sheep flocks comprising 343 animals, all with a previously recognized problem of abscesses due to Corynebacterium pseudotuberculosis, were examined for the presence of abscesses and antibody titers to C. pseudotuberculosis as detected by direct microagglutination assay. In sheep there was a strong positive relationship between age and titer (p less than 0.0001). However, the relationship in goats between age and titer could not be determined due to a strong interaction between flock and age. When the relationship between abscesses and titer was examined, it was found that goats with abscesses had higher titers than those that did not (p less than 0.05), whereas there was no difference in titer between sheep with abscesses and those without (p = 0.5753). The sensitivity of the microagglutination test was poor to good for both species (52.3% for goats and 89.7% for sheep). The specificity of the test was fair to poor (64.9% for goats and 21.7% for sheep). Given a disease prevalence of 13.5% for goats and 8.5% for sheep the predictive value of the positive test was very poor (18.9% for goats and 9.6% for sheep) but the predictive value of the negative test was good to excellent (89.7% for goats and 95.8% for sheep). The poor specificity of the test and therefore the positive predictive value may be due in part to the criterion of classification of presence of disease, i.e. presence of an abscess at the time of sampling.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2766152

  18. Characterization of size, structure and purity of serogroup X Neisseria meningitidis polysaccharide, and development of an assay for quantification of human antibodies.

    PubMed

    Xie, Ouli; Bolgiano, Barbara; Gao, Fang; Lockyer, Kay; Swann, Carolyn; Jones, Christopher; Delrieu, Isabelle; Njanpop-Lafourcade, Berthe-Marie; Tamekloe, Tsidi Agbeko; Pollard, Andrew J; Norheim, Gunnstein

    2012-08-31

    Serogroup X Neisseria meningitidis (MenX) has recently emerged as a cause of localized disease outbreaks in sub-Saharan Africa. In order to prepare for vaccine development, MenX polysaccharide (MenX PS) was purified by standard methods and analyzed for identity and structure by NMR spectroscopy. This study presents the first full assignment of the structure of the MenX PS using (13)C, (1)H and (31)P NMR spectroscopy and total correlation spectroscopy (TOCSY) and (1)H-(13)C heteronuclear single quantum coherence (HSQC). Molecular size distribution analysis using HPLC-SEC with multi-angle laser light scattering (MALLS) found the single peak of MenX PS to have a weight-average molar mass of 247,000g/mol, slightly higher than a reference preparation of purified serogroup C meningococcal polysaccharide. MenX PS tended to be more thermostable than serogroup A PS. A method for the quantification of MenX PS was developed by use of high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). A novel and specific ELISA assay for quantification of human anti-MenX PS IgG based on covalent linkage of the MenX PS to functionally modified microtitre plates was developed and found valid for the assessment of the specific antibody concentrations produced in response to MenX vaccination or natural infection. The current work thus provides the necessary background for the development of a MenX PS-based vaccine to prevent meningococcal infection caused by bacteria bearing this capsule.

  19. Risk factors for herds to test positive for Mycobacterium avium ssp. paratuberculosis-antibodies with a commercial milk enzyme-linked immunosorbent assay (ELISA) in Ontario and western Canada.

    PubMed

    Sorge, Ulrike S; Lissemore, Kerry; Godkin, Ann; Jansen, Jocelyn; Hendrick, Steven; Wells, Scott; Kelton, David F

    2012-09-01

    The objectives of this study were to identify risk factors associated with i) a Mycobacterium avium subsp. paratuberculosis (MAP)-antibody milk enzyme-linked immunosorbent assay (MAP milk ELISA)-positive herd status, and ii) the within-herd MAP milk ELISA-positive prevalence in Canadian dairy herds. This prospective cohort study was conducted between 2005 and 2009 on 226 herds in Ontario and western Canada, which participated in a voluntary risk assessment (RA)-based Johne's disease control program. Two MAP milk ELISA and risk assessments and a previsit survey were available per herd. The overall farm RA scores alone could not be used to predict whether a herd would test positive for MAP antibodies. However, the results of this study indicated that increasing the likelihood of exposing calves to MAP through certain management practices, as assessed with the RA, increased the likelihood of a herd being test-positive for MAP antibodies.

  20. Effect of gamma irradiation on reactivity of rinderpest virus antigen with bovine immune serum in enzyme-linked immunosorbent assays and virus neutralization and indirect fluorescent-antibody tests.

    PubMed Central

    Saliki, J T; Berninger, M L; Torres, A; House, J A; Mebus, C A; Dubovi, E J

    1993-01-01

    Gamma irradiation effectively inactivated gradient-purified rinderpest virus. Irradiated antigen and sera remained functional in enzyme-linked immunosorbent assays, virus neutralization tests, and indirect fluorescent-antibody tests. Irradiation, however, led to a dose-dependent decrease in reactivity, particularly significant (P < 0.05) when both reagents were irradiated. To avoid false-positive reactions, only one reagent (serum or antigen) may be irradiated. PMID:8432831

  1. Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in (51)Cr-release and CD107a assays.

    PubMed

    Mata, Mariana M; Mahmood, Fareeha; Sowell, Ryan T; Baum, Linda L

    2014-04-01

    Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in (51)Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16(+)CD56(dim) NK cells and CD14(+) monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56(dim) NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a (51)Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.

  2. Interpretations of antibody responses to Salmonella enterica serotype enteritidis gm flagellin in poultry flocks are enhanced by a kinetics-based enzyme-linked immunosorbent assay.

    PubMed

    McDonough, P L; Jacobson, R H; Timoney, J F; Mutalib, A; Kradel, D C; Chang, Y F; Shin, S J; Lein, D H; Trock, S; Wheeler, K

    1998-07-01

    Many regulatory and diagnostic programs for the detection of Salmonella enterica serotype Enteritidis infection in commercial poultry flocks have relied on rapid Pullorum agglutination tests to screen birds because of the shared antigens of S. enterica Enteritidis and S. enterica Pullorum and Gallinarum; however, the use of the enzyme-linked immunosorbent assay (ELISA) format affords better analytical sensitivity than crude agglutination tests. In this study, we adapted our earlier conventional indirect ELISA, using gm flagellin as the antigen, to a kinetics-based, computer-controlled ELISA (KELA). The KELA was used to screen for flagellin antibody from three commercial flocks: (i) a large flock involved in a U.S. Department of Agriculture trace back from a human S. enterica Enteritidis foodborne outbreak (n = 3,209), (ii) a flock infected with the endemic S. enterica Enteritidis serotype but which also had multiple other Salmonella serotypes (n = 65), and (iii) an S. enterica Pullorum-infected flock (n = 12). The first flock (S. enterica Enteritidis prevalence of 2.45% based on culture) provided a field test of the KELA and allowed the calculation of diagnostic sensitivity (D-Sn) and diagnostic specificity (D-Sp). With a cutoff of 10 (used for screening flocks [i.e., high sensitivity]), the KELA has a D-Sn of 95.2% and a D-Sp of 18.5%; with a cutoff of 140 (used in confirmatory flock testing [i.e., high specificity]), the KELA has a D-Sn of 28.0% and a D-Sp of 99.1%. We found that with a cutoff of 60 (D-Sn = 63.1%; D-Sp = 91.6%), we could eliminate reactions in the KELA caused by other non-S. enterica Enteritidis salmonellae. The KELA was also compared to two commercial rapid Pullorum tests, the Solvay (D-Sn = 94.9%; D-Sp = 55.5%) and the Vineland (D-Sn = 62.0%; D-Sp = 75.3%).

  3. Validation of an indirect enzyme-linked immunosorbent assay for the detection of antibody against Brucella abortus in cattle sera using an automated ELISA workstation.

    PubMed

    Paweska, J T; Potts, A D; Harris, H J; Smith, S J; Viljoen, G J; Dungu, B; Brett, O L; Bubb, M; Prozesky, L

    2002-03-01

    that for the CFT (0.833). Analysis of distribution of PP values in sera from vaccinated and naturally infected cattle shows that in vaccinated animals all readings were below 31 PP where in infected ones these values represented 43%. Therefore, it appears that I-ELISA could be of use in identifying some naturally infected animals (with values > 31 PP), but more sera from reference vaccinated and infected animals need to be tested to further substantiate this statistically. Of 834 sera positive by RBT, SAT and CFT, 825 (98.9%) were positive in the I-ELISA. Compared to C-ELISA the relative diagnostic sensitivity of the I-ELISA was 94% and of the CFT 88% when testing 100 Canadian cattle sera. Of 258 South African cattle sera, of which 183 (70.9 %) were positive by the I-ELISA and 148 (57.4 %) by the CFT, 197 (76.4%) were positive by C-ELISA when re-tested in Canada. One has to stress, however, that Canadian C-ELISA has not been optimised locally. Thus, the C-ELISA was probably not used at the best diagnostic threshold for testing South African cattle sera. This study shows that the I-ELISA performed on an automated ELISA workstation provides a rapid, simple, highly sensitive and specific diagnostic system for large-scale detection of antibodies against B. abortus. Based on the diagnostic accuracy of this assay reported here, the authors suggest that it could replace not only the currently used confirmatory CFT test, but also the two in-use screening tests, namely the RBT and SAT.

  4. Preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by double-antigen sandwich enzyme-linked immunosorbent assay.

    PubMed

    Jiao, Yongjun; Zeng, Xiaoyan; Guo, Xiling; Qi, Xian; Zhang, Xiao; Shi, Zhiyang; Zhou, Minghao; Bao, Changjun; Zhang, Wenshuai; Xu, Yan; Wang, Hua

    2012-02-01

    The recent emergence of the human infection confirmed to be caused by severe fever with thrombocytopenia syndrome virus (SFTSV) in China is of global concern. Safe diagnostic immunoreagents for determination of human and animal seroprevalence in epidemiological investigations are urgently needed. This paper describes the cloning and expression of the nucleocapsid (N) protein of SFTSV. An N-protein-based double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) system was set up to detect the total antibodies in human and animal sera. We reasoned that as the double-antigen sandwich ELISA detected total antibodies with a higher sensitivity than traditional indirect ELISA, it could be used to detect SFTSV-specific antibodies from different animal species. The serum neutralization test was used to validate the performance of this ELISA system. All human and animal sera that tested positive in the neutralization test were also positive in the sandwich ELISA, and there was a high correlation between serum neutralizing titers and ELISA readings. Cross-reactivity was evaluated, and the system was found to be highly specific to SFTSV; all hantavirus- and dengue virus-confirmed patient samples were negative. SFTSV-confirmed human and animal sera from both Anhui and Hubei Provinces in China reacted with N protein in this ELISA, suggesting no major antigenic variation between geographically disparate virus isolates and the suitability of this assay in nationwide application. ELISA results showed that 3.6% of the human serum samples and 47.7% of the animal field serum samples were positive for SFTSV antibodies, indicating that SFTSV has circulated widely in China. This assay, which is simple to operate, poses no biohazard risk, does not require sophisticated equipment, and can be used in disease surveillance programs, particularly in the screening of large numbers of samples from various animal species.

  5. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, Bernard F.; Chen, Bi-Xing

    1997-01-01

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest.

  6. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, B.F.; Chen, B.X.

    1997-07-22

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest. 8 figs.

  7. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, Bernard F.; Chen, Bi-Xing

    1999-01-01

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example, detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest, detecting a polypeptide such as those expressed by infectious agents, fungi or parasites.

  8. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, B.F.; Chen, B.

    1999-07-20

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example, detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest, detecting a polypeptide such as those expressed by infectious agents, fungi or parasites. 25 figs.

  9. Expression of Epstein-Barr virus antibodies EA-IgG, Rta-IgG, and VCA-IgA in nasopharyngeal carcinoma and their use in a combined diagnostic assay.

    PubMed

    Li, Y; Wang, K; Yin, S K; Zheng, H L; Min, D L

    2016-03-18

    Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma, which can be monitored by the levels of Rta protein antibody IgG (Rta-IgG), early antigen antibody (EA-IgG), and viral capsid antibody (VCA-IgA). In the present study, we investigated the serum levels of Rta-IgG, EA-IgG, and VCA-IgA in nasopharyngeal cancer patients, and the diagnostic value of a combined assay that includes these antibodies in addition to the EBV-DNA. A total of 56 nasopharyngeal cancer patients were recruited as the study population, along with 48 benign rhinitis patients and 42 healthy individuals. Serum EA-IgG, Rta-IgG, and VCA-IgA levels were measured by enzyme-linked immunosorbent assay, and EBV-DNA was quantified with PCR. The diagnostic value of these indices was further evaluated by ROC curve analysis. The expression levels of EA-IgG, Rta-IgG, VCA-IgA, and EBV-DNA were elevated in the nasopharyngeal cancer patients, who had higher levels of these antibodies than those in the rhinitis patients, followed by the healthy individuals. These indices were also increased with advanced TNM stage. The overall diagnostic efficacy was ranked as follows: VCA-IgA, Rta-IgA, EA-IgA, and EBV-DNA. The combined diagnosis using these four indices increased the sensitivity to 98.21% and the negative predictive value to 98.61%, without any significant compromise on the test specificity. In conclusion, EA-IgG, Rta-IgG, VCA-IgA, and EBV-DNA expression levels were elevated in nasopharyngeal patients. The combined diagnostic value of these serum indices has important implications in nasopharyngeal carcinoma.

  10. Multimodal imaging and detection strategy with 124 I-labeled chimeric monoclonal antibody cG250 for accurate localization and confirmation of extent of disease during laparoscopic and open surgical resection of clear cell renal cell carcinoma.

    PubMed

    Povoski, Stephen P; Hall, Nathan C; Murrey, Douglas A; Sharp, David S; Hitchcock, Charles L; Mojzisik, Cathy M; Bahnson, Eamonn E; Knopp, Michael V; Martin, Edward W; Bahnson, Robert R

    2013-02-01

    Renal cell carcinoma (RCC) accounts for approximately 85% to 90% of all primary kidney malignancies, with clear cell RCC (ccRCC) constituting approximately 70% to 85% of all RCCs. This study describes an innovative multimodal imaging and detection strategy that uses (124)I-labeled chimeric monoclonal antibody G250 ((124)I-cG250) for accurate preoperative and intraoperative localization and confirmation of extent of disease for both laparoscopic and open surgical resection of ccRCC. Two cases presented herein highlight how this technology can potentially guide complete surgical resection and confirm complete removal of all diseased tissues. This innovative (124)I-cG250 (ie, (124)I-girentuximab) multimodal imaging and detection approach, which would be clinically very useful to urologic surgeons, urologic medical oncologists, nuclear medicine physicians, radiologists, and pathologists who are involved in the care of ccRCC patients, holds great potential for improving the diagnostic accuracy, operative planning and approach, verification of disease resection, and monitoring for evidence of disease recurrence in ccRCC patients.

  11. Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay

    PubMed Central

    Cha, Sang-Ho; Kim, Sung-Hee; Bischoff, Karyn; Kim, Hyun-Jeong; Son, Seong-Wan

    2012-01-01

    A monoclonal antibody (mAb) against zearalenone (ZEN) was produced using ZEN-carboxymethoxylamine and -BSA conjugates. Antibody produced by one clone showing a very high binding ability was selected and found to have a higher affinity for ZEN compared to a commerciall ZEN antibody. We developed two direct competitive ELISA systems using the selected antibody (ZEN-coated and anti-ZEN antibody-coated ELISA). Quantitative ranges for the anti-ZEN antibody-coated ELISA and ZEN-coated ELISA were from 25 to 750 ppb and from 12.5 to 100 ppb, respectively. The detection limit of both methods as measured with standard solutions was 10 ppb. The intra-plate and inter-well variation of both ELISAs were less than 10%. The IC50 values for α-zearalenol, β-zearalenol, α-zearalanol, and β-zearalanol compared to ZEN were 108.1, 119.3, 114.1, and 130.3% for the ZEN-coated ELISA. These values were 100.7, 120.7, 121.6, and 151.6% for the anti-ZEN antibody-coated ELISA. According to the anti-ZEN antibody-coated ELISA, the average recovery rates of ZEN from spiked animal feed containing 150 to 600 ng/mL of ZEN ranged from 106.07 to 123.00% with 0.93 to 2.28% coefficients of variation. Our results demonstrate that the mAb developed in this study could be used to simultaneously screen for ZEN and its metabolites in feed. PMID:22705733

  12. A comparison of a blocking ELISA and a haemagglutination inhibition assay for the detection of antibodies to Avibacterium (Haemophilus) paragallinarum in sera from artificially infected chickens.

    PubMed

    Sun, H; Miao, D; Zhang, P; Gong, Y; Blackall, P J

    2007-10-01

    The ability of blocking ELISAs and haemagglutination-inhibition (HI) tests to detect antibodies in sera from chickens challenged with either Avibacterium (Haemophilus) paragallinarum isolate Hp8 (serovar A) or H668 (serovar C) was compared. Serum samples were examined weekly over the 9 weeks following infection. The results showed that the positive rate of serovar A specific antibody in the B-ELISA remained at 100% from the second week to the ninth week. In chickens given the serovar C challenge, the highest positive rate of serovar C specific antibody in the B-ELISA appeared at the seventh week (60% positive) and was then followed by a rapid decrease. The B-ELISA gave significantly more positives at weeks 2, 3, 7, 8 and 9 post-infection for serovar A and at week 7 post-infection for serovar C. In qualitative terms, for both serovar A and serovar C infections, the HI tests gave a lower percentage of positive sera at all time points except at 9 weeks post-infection with serovar C. The highest positive rate for serovar A HI antibodies was 70% of sera at the fourth and fifth weeks post-infection. The highest rate of serovar C HI antibodies was 20% at the fifth and sixth weeks post-infection. The results have provided further evidence of the suitability of the serovar A and C B-ELISAs for the diagnosis of infectious coryza.

  13. Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

    PubMed

    Dong, Sa; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Xie, Yajing; Zhong, Jianfeng; Xu, Chongxin; Liu, Xianjin

    2017-03-01

    Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10(-8) M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL(-1), respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL(-1)), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

  14. Expression of EBV antibody EA-IgA, Rta-IgG and VCA-IgA and SA in serum and the implication of combined assay in nasopharyngeal carcinoma diagnosis.

    PubMed

    Xia, Cui; Zhu, Kang; Zheng, Guoxi

    2015-01-01

    Epstein-Barr virus (EBV) is an important non-invasive index for nasopharyngeal carcinoma. Serum sialic acid (SA) level was known to be related with tumor progression. Rta protein antibody IgG (Rta-IgG), early antigen antibody (EA-IgA) and viral capsid antibody (VCA-IgA) levels in serum can also be used to effectively monitor the progression of cancer. This study investigated serum level of SA, Rta-IgG, EA-IgA and VCA-IgA in nasopharyngeal cancer patients and the diagnostic value of combined assay. A total of 64 nasopharyngeal cancer patients were recruited, in parallel with 60 benign rhinitis and 60 healthy individuals. Serum SA, EA-IgA, Rta-IgG and VCA-IgA levels were measured by enzyme-linked immunosorbent assay (ELISA). The diagnostic value of these indexes was further evaluated by ROC curve analysis. Logistic regression model was used to analyze the diagnostic implication of combined assay. The expression levels of SA, EA-IgA, Rta-IgG, and VCA-IgA were highest in nasopharyngeal cancer patients. Those indexes were also increased with advanced TNM stage of cancer. The overall diagnostic efficacy was ranked as: VCA-IgA, Rta-IgA, EA-IgA and SA. The combined diagnosis increased the sensitivity to 98.44% and the negative predictive value to 99.03%, without compromising specificity. SA, EA-IgA, Rta-IgG and VCA-IgA expression levels were elevated in nasopharyngeal patients. The combined diagnosis of those serum indexes may improve the diagnostic efficacy of nasopharyngeal carcinoma.

  15. Assessment of Neutralising Activity of Colostrum-Derived, Polyclonal, Bovine Antibodies: Use of the J774A.1 Anthrax Lethal Toxin Cytototoxity Assay

    DTIC Science & Technology

    2005-12-01

    antibodies were isolated from pooled, first milking colostrum. Caseins were removed from colostrum by pH adjustment to 4.6 with acetate buffer...Colostrum whey was subsequently centrifuged, filtered through 0.45 µm filters, adjusted to pH 7.5 and dialysed against PBS. Antibodies recognising...activated Sepharose 4B (Amersham Biosciences, Uppsala, Sweden) at 3 mg/mL of hydrated gel. After DSTO-TR-1832 3 whey was applied to the PA/LF

  16. Evaluation of a Novel Enzyme-Linked Immunosorbent Assay To Detect Immunoglobulin G Antibody to Enolase for Serodiagnosis of Invasive Candidiasis▿

    PubMed Central

    Laín, Ana; Moragues, María D.; Ruiz, Juan Carlos García; Mendoza, Joaquín; Camacho, Ana; del Palacio, Amalia; Pontón, José

    2007-01-01

    The performance of a new test to detect antibodies to Candida albicans recombinant enolase was investigated in 47 immunocompromised and 51 immunocompetent patients. The sensitivity, specificity, and positive and negative predictive values of the test for the diagnosis of invasive candidiasis were 81.0, 83.9, 79.1, and 85.5%, respectively. PMID:17229884

  17. An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

  18. Diagnostic performance of an indirect enzyme-linked immunosorbent assay (ELISA) to detect bovine leukemia virus antibodies in bulk-tank milk samples

    PubMed Central

    Nekouei, Omid; Durocher, Jean; Keefe, Greg

    2016-01-01

    This study assessed the diagnostic performance of a commercial ELISA for detecting bovine leukemia virus antibodies in bulk-tank milk samples from eastern Canada. Sensitivity and specificity of the test were estimated at 97.2% and 100%, respectively. The test was recommended as a cost-efficient tool for large-scale screening programs. PMID:27429469

  19. Localization and distribution of ‘Candidatus Liberibacter asiaticus’ in citrus and periwinkle by direct tissue blot immuno assay with an anti-OmpA polyclonal antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the a-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA poly...

  20. Measuring IgA Anti-β2-Glycoprotein I and IgG/IgA Anti-Domain I Antibodies Adds Value to Current Serological Assays for the Antiphospholipid Syndrome

    PubMed Central

    Pericleous, Charis; Ferreira, Isabel; Borghi, Orietta; Pregnolato, Francesca; McDonnell, Thomas; Garza-Garcia, Acely; Driscoll, Paul; Pierangeli, Silvia; Isenberg, David; Ioannou, Yiannis; Giles, Ian; Meroni, Pier Luigi; Rahman, Anisur

    2016-01-01

    Introduction Currently available clinical assays to detect antiphospholipid antibodies (aPL) test for IgG and IgM antibodies to cardiolipin (aCL) and β2-glycoprotein I (aβ2GPI). It has been suggested that testing for IgA aPL and for antibodies to Domain I (DI), which carries the key antigenic epitopes of β2GPI, could add value to these current tests. We performed an observational, multicenter cohort study to evaluate the utility of IgG, IgM and IgA assays to each of CL, β2GPI and DI in APS. Methods Serum from 230 patients with APS (n = 111), SLE but not APS (n = 119), and 200 healthy controls were tested for IgG, IgM and IgA aCL, aβ2GPI and aDI activity. Patients with APS were further classified into thrombotic or obstetric APS. Logistic regression and receiver operator characteristic analyses were employed to compare results from the nine different assays. Results All assays displayed good specificity for APS; IgG aCL and IgG aβ2GPI assays however, had the highest sensitivity. Testing positive for IgA aβ2GPI resulted in a higher hazard ratio for APS compared to IgM aβ2GPI. Positive IgG, IgM or IgA aDI were all associated with APS, and in subjects positive for aCL and/or aβ2GPI, the presence of aDI raised the hazard ratio for APS by 3–5 fold. IgG aCL, aβ2GPI, aDI and IgA aDI were associated with thrombotic but not obstetric complications in patients with APS. Conclusion Measuring IgG aDI and IgA aβ2GPI and aDI may be useful in the management of patients with APS, particularly thrombotic APS. PMID:27253369

  1. Analysis of the serologic relationship among San Miguel sea lion virus and vesicular exanthema of swine virus isolates. Application of the western blot assay for detection of antibodies in swine sera to these virus types.

    PubMed

    Seal, B S; House, J A; Whetstone, C A; Neill, J D

    1995-04-01

    Caliciviruses are positive-sense single-stranded RNA viruses with a single capsid protein. The serotypes of the marine mammal calicivirus, San Miguel sea lion virus (SMSV), are antigenically related to vesicular exanthema of swine virus (VESV) and are potentially hazardous to swine. Western blot assays using purified SMSV serotypes 1 and 4 were used to further examine the serologic relationship among SMSV and VESV isolates. With the exception of SMSV 8 and SMSV 12, rabbit polyclonal antisera generated against all the available SMSV and VESV isolates reacted positively, as assessed by western blot, with purified capsid protein from SMSV 1 and SMSV 4. Consequently, the SMSV 8 and SMSV 12 virus isolates may not be members of the SMSV/VESV calicivirus group. Using antisera from pigs experimentally inoculated with SMSV and VESV as positive controls, a western blot assay for these virus types was utilized to check for the presence of antibodies to calciviruses in swine sera. Sera from colostrum-deprived gnotobiotic pigs were used as a negative control in all experiments. Examination of sera from domestic and feral swine collected in Iowa, California, and Florida was completed using this technique. The presence of antibodies to these virus types was not detected in any of the porcine sera tested.

  2. Pseudotype-Based Neutralization Assays for Influenza: A Systematic Analysis

    PubMed Central

    Carnell, George William; Ferrara, Francesca; Grehan, Keith; Thompson, Craig Peter; Temperton, Nigel James

    2015-01-01

    The use of vaccination against the influenza virus remains the most effective method of mitigating the significant morbidity and mortality caused by this virus. Antibodies elicited by currently licensed influenza vaccines are predominantly hemagglutination-inhibition (HI)-competent antibodies that target the globular head of hemagglutinin (HA) thus inhibiting influenza virus entry into target cells. These antibodies predominantly confer homosubtypic/strain specific protection and only rarely confer heterosubtypic protection. However, recent academia or pharma-led R&D toward the production of a “universal vaccine” has centered on the elicitation of antibodies directed against the stalk of the influenza HA that has been shown to confer broad protection across a range of different subtypes (H1–H16). The accurate and sensitive measurement of antibody responses elicited by these “next-generation” influenza vaccines is, however, hampered by the lack of sensitivity of the traditional influenza serological assays HI, single radial hemolysis, and microneutralization. Assays utilizing pseudotypes, chimeric viruses bearing influenza glycoproteins, have been shown to be highly efficient for the measurement of homosubtypic and heterosubtypic broadly neutralizing antibodies, making them ideal serological tools for the study of cross-protective responses against multiple influenza subtypes with pandemic potential. In this review, we will analyze and compare literature involving the production of influenza pseudotypes with particular emphasis on their use in serum antibody neutralization assays. This will enable us to establish the parameters required for optimization and propose a consensus protocol to be employed for the further deployment of these assays in influenza vaccine immunogenicity studies. PMID:25972865

  3. Fold Rise in Antibody Titers by Measured by Glycoprotein-Based Enzyme-Linked Immunosorbent Assay Is an Excellent Correlate of Protection for a Herpes Zoster Vaccine, Demonstrated via the Vaccine Efficacy Curve

    PubMed Central

    Gilbert, Peter B.; Gabriel, Erin E.; Miao, Xiaopeng; Li, Xiaoming; Su, Shu-Chih; Parrino, Janie; Chan, Ivan S. F.

    2014-01-01

    Background. The phase III Zostavax Efficacy and Safety Trial of 1 dose of licensed zoster vaccine (ZV; Zostavax; Merck) in 50–59-year-olds showed approximately 70% vaccine efficacy (VE) to reduce the incidence of herpes zoster (HZ). An objective of the trial was to assess immune response biomarkers measuring antibodies to varicella zoster virus (VZV) by glycoprotein-based enzyme-linked immunosorbent assay as correlates of protection (CoPs) against HZ. Methods. The principal stratification vaccine efficacy curve framework for statistically evaluating immune response biomarkers as CoPs was applied. The VE curve describes how VE against the clinical end point (HZ) varies across participant subgroups defined by biomarker readout measuring vaccine-induced immune response. The VE curve was estimated using several subgroup definitions. Results. The fold rise in VZV antibody titers from the time before immunization to 6 weeks after immunization was an excellent CoP, with VE increasing sharply with fold rise: VE was estimated at 0% for the subgroup with no rise and at 90% for the subgroup with 5.26-fold rise. In contrast, VZV antibody titers measured 6 weeks after immunization did not predict VE, with similar estimated VEs across titer subgroups. Conclusions. The analysis illustrates the value of the VE curve framework for assessing immune response biomarkers as CoPs in vaccine efficacy trials. Clinical Trials Registration. NCT00534248. PMID:24823623

  4. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell-core silica nanospheres based on enzyme-linked immunosorbent assay.

    PubMed

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou

    2015-08-05

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL(-1) with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics.

  5. Detection and Quantification of Antibodies to Newcastle Disease Virus in Ostrich and Rhea Sera Using a Liquid Phase Blocking Enzyme-Linked Immunosorbent Assay

    PubMed Central

    de Sousa, Ricardo Luiz Moro; Montassier, Helio José; Pinto, Aramis Augusto

    2000-01-01

    A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method. The cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r = 0.875). The two tests showed good agreement (κ = 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable. PMID:11063502

  6. [Significance of the control serum measurement in infectious disease tests--a case of lot-to-lot variation of anti-HIV antibody assay kit].

    PubMed

    Sasaki, Miho; Sasaki, Kenji

    2002-07-01

    We are using infectious disease test kits consisting of positive serum diluted with negative pooled serum (P-S) and positive control (P-C). In two anti-HIV antibody tests the results for both P-S and P-C fluctuated between positive and negative depending on the lot No. of the reagent. In Western blot tests carried out to confirm the tests, the P-C was found to be positive and the P-S tests were both inconclusive. We speculated that the P-S had very weak antibodies that reacted differently from patient samples. Manufacturers of such kits, however, must supply reagents with appropriate reactivity, so it is important that they be informed of inconsistencies that could invalidate cut-off values and lead to false-positives and false-negatives.

  7. Evaluation of Multiple Immunoassay Technology Platforms to Select the Anti-Drug Antibody Assay Exhibiting the Most Appropriate Drug and Target Tolerance

    PubMed Central

    Collet-Brose, Justine

    2016-01-01

    The aim of this study was, at the assay development stage and thus with an appropriate degree of rigor, to select the most appropriate technology platform and sample pretreatment procedure for a clinical ADA assay. Thus, ELISA, MSD, Gyrolab, and AlphaLISA immunoassay platforms were evaluated in association with target depletion and acid dissociation sample pretreatment steps. An acid dissociation step successfully improved the drug tolerance for all 4 technology platforms and the required drug tolerance was achieved with the Gyrolab and MSD platforms. The target tolerance was shown to be better for the ELISA format, where an acid dissociation treatment step alone was sufficient to achieve the desired target tolerance. However, inclusion of a target depletion step in conjunction with the acid treatment raised the target tolerance to the desired level for all of the technologies. A higher sensitivity was observed for the MSD and Gyrolab assays and the ELISA, MSD, and Gyrolab all displayed acceptable interdonor variability. This study highlights the usefulness of evaluating the performance of different assay platforms at an early stage in the assay development process to aid in the selection of the best fit-for-purpose technology platform and sample pretreatment steps. PMID:27243038

  8. Binding Assays for the Quantitative Detection of P. brevis Polyether Neurotoxins in Biological Samples and Antibodies as Therapeutic Aids for Polyether Marine Intoxication

    DTIC Science & Technology

    1990-05-15

    antibodies, sodium channels 0 6 01 receptor binding, brevetoxins. 1 ABSTRACT (Continue on reverse if necessary and identify by block number) The...redissolved in 0.3 volumes of the original volume 6 in phosphate buffered saline containing 0.01% sodium azide. IgG is stored for longer periods of time... sodium borotritiide used for reduction. Using 1 Ci of tritiated sodium borotritiide, we have been successful in producing between 200 and 250 mCi of

  9. Binding Assays for the Quantitative Detection of P. brevis Polyether Neurotoxins in Biological Samples and Antibodies as Therapeutic Aids for Polyether Marine Intoxication

    DTIC Science & Technology

    1988-12-15

    each in the presence and absence of 0.01% tween; [31 urease enzyme -toxin conjugate lost activity with time (over a 6-month period). Results...specific antibodies. As described earlier, however, problems with stability and sensitivity of the urease enzyme preclude firther work. AntigoatlgG...linked protein A; [51 Immunoassays employing urease enzyme are unstable and exhibit a high degree of backgrow..d activity due to nonspecific adsorption

  10. [Comparison of antibody responses to hepatitis B surface antigen among four recipient groups of hepatitis B vaccines that have been approved in Japan: evaluation using passive hemagglutination assay and chemiluminescent immunoassay].

    PubMed

    Ogata, Norio

    2009-10-01

    In hepatitis B virus (HBV) infection-preventing programs, serum or plasma levels of antibody to hepatitis B surface antigen (anti-HBs) are important to determine whether individuals are protective or not. We compared anti-HBs responses using passive hemagglutination assay (Mycell) and chemiluminescent immunoassay (Architect) among four recipient groups of HB vaccines, Meinyu, HBY, Bimmugen and Heptavax II, that have been approved in Japan. Overall, in a total of 1875 vaccinees Mycell results showed recipient groups of Meinyu and HBY acquired higher anti-HBs levels than those of Bimmugen and Heptavax II. Comparison of anti-HBs responses by both Mycell and Architect in recipient groups of Meinyu (n=150), HBY (n=218), Bimmugen (n=260), and Heptavax II (n=47) demonstrated the order of vaccinees' responses, such as geometric mean titers, ratios of acquiring high antibody levels (Mycell titers over 1024, Architect measurements over 1000 mIU/mL), and ratios of having unsuccessful antibody responses (Mycell titers under 8, Architect measurements under 10 mIU/mL), were somewhat different between the two assays. Comparison of Architect measurements at given Mycell titers revealed Bimmugen-recipients showed significantly lower values than HBY- or Heptavax II-recipients. Around critical protective levels, 5 of 22 Bimmugen-recipients with Mycell titers 16 or 32 showed Architect measurements under 10 mIU/mL, while 8 of 11 Heptavax II-recipients with Mycell titers below 8 demonstrated Architect measurements over 10 mIU/mL. Thus, discrepancies in anti-HBs evaluation between Mycell and Architect seemed to partly depend on administered vaccines. These results indicate anti-HBs concentration should be evaluated carefully so that we could completely prevent HBV infection.

  11. Accurate quantitation for in vitro refolding of single domain antibody fragments expressed as inclusion bodies by referring the concomitant expression of a soluble form in the periplasms of Escherichia coli.

    PubMed

    Noguchi, Tomoaki; Nishida, Yuichi; Takizawa, Keiji; Cui, Yue; Tsutsumi, Koki; Hamada, Takashi; Nishi, Yoshisuke

    2017-03-01

    Single domain antibody fragments from two species, a camel VHH (PM1) and a shark VNAR (A6), were derived from inclusion bodies of E. coli and refolded in vitro following three refolding recipes for comparing refolding efficiencies: three-step cold dialysis refolding (TCDR), one-step hot dialysis refolding (OHDR), and one-step cold dialysis refolding (OCDR), as these fragments were expressed as 'a soluble form' either in cytoplasm or periplasm, but the amount were much less than those expressed as 'an insoluble form (inclusion body)' in cytoplasm and periplasm. In order to verify the refolding efficiencies from inclusion bodies correctly, proteins purified from periplasmic soluble fractions were used as reference samples. These samples showed far-UV spectra of a typical β-sheet-dominant structure in circular dichroism (CD) spectroscopy and so did the refolded samples as well. As the maximal magnitude of ellipticity in millidegrees (θmax) observed at a given wave length was proportional to the concentrations of the respective reference samples, we could draw linear regression lines for the magnitudes vs. sample concentrations. By using these lines, we measured the concentrations for the refolded PM1 and A6 samples purified from solubilized cytoplasmic insoluble fractions. The refolding efficiency of PM1 was almost 50% following TCDR and 40% and 30% following OHDR and OCDR, respectively, whereas the value of A6 was around 30% following TCDR, and out of bound for quantitation following the other two recipes. The ELISA curves, which were derived from the refolded samples, coincided better with those obtained from the reference samples after converting the values from the protein-concentrations at recovery to the ones of refolded proteins using recovery ratios, indicating that such a correction gives better results for the accurate measure of the ELISA curves than those without correction. Our method require constructing a dual expression system, expressed both in

  12. Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae.

    PubMed

    Okada, Munenori; Asai, Tetsuo; Futo, Satoshi; Mori, Yasuyuki; Mukai, Tetsuya; Yazawa, Shigeto; Uto, Takehiko; Shibata, Isao; Sato, Shizuo

    2005-02-25

    To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.

  13. Production of pseudorabies virus recombinant glycoprotein B and its use in an agar gel immunodiffusion (AGID) test for detection of antibodies with sensitivity and specificity equal to the virus neutralization assay.

    PubMed

    Serena, María Soledad; Geisler, Christoph; Metz, Germán Ernesto; Mórtola, Eduardo Carlos; Echeverría, María Gabriela

    2016-04-01

    Pseudorabies virus (PrV) causes Aujeszky's disease (AD), which affects mainly swine, but also cattle, sheep, and wild animals, resulting in substantial economic losses due to animal mortality and lost productivity worldwide. To combat PrV, eradication programs using PrV strains lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable, easy-to-use, and sensitive tests that can detect PrV infection in pigs infected with either wild-type virus or vaccine strain (gE-deleted) virus. To meet this demand, we used the baculovirus-insect cell system to produce recombinant glycoprotein B (gB) as antigen for an immune assay. The high GC-content (70% average) of the gB gene from the Argentinian PrV CL15 strain necessitated the use of betaine as a PCR enhancer to amplify the extracellular domain. Recombinant gB was expressed at high levels and reacted strongly with sera from PrV infected pigs. We used the recombinant gB to develop an agar gel immunodiffusion (AGID) test for detection of PrV antibodies. Compared to the gold standard virus neutralization (VN) assay, the AGID sensitivity and specificity were 95% and 96.6% respectively. Thus, recombinant gB produced in the baculovirus-insect cell system is a viable source of antigen for the detection of PrV antibodies in AGID tests. Considering its relatively lower cost, simplicity of use and result interpretation, our AGID is a valuable alternative tool to the VN assay.

  14. Profile of the MP Diagnostics HTLV Blot 2.4 test: a supplemental assay for the confirmation and differentiation of antibodies to HTLV-1 and HTLV-2.

    PubMed

    Miller, Liane

    2016-01-01

    As the first US FDA-approved assay for supplemental HTLV testing, the MP Diagnostics HTLV Blot 2.4 is an effective and efficient method for confirming and differentiating HTLV type infection in repeatedly reactive samples. Novel and patented antigens added increased sensitivity in identifying specimens from infected individuals while differentiating those from uninfected individuals with false reactivity.

  15. Enzyme-linked immunosorbent assay to differentiate the antibody responses of animals infected with Brucella species from those of animals infected with Yersinia enterocolitica O9.

    PubMed

    Erdenebaatar, Janchivdorj; Bayarsaikhan, Balgan; Watarai, Masahisa; Makino, Sou-ichi; Shirahata, Toshikazu

    2003-07-01

    Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.

  16. Rapid detection of serum antibody by dual-path platform VetTB assay in white-tailed deer infected with Mycobacterium bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine tuberculosis (TB) in various cervid species remains a significant problem affecting both farmed herds and free-ranging populations. Traditional skin testing was demonstrated to have serious limitations in certain deer species, whereas emerging serological assays showed promising diagnostic pe...

  17. The application of monoclonal antibodies in cancer diagnosis.

    PubMed

    Zhang, Xuemei; Soori, Gamini; Dobleman, Thomas J; Xiao, Gary G

    2014-01-01

    Cancer becomes the second leading cause of death in the world. An effective strategy for early diagnosis of the disease is key to reduce the mortality and morbidity. Development of effective monoclonal antibody (mAb)-based assays or diagnostic imaging techniques for detection of antigens and small molecules that are released from cancerous cells will enhance modern diagnostic medicine of cancer significantly. Although mAb technology is still under development, recent advances in preparation of recombinant antigen and antibody engineering techniques have dramatically enhanced the applications of this technology in cancer diagnosis. Compared with other methods, mAb-based assays may provide spatial, temporal, accurate and quantitative measurement for diagnosis of the disease. This review summarizes the progress of the mAb-based assays in the field of molecular diagnosis of cancer.

  18. Design and validation of a qPCR assay for accurate detection and initial serogrouping of Legionella pneumophila in clinical specimens by the ESCMID Study Group for Legionella Infections (ESGLI).

    PubMed

    Mentasti, M; Kese, D; Echahidi, F; Uldum, S A; Afshar, B; David, S; Mrazek, J; De Mendonça, R; Harrison, T G; Chalker, V J

    2015-07-01

    Prompt detection of Legionella pneumophila is essential for rapid investigation of legionellosis. Furthermore, as the majority of L. pneumophila infections are caused by serogroup 1 (sg1) strains, rapid identification of such strains can be critical in both routine and outbreak scenarios. The ESCMID Study Group for Legionella Infections (ESGLI) was established in 2012 and immediately identified as a priority the validation of a reliable, easy to perform and interpret, cost-effective qPCR assay to standardise the detection of L. pneumophila DNA amongst members. A novel L. pneumophila assay targeting the mip gene was designed and combined with previously published methodologies amplifying the sg1 marker (wzm) and the green fluorescent protein gene (gfp) internal process control. The resulting triplex assay was validated internationally on the three qPCR platforms used by the majority of European Legionella reference laboratories: ABI 7500 (Life Technologies), LightCycler 480 Instrument II (Roche) and Rotor-Gene Q (Qiagen). Clinical and EQA specimens were tested together with a large panel of strains (251 in total) to validate the assay. The assay proved to be 100% specific for L. pneumophila and sg1 DNA both in silico and in vitro. Efficiency values for mip and wzm assays ranged between 91.97 and 97.69%. Limit of detection values estimated with 95% confidence were adopted for mip and wzm assays on all three qPCR platforms. Inhibition was not observed. This study describes a robust assay that could be widely implemented to standardise the molecular detection of L. pneumophila among ESGLI laboratories and beyond.

  19. A survey for antibodies to equine arteritis virus in donkeys, mules and zebra using virus neutralisation (VN) and enzyme linked immunosorbent assay (ELISA).

    PubMed

    Paweska, J T; Binns, M M; Woods, P S; Chirnside, E D

    1997-01-01

    A seroepidemiological survey of donkeys in South Africa (n = 4300) indicated a wide distribution and increasing prevalence of antibodies to equine arteritis virus (EAV). Donkey sera inhibited equine arteritis virus infection in virus neutralisation (VN) tests and in ELISA specifically bound to a recombinant antigen derived from the Bucyrus isolate of EAV. These results suggest that donkeys have been exposed to the same serotype of this virus as circulates among horses. A good correlation existed between EAV neutralising antibody titres and ELISA absorbance values (0.8631); the ELISA was sensitive and specific (99.2% and 80.3% respectively) for donkey sera when compared to the VN test and the recombinant ELISA antigen did not cross-react with sera positive for common African equine pathogens. VN+ ELISA+ donkeys were also found in Morocco and Zimbabwe and seropositive mules in both South Africa and Morocco. No seropositive zebra (n = 266) were detected from game reserves or zoos in 9 countries. The results confirm that in addition to horses and donkeys, mules are naturally infected with EAV.

  20. Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins

    PubMed Central

    Rosen, Osnat; Ozeri, Eyal; Barnea, Ada; David, Alon Ben; Zichel, Ran

    2016-01-01

    Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV < 13%. Most importantly, the antitoxin potency measured by the in vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p < 0.0001). Thus, this new in vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval. PMID:27669303

  1. Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

    PubMed Central

    Sharma, Shashi K.; Ferreira, Joseph. L.; Eblen, Brian S.; Whiting, Richard C.

    2006-01-01

    An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event. PMID:16461671

  2. Accuracy of AccessBio Immunoglobulin M and Total Antibody Rapid Immunochromatographic Assays for the Diagnosis of Acute Scrub Typhus Infection

    DTIC Science & Technology

    2010-02-01

    Median no. of Median (range) reciprocal Infection group or specimens days of fever Verification scrub typhus lgM titer on Geographical source...five for the murine typhus group, seven for the leptospirosis group, nine for the malaria group, four for the dengue fever group, and five for the...diagnoses of a variety of acute tropical fevers prevalent in Southeast Asia suggest that the AccessBio scrub typhus IgM ICT is suitably accurate for

  3. Proposed mechanisms whereby the T3U, dialyzed fraction (%FT4) and antibody extracted (AE) T4 normalize binding protein effects in free T4 (FT4) assays do not explain the findings in monthyroidally ill (NTI) patients

    SciTech Connect

    Witherspoon, L.R.; Shuler, S.E.; Gilbert, S.

    1984-01-01

    Apparently falsely low FT4 results are observed in patients with NTI. FT4 estimates in NTI using some AE assays are lower than corresponding FTI. Both are lower than equilibrium dialysis (ED) results. These three approaches to FT4 estimation are similar - the T3U, %FT4 and %AE all are inversely related to TBG concentration. A FT4 estimate may be obtained by multiplying any of these x total T4 concentration. The mass of AE T4 may be quantitated by competitive binding, either after separation of AE T4 from serum (two step) or by employing a radiolabeled T4 analog recognized by the antibody but not by TBG. The authors made FT4 estimates in euthyroid patients with high, normal or low TBG concentrations; in hyper- and hypothyroid patients; and in NTI patients presumed euthyroid. Each approach produces similar results in euthyroid patients. The T3U and some AE assays display TBG dependence at TBG <10 and>50 ..mu..g/ml. In hyperthyroidism, the T3U is high, %FT4 normal or high, while the %AE is low. All FT4 results are high in hyperthyroidism. In hypothyroidism the T3U is low, %FT4 normal or low, while the %AE is high. All FT4 results are low in hypothyroidism. In NTI, the T3U is appropriate for the TBG concentration, %FT4 is often high and %AE is low. Furthermore, methods for quantifying the AE mass do not agree - index results are low, analog results lowers, such that %B/B is>100%, while the two step results are normal. The suggestion that the T3U, %FT4 and %AE all serve to normalize TBG effects in FT4 assays is inadequate. AE assays should be employed clinically with caution until the apparent anomalies, especially those seen in the analog systems, are explained.

  4. Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans.

    PubMed

    Galula, Jedhan U; Chang, Gwong-Jen J; Chuang, Shih-Te; Chao, Day-Yu

    2016-02-01

    The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Paired-receiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP- and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP- and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great

  5. Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule

    PubMed Central

    Chen, Weiye; Li, Cuicui; Xie, Meimei; Bu, Zhigao

    2016-01-01

    From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP) (rPPRV-GFP), an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT). Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field. PMID:27768770

  6. Development and validation of an enzyme-linked immunosorbent assay for the detection of circulating antibodies raised against growth hormone as a consequence of rbST treatment in cows.

    PubMed

    Rochereau-Roulet, Sandrine; Gaudin, Isabelle; Chéreau, Sylvain; Prévost, Stéphanie; André-Fontaine, Geneviève; Pinel, Gaud; Le Bizec, Bruno

    2011-08-26

    The recombinant bovine growth hormone (rbST) is used to increase lactating performances of dairy cows. Administration of rbST is banned in the European Union; nevertheless, its use is probable. Until now, efficient analytical strategies to detect such practice are based on the direct detection by mass spectrometry of the presence of rbST in biological fluids, which suits for confirmatory purposes. Current screening strategies do not offer satisfactory performances; therefore, alternative screening strategies are required. The aim of the present work is to develop and validate an ELISA to measure the production of specific antibodies upon rbST in bovine sera. In this immunoassay, rbST is absorbed onto microtiter plate. After specific purification of the antibodies in serum, samples are analysed and the presence of antibodies anti-rbST is detected by Protein G peroxidase conjugate and 2-2'-azino di-ethyl benz-thiazoline-6-sulphonic acid (ABTS). The mean reproducibility of the OD (λ=405 nm) measurement was calculated with a CV of 13%. The intra- and inter-assay CVs ranged from 0.79% to 7.91% and from 2.69% to 20% respectively. The test presents cross-reaction with other growth hormones such as the recombinant equine (reST) and porcine (pST) (100% and 80% respectively). The specificity of the test toward rbST anabolic treatment was confirmed through the analysis of sera samples collected on animals administered with other anabolic compounds (steroids). The performances of the present anti-rbST ELISA proves its efficiency as a new screening tool to highlight illegal administration of rbST in cattle up to at least 3 weeks after treatment.

  7. Efficacy of IgM anti-blood type antibody monitoring by enzyme-linked immunosorbent assay after renal transplantation across the blood barrier: high-dose immunoglobulin administration blocks IgM rather than IgG anti-blood type antibodies.

    PubMed

    Ishida, H; Tanabe, K; Furusawa, M; Isizuka, T; Tokumoto, T; Shimmura, H; Shimizu, T; Miyamoto, N; Hayashi, T; Toma, H

    2004-09-01

    We used an enzyme linked immunosorbent assay (ELISA) to investigate the presence of subtypes of anti-blood-type antibodies in patients with biopsy-proven humoral rejection after ABO-incompatible renal transplantation. High agglutinin IgG and IgM anti-blood type antibodies from 12 ABO-incompatible recipients with vascular rejection were separately assessed using an ELISA. Patients who exhibited excellent renal function despite high agglutinin titers of anti-blood-type antibodies(n = 8) were also examined. All 12 rejection patients exhibited highly elevated titers of IgG and IgM, while the eight stable patients exhibited only slightly elevated IgG titers, but not IgM. IgG and IgM titers did not change after plasmapheresis and steroid pulse therapy, whereas IVIg treatment significantly blocked both IgG and IgM, with IgM being blocked to a larger extent than IgG. Blocking of IgM seems to play an important role in improving ABO-incompatible grafts.

  8. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

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

  9. Clinical laboratory applications of monoclonal antibodies.

    PubMed Central

    Payne, W J; Marshall, D L; Shockley, R K; Martin, W J

    1988-01-01

    Monoclonal antibody (MAb) technology is well recognized as a significant development for producing specific serologic reagents to a wide variety of antigens in unlimited amounts. These reagents have provided the means for developing a number of highly specific and reproducible immunological assays for rapid and accurate diagnosis of an extensive list of diseases, including infectious diseases. The impact that MAbs have had in characterizing infectious disease pathogens, as well as their current and future applications for use in clinical microbiology laboratories, is reviewed. In addition, the advantages (and disadvantages) of the use of MAbs in a number of immunoassays, such as particle agglutination, radioimmunoassays, enzyme-linked immunosorbent assays, immunofluorescent-antibody assays, and immunohistology, are explored, including the use of these reagents in novel test system assays. Also, nucleic acid probe technology is compared with the use of MAbs from the perspective of their respective applications in the diagnosis of infectious disease agents. There is no question that hybridoma technology has the potential to alter significantly the methods currently used in most clinic