Sample records for accurate antibody assays

  1. Standardization of anti-DNA antibody assays. (United States)

    Pisetsky, David S


    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.

  2. Production and assay of forskolin antibodies

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


    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.

  3. A novel assay for monitoring internalization of nanocarrier coupled antibodies

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    Pickering Edward M


    Full Text Available Abstract Background Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA. Results The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv (F5 antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol-linked lipid (DSPE-PEG-NTA-Ni was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay. Conclusion The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.

  4. Monoclonal antibody technologies and rapid detection assays (United States)

    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...

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

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    Chao Shan


    Full Text Available 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.

  6. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

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    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))


    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

  7. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity. (United States)

    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


    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.

  8. Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo. (United States)

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


    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.

  9. 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. (United States)

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


    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.

  10. ABAP: antibody-based assay for peptidylarginine deiminase activity. (United States)

    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


    Members of the family of peptidylarginine deiminases (PADs, EC 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.

  11. Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.

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    Christine Bee

    Full Text Available Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (K(D values spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent K(D and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.

  12. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification (United States)

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


    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.

  13. Antibody-Based Assays for Phenotyping of Extracellular Vesicles

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    Lotte Hatting Pugholm


    Full Text Available 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.

  14. ELISPOT Assay for Measurement of Antigen-Specific and Polyclonal Antibody Responses. (United States)

    Lycke, Nils; Coico, Richard


    The enzyme-linked immunospot (ELISPOT) assay for detection of antigen-specific and polyclonal antibody responses by single antibody-secreting cells has become the method of choice due to its cell-based quantitative value. Antigen stability and specificity and the diversity of antigens that can be used in the assay have contributed to the translational application of ELISPOT as demonstrated by many FDA-approved clinical tests that employ this technique. In addition, the ELISPOT assay can be used to detect two antigenically different secreted antibodies simultaneously by two-color analysis and offers the unique possibility of quantifying the number of antibody molecules secreted per cell.

  15. Multicentre comparison of a diagnostic assay: Aquaporin-4 antibodies in neuromyelitis optica

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    P. Waters (Patrick); M. Reindl (Markus); A. Saiz (Albert Abe); K. Schanda (Kathrin); F. Tuller (Friederike); V. Kral (Vlastimil); P. Nytrova (Petra); O. Sobek (Ondrej); H.H. Nielsen (Helle Hvilsted); T. Barington (Torben); S.T. Lillevang (Søren T.); Z. Illes (Zsolt); K. Rentzsch (Kristin); A. Berthele (Achim); T. Berki (Tímea); L. Granieri; A. Bertolotto (Antonio); B. Giometto; L. Zuliani (Luigi); D. Hamann (Dörte); J.L. Van Pelt (Joost L.); R.Q. Hintzen (Rogier); R. Höftberger (Romana); C. Costa (Carme); M. Comabella (Manuel); X. Montalban (Xavier); M. Tintoré; A. Siva (Aksel); A. Altintas (Ayse); G. Deniz (Gunnur); M. Woodhall (Mark); J. Palace (Jacqueline); F. Paul (Friedemann); H.P. Hartung; O. Aktas (Orhan); S. Jarius (Sven); B. Wildemann (Brigitte); C. Vedeler (Christian); A. Ruiz (Anne); M.I. Leite (M. Isabel); P. Trillenberg (Peter); M. Probst (Monika); S. Saschenbrecker (Sandra); A.J.P.E. Vincent (Arnoud); R. Marignier (Romain)


    textabstractObjective 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 (

  16. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C


    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...

  17. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification. (United States)

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


    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.

  18. The line blot assay: problems with titrating first and second antibodies for Western blot and immunohistochemistry assays? (United States)

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


    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.

  19. A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

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    Wychowski Czeslaw


    Full Text Available Abstract Background/Aim The role of humoral immunity in hepatitis C virus (HCV infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. Methods The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. Results The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively. The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134 for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85 for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles

  20. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye. (United States)

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


    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. Enzyme-linked immunosorbent assay for detection of streptolysin O antibodies.


    Reitano, M; Pisano, M. A.; Eriquez, L A; D'Amato, R F


    An enzyme-linked immumosorbent assay (ELISA), based upon the detection of streptolysin O antibodies in human sera, was developed. Disposable polystyrene tubes, sensitized with streptolysin O antigen, were used as the test vehicles. Corresponding antibodies, present in test sera, were detected by binding of the antibodies to goat anti-human immunoglobulin G conjugated to horseradish peroxidase. Demonstration of bound conjugate was accomplished by monitoring peroxidase activity spectrophotometr...

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


    PARTRIDGE, MICHAEL A.; Shobha Purushothama; Chinnasamy Elango; Yanmei Lu


    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, a...

  3. Detection of infliximab levels and anti-infliximab antibodies : a comparison of three different assays

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    Casteele, N. Vande; Buurman, D. J.; Sturkenboom, M. G. G.; Kleibeuker, J. H.; Vermeire, S.; Rispens, T.; van der Kleij, D.; Gils, A.; Dijkstra, G.


    Background Formation of antibodies to infliximab (ATI) inversely correlates with functional drug levels and clinical outcome. Comparison of drug levels and anti-drug antibody monitoring is hampered by lack of standardisation. Aim To determine the correlation between three different assays for measur

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

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


    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. Solid phase radioimmunoassay using labelled antibodies: a conceptual framework for designing assays

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    Kalmakoff, J.; Parkinson, A.J.; Crawford, A.M.; Williams, B.R.


    A simple theoretical model for the antigen-antibody reaction is presented and used to evaluate the optimum conditions for designing solid phase radioimmunoassay (RIA) using labelled antibodies. Both theoretical and experimental data are presented, using a wide variety of antigens and their corresponding antibodies. The types of RIA described include the direct, the indirect, and sandwich assays for detecting either antigen or antibody. The experimental results confirm in a semiquantitative manner that the greatest sensitivity of the RIA is achieved when the smallest amount of labelled antibody is used, that whenever possible the antigen/antibody ratio should be greater than unity (greater than 1), and that the formation of the antigen-antibody complex is dependent on the mass action effect.

  6. Switching assay as a novel approach for specific antigen- antibody interaction analysis using magnetic nanoparticles (United States)

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


    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.

  7. Indirect ELISA and indirect immunofluorescent antibody assay for detecting the antibody against murine norovirus S7 in mice. (United States)

    Kitagawa, Yota; Tohya, Yukinobu; Ike, Fumio; Kajita, Ayako; Park, Sang-Jin; Ishii, Yoshiyuki; Kyuwa, Shigeru; Yoshikawa, Yasuhiro


    To evaluate murine norovirus (MNV) infection in laboratory mice, we attempted to develop an enzyme-linked immunosorbent assay (ELISA) system and an indirect immunofluorescent antibody (IFA) assay for detecting the anti-MNV-S7 antibody in mice. MNV-S7, which was isolated in Japan, was used in both assays. The antigen for ELISA was prepared by ultracentrifugation of culture supernatants of RAW 264 cells infected with MNV-S7. Positive sera were obtained from 6-week-old, female C57BL/6JJcl mice inoculated orally with MNV-S7. IFA against infected RAW 264 cells was able to discriminate positive sera from negative sera. Indirect ELISA was performed using 96-well ELISA plates coated with formalin-treated MNV-S7 antigen. In this ELISA system, mouse sera obtained 2 weeks after infection or later showed significantly high OD values and were judged positive. An equal level of anti-MNV-S7 antibody response was observed in BALB/cAJcl, C57BL/6JJcl, DBA/2JJcl, and Jcl:ICR mice; whereas, C3H/HeJJcl mice demonstrated slightly lower antibody production 4 weeks after infection. We also used this ELISA system to evaluate 77 murine serum samples obtained from 15 conventional mouse rooms in research facilities in Japan and found that approximately half of the serum samples contained antibody to MNV-S7. We found that some serum samples were negative for antibodies to mouse hepatitis virus and Mycoplasma pulmonis but positive for antibody to MNV-S7. The results suggest that the MNV infection is more prevalent than other infections such as mouse hepatitis virus and Mycoplasma pulmonis in conventional mouse colonies in Japan, as is the case in other areas of the world.

  8. Dye Labelled Monoclonal Antibody Assay for Detection of Toxic Shock Syndrome Toxin -1 from Staphylococcus Aureus

    Directory of Open Access Journals (Sweden)

    V Javid Khojasteh


    Full Text Available Objective: The aim of study was to develop a rapid assay, dye labelled monoclonal antibody assay (DLMAA, using non-radioactive organic synthetic dyes for identification of Toxic Shock Syndrome Toxin-1 (TSST-1 producing strains of Staphylococcus aureus.Materials and Methods: The assay protocol required only two simple steps; addition of TSST-1 antigen to a nitrocellulose membrane and then adding a colloidal dye labelled antibody (D/A suspension detection reagent.Results: The sensitivity and specificity of the assay was determined relative to positive and negative strains compared to an ELISA assay. Overall 100% agreement was found between both assays. The sensitivity for detection of TSST-1 was 30 ng.Conclusion: The DLMAA did not require handling and disposal of radioactive materials. It is a rapid qualitative technique for detection of TSST-1 toxin at room temperature within a short time.

  9. A rapid and convenient dot-immunobinding assay for chicken egg-yolk antibodies

    Institute of Scientific and Technical Information of China (English)



    The dot-immunobinding assay was applied to investigate the characteristics of chicken egg yolk antibodies. This method of assay was proved to be a rapid and simple method to demonstrate and characterize the egg-yolk antibody IgY in comparison with the traditional ELISA assay. By using the BandScan software, the gray scale value of dots and the background could be determined. According to the intensity of dots (gray scale value) compared to the standard sample of 10 μg, how much IgY remained can be determined in a shorter time.

  10. Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Pierce, E.A.; Dame, M.C.; DeLuca, H.F.


    A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D/sub 3/ and should be useful for the detection of antibodies to ligand-binding proteins in general.

  11. Screening a hybridoma producing a specific monoclonal antibody to HLA-A24+Bw4 antigen by cytotoxicity inhibition assay. (United States)

    Hiroishi, S; Kaneko, T; Arita, J


    A hybridoma secreting a monoclonal antibody (Tsa-1, IgG3) reacting specifically to HLA-A24+Bw4 was screened by cytotoxicity inhibition assay and micrototoxicity test. The R value of the antibody was 0.843.

  12. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor (United States)

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique


    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

  13. Identification of Haemophilus influenzae type b by a monoclonal antibody coagglutination assay.


    Hamel, J.; Brodeur, B R; Belmaaza, A; Montplaisir, S; Musser, J M; Selander, R K


    A coagglutination assay using monoclonal antibody is described for the identification of Haemophilus influenzae type b. An immunoglobulin G2a monoclonal antibody, Hb-2, directed against a serotype-specific outer membrane protein of H. influenzae type b was adsorbed to Staphylococcus aureus Cowan 1 cells. In a dot enzyme immunoassay, Hb-2 reacted with 453 of 455 H. influenzae type b isolates and did not react with H. influenzae of other serotypes, untypeable H. influenzae strains, or other bac...

  14. Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Wu; Zhangyuan Liao; Jing Ye; Huiqing Dong; Chaodong Wang; Piu Chan


    A total of 66 samples (from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, and 13 cases with optic neuritis) were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay.The sensitivities and specificities of the two assays were similar.We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay.A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the sera of neuromyelitis optica patients.No significant correlations were identified with onset age or disease duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica.The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

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


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


    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. Antibod...

  16. 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. (United States)

    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


    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.

  17. Impact of immunization technology and assay application on antibody performance--a systematic comparative evaluation.

    Directory of Open Access Journals (Sweden)

    Michael C Brown

    Full Text Available Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC, and enzyme-linked immunosorbent assays (ELISA, among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein, DNA prime/protein fragment-boost ("DNA immunization"; 3 per protein, and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot. Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry, although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.

  18. Activity assays and immunoassays for plasma Renin and prorenin: information provided and precautions necessary for accurate measurement

    DEFF Research Database (Denmark)

    Campbell, Duncan J; Nussberger, Juerg; Stowasser, Michael;


    BACKGROUND: Measurement of plasma renin is important for the clinical assessment of hypertensive patients. The most common methods for measuring plasma renin are the plasma renin activity (PRA) assay and the renin immunoassay. The clinical application of renin inhibitor therapy has thrown...... into focus the differences in information provided by activity assays and immunoassays for renin and prorenin measurement and has drawn attention to the need for precautions to ensure their accurate measurement. CONTENT: Renin activity assays and immunoassays provide related but different information....... Whereas activity assays measure only active renin, immunoassays measure both active and inhibited renin. Particular care must be taken in the collection and processing of blood samples and in the performance of these assays to avoid errors in renin measurement. Both activity assays and immunoassays...

  19. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies (United States)

    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...

  20. Safe and Objective Assay of Enterovirus 71 Neutralizing Antibodies via Pseudovirus

    Institute of Scientific and Technical Information of China (English)

    JIN Jun; XU Lin; GUO Shi-jie; SUN Shi-yang; ZHANG Shu; ZHU Chang-lin; KONG Wei; JIANG Chun-lai


    Current serum neutralization assays based on the inhibition of the eytopathic effect(Nt-CPE) need to manipulate live viruses,which are time-consuming,labor-intensive,and have the potential exposure to infectious agents,so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71(EV71 ) neutralizing antibodies was developed.First,we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome.Vero cells infected with 200 CCID50(50% cell culture infective dose) of EV71 pseudovirus for 24 h were found to have the best performance.Seval sera were measured by EV71 pseudoparticle neutralization assay(Nt-PPN) and the conventional serological method Nt-CPE.Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods.Overall,the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing antibodies.This method can be used for detecting neutralizing antibodies of other picornaviruses,such as hepatitis A virus(HAV) and coxsackievirus 16(CVA16),and make it possible to determine whether there is cross-reactivity between EV71 and CVA16.

  1. Indirect solid-phase immunosorbent assay for detection of arenavirus antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, A.P.; Rezapkin, G.V.; Dzagurova, T.K.; Tkachenko, E.A. (Institute of Poliomyelitis anU Viral Encephalities of the U.S.S.R. Academy of Medical Sciences, Moscow)


    Indirect enzyme-linked immunosorbent assay (ELISA) and solid phase radioimmunoassay (SPRIA) using either enti-human or anti-mouse IgG labelled with horseradish peroxidase and /sup 125/I, respectively, were developed for the detection of Junin, Machupo, Tacaribe, Amapari, Tamiami, Lassa and LCM arenaviruses. Both methods allow high sensitivity detection of arenavirus antigens and antibodies.

  2. Effect of treatment on serum antibody to Hymenolepis nana detected by enzyme-linked immunosorbent assay. (United States)

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


    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.

  3. Effect of treatment on serum antibody to Hymenolepis nana detected by enzyme-linked immunosorbent assay. (United States)

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


    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

  4. Comparison of the latex agglutination test with the hemagglutination inhibition test, enzyme-linked immunosorbent assay, and neutralization test for detection of antibodies to rubella virus.


    Meegan, J M; Evans, B. K.; Horstmann, D. M.


    The ability of a rapid, latex agglutination test to diagnose rubella infection and to measure immune status was evaluated by comparison with the hemagglutination-inhibition (HAI) test, enzyme-linked immunosorbent assay (ELISA), and the neutralization (NT) test. The latex agglutination test accurately detected serological conversions in 74 pairs of sera representing 21 natural infections and 53 immunizations. The antibody levels of 276 sera from the general population were determined by latex ...

  5. High-throughput immunoturbidimetric assays for in-process determination of polyclonal antibody concentration and functionality in crude samples

    DEFF Research Database (Denmark)

    Bak, Hanne; Kyhse-Andersen, J.; Thomas, O.R.T.


    We present fast, simple immunoturbidimetric assays suitable for direct determination of antibody 'concentration' and 'functionality' in crude samples, such as in-process samples taken at various stages during antibody purification. Both assays display excellent linearity and analytical recovery....... The 'functionality' assay displayed concentration dependent sensitivity to interference for ammonium sulphate and Tris(hydroxymethyl)-amino-methane, but was essentially unaffected by all other salts and buffer combinations tested. The immunoturbidimetric assays described here are generically applicable to polyclonal...... antibodies, require only basic laboratory equipment, are robust, fast, cheap, easy to perform, and readily adapted to automation....

  6. Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay. (United States)

    Toh, Saw Yi; Citartan, Marimuthu; Gopinath, Subash C B; Tang, Thean-Hock


    The application of antibodies in enzyme-linked immunosorbent assay (ELISA) is the basis of this diagnostic technique which is designed to detect a potpourri of complex target molecules such as cell surface antigens, allergens, and food contaminants. However, development of the systematic evolution of Ligands by Exponential Enrichment (SELEX) method, which can generate a nucleic acid-based probe (aptamer) that possess numerous advantages compared to antibodies, offers the possibility of using aptamers as an alternative molecular recognition element in ELISA. Compared to antibodies, aptamers are smaller in size, can be easily modified, are cheaper to produce, and can be generated against a wide array of target molecules. The application of aptamers in ELISA gives rise to an ELISA-derived assay called enzyme-linked apta-sorbent assay (ELASA). As with the ELISA method, ELASA can be used in several different configurations, including direct, indirect, and sandwich assays. This review provides an overview of the strategies involved in aptamer-based ELASA.

  7. Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics (United States)

    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


    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

  8. Prevalence of antibodies to type A influenza virus in wild avian species using two serologic assays (United States)

    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.


    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.

  9. Development and validation of a homogeneous mobility shift assay for the measurement of infliximab and antibodies-to-infliximab levels in patient serum. (United States)

    Wang, Shui-Long; Ohrmund, Linda; Hauenstein, Scott; Salbato, Jared; Reddy, Rukmini; Monk, Patrick; Lockton, Steven; Ling, Nicholas; Singh, Sharat


    Antibody-based drugs such as infliximab (IFX) are effective for the treatment of inflammatory bowel disease (IBD) and other immune-mediated disorders. The development of antibodies against these drugs may result in unfavorable consequences, including the loss of drug efficacy, hypersensitivity reactions, and other adverse events. Therefore, accurate monitoring of serum drug and anti-drug antibody levels should be an important part of therapy for patients being treated with an antibody-based drug. Current methods for the assessment of anti-drug antibodies and drug levels, involving various bridging ELISA and radioimmunoassay techniques, are limited by their sensitivity, interference, and/or complexity. To overcome these limitations, we have developed a non-radiolabeled homogeneous mobility shift assay (HMSA) to measure the antibodies-to-infliximab (ATI) and IFX levels in serum samples. Full method validation was performed on both the ATI- and IFX-HMSA, and the clinical sample test results were also compared with those obtained from a bridging ELISA method to evaluate the difference in performance between the two assays. Validation of the ATI-HMSA revealed a lower limit of quantitation of 0.012 μg/mL in serum. The linear range of quantitation was 0.029-0.54 μg/mL. The intra- and inter-assay precision was less than 20% of coefficient of variation (CV), and the accuracy (% error) of the assay was less than 20%. In serum samples, ATI as low as 0.036 μg/mL can be measured, even in the presence of 60 μg/mL of IFX in the serum. Sera from 100 healthy subjects were tested to determine the cut point of the assay. ATI-positive samples that had been previously analyzed by using a bridging ELISA from 100 patients were also measured by the new method. There was a high correlation between the two methods for ATI levels (pELISA method. Validation of the mobility shift IFX assay also showed high assay sensitivity, precision and accuracy. The HMSA method may also be applied to

  10. Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

    Directory of Open Access Journals (Sweden)

    Kui Zhu


    Full Text Available Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv, as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs, magnetic nanoparticles (MNPs, quantum dots (QDs and carbon nanomaterials (graphene and carbon nanotube, for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC, which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT, will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.

  11. Recent developments in antibody-based assays for the detection of bacterial toxins. (United States)

    Zhu, Kui; Dietrich, Richard; Didier, Andrea; Doyscher, Dominik; Märtlbauer, Erwin


    Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.

  12. An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C


    Recent advances in the development of conjugate polysaccharide vaccines for human use have stimulated interest in the use of assays detecting antibody-secreting cells (AbSC) with specificity for bacterial antigens. Here we present improved haemolytic plaque-forming cell (PFC) assays detecting Ab......SC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti......-polysaccharide AbSC of the IgG isotype, the increase was as high as 7.4-11.8 times. Evidence is presented that the pronounced improvement in the detection of the latter is due to the presence of aggregating anti-IgG antibody from the beginning of the assay. It is proposed that in the case of low affinity of anti...

  13. Newly established monoclonal antibody diagnostic assays for Schistosoma mansoni direct detection in areas of low endemicity.

    Directory of Open Access Journals (Sweden)

    Rafaella Fortini Queiroz Grenfell

    Full Text Available BACKGROUND: Current available methods for diagnosis of schistosomiasis mansoni lack sufficient sensitivity, which results in underreporting of infectious in areas of low endemicity. METHODOLOGY/PRINCIPAL FINDINGS: We developed three novel diagnostic methodologies for the direct detection of schistosome infection in serum samples. These three new methods were evaluated with positive patients from a low endemicity area in southeast Brazil. The basis of the assay was the production of monoclonal antibodies against the protein backbone of heavily glycosylated Circulating Cathodic Antigen (CCA. The antibodies were also selected for having no specificity to repeating poly-Lewis x units. Assays based on the detection CCA-protein should not encounter a limitation in sensitivity due to a biological background of this particular epitope. Three diagnostic methodologies were developed and validated, (i Immunomagnetic Separation based on improved incubation steps of non-diluted serum, (ii Direct Enzyme-linked Immunosorbent Assay and (iii Fluorescent Microscopy Analysis as a qualitative assay. The two quantitative assays presented high sensitivity (94% and 92%, respectively and specificity (100%, equivalent to the analysis of 3 stool samples and 16 slides by Kato-Katz, showing promising results on the determination of cure. CONCLUSIONS/SIGNIFICANCE: The Immunomagnetic Separation technique showed excellent correlation with parasite burden by Cohen coefficient. The qualitative method detected 47 positive individuals out of 50 with the analysis of 3 slides. This easy-to-do method was capable of discriminating positive from negative cases, even for patients with low parasite burden.

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

    DEFF Research Database (Denmark)

    Sciascia, Savino; Baldovino, Simone; Schreiber, Karen


    . 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...

  15. Highly Accurate Antibody Assays for Early and Rapid Detection of Tuberculosis in African and Asian Elephants (United States)

    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...

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  17. Specific immunoradiometric assay of insulin-like growth factor I with use of monoclonal antibodies. (United States)

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


    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.

  18. Flow cytometry-based assay to evaluate human serum MUC1-Tn antibodies

    DEFF Research Database (Denmark)

    Van Elssen, Catharina H M J; Clausen, Henrik; Germeraad, Wilfred T V;


    to the underglycosylation of MUC1, cancer-specific MUC1-Tn/STn antigens, which are highly immunogenic, become exposed. We aimed at developing a system that allows detection of antibodies directed to the native form of MUC1 and the underglycosylated MUC1-Tn epitopes. To this end, we made use of the Chinese Hamster Ovary......-ldlD MUC1 system was used to detect serum MUC1 and MUC1-Tn antibodies. Using this system, we could confirm the presence of MUC1-Tn antibodies in the serum of a patient vaccinated with a truncated MUC1 peptide. This indicates that the CHO-ldlD MUC1 system represents a flow cytometry-based technique...... in vaccination studies as well as for functional assays....

  19. HLA antibody detection with solid phase assays: great expectations or expectations too great? (United States)

    Gebel, H M; Bray, R A


    Alloantibodies directed against HLA antigens, are a barrier to long-term solid organ allograft survival. The clinical impact of preformed, donor-directed HLA alloantibodies range from acceptable risk to unequivocal contraindication for organ transplantation. HLA antibodies are key factors that limit patient access to donor organs. Serological methods were once the only approach to identify HLA antigens and antibodies. Limitations in these technologies led to the development of solid phase approaches. In the early 1990s, the development of the polymerase chain reaction enabled DNA-based HLA antigen testing to be performed. By the mid-1990s, microparticle-based technology that utilized flow cytometry for analysis was developed to detect both classes I and II HLA antibodies. These methodologies revolutionized clinical histocompatibility testing. The strengths and weaknesses of these assays are described in detail in this review.

  20. Detection of anti-infliximab antibodies is impacted by antibody titer, infliximab level and IgG4 antibodies: a systematic comparison of three different assays (United States)

    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


    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

  1. Thrombotic risk assessment in antiphospholipid syndrome: the role of new antibody specificities and thrombin generation assay. (United States)

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


    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.

  2. High yield purification of Plasmodium falciparum merozoites for use in opsonizing antibody assays. (United States)

    Hill, Danika L; Eriksson, Emily M; Schofield, Louis


    Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts.  Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies.  

  3. An improved Bathocuproine assay for accurate valence identification and quantification of copper bound by biomolecules. (United States)

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


    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.

  4. Measurement of antibodies to pneumococcal, meningococcal and haemophilus polysaccharides, and tetanus and diphtheria toxoids using a 19-plexed assay. (United States)

    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


    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.

  5. Comparison of antibody responses to human papillomavirus vaccination as measured by three assays

    Directory of Open Access Journals (Sweden)

    Hilary Ann Robbins


    Full Text Available 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 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 3 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 1 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.

  6. Assessment of assay sensitivity and precision in a malaria antibody ELISA. (United States)

    Rajasekariah, G Halli R; Kay, Graeme E; Russell, Natrice V; Smithyman, Anthony M


    Many types of ELISA-based immunodiagnostic test kits are commercially available in the market for specific indications. These kits provide necessary assay components, reagents, and guidelines to perform the assay under designated optimal conditions. By using these kits, any unknown or test sample can be assessed as negative or positive based on the results of referral calibrator (Ref+ve and Ref-ve) samples. It is essential to provide reliable test kits to end-users with adequate quality control analysis. Therefore, it is necessary to check the kit for any variations in its performance. While developing a malaria antibody ELISA test-kit, we optimized assay conditions with chequer-board analyses and developed an assay protocol. We have taken out kits randomly from the assembly line and had them evaluated by operators who are new to the test-kits. Assays are performed as per the test guidelines provided. Sera, diluted serially, have shown a clear discriminatory signal between a negative vs. positive sample. A COV is determined by evaluating the Ref-ve calibrator in replicate antigen-coated wells from 6 different plates. This COV is used as a tool to determine S/N ratio of test samples. Besides Ref-ve and Ref+ve calibrators, additional field serum samples are tested with the test kit. Several performance indices, such as mean, standard deviation, %CV are calculated, and the inter- and intra-assay variations determined. The assay precision is determined with large and small replicate samples. In addition, assays are performed concurrently in triplicate-, duplicate-, and single-wells, and the results are analyzed for any assay variations. Different plate areas are identified in antigen-coated 96-well plates and tested blind to detect any variations. The S/N ratio is found to be a very effective tool in determining the assay sensitivity. The %CV was within 10-15%. Variations seen in the assays are found to be due to operator errors and not due to kit reagents. These

  7. Sensitive detection of p53 antibodies in a homogeneous fluorescence assay format (United States)

    Neuweiler, Hannes; Schulz, Andreas; Wolfrum, Juergen M.; Sauer, Markus


    Circulating p53 autoantibodies are found to be a universal and highly specific tumor marker for malignant diseases. Hence, sereological screening for p53 autoantibodies at low concentration levels has become increasingly relevant for early-stage and follow-up of tumor diagnostics. We developed a new method for the highly sensitive detection of p53 antibodies in a homogeneous fluorescence assay format. Short, linear peptide derived form antibody recognition sequences so human p53 were labeled with an oxazine dye. Hydrophobic interactions constrain a conformation, where the dye interacts selectively with a tryptophan residue in the peptide sequence. Subsequently, the fluorescence of the dye is quenched efficiently due to electron transfer from the indole derivative to the dye in the excited state. Specific antibody recognition induces a conformational change in the peptide structure, repealing the dye-tryptophan interaction. Consequently, a fluorescence increase upon antibody binding signals the binding event. The long-wavelength absorption and emission characteristics of the probe and the use of a red pulsed diode laser as excitation source in a confocal fluorescence microscopic set-up allows ultra sensitive antibody detection at the single-molecule level. The effectiveness of the probes are highlighted by the detection of individual p53 autoantibodies directly in serum dilutions of cancer patients.

  8. Detection of antibodies to Actinobacillus pleuropneumoniae serotype 12 in pig serum using a blocking enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Andresen, Lars Ole; Klausen, Joan; Barfod, Kristen


    in samples of pig serum were detected by inhibition of the binding of polyclonal rabbit antibodies raised against Ap serotype 12. The assay was evaluated against sera from experimentally infected pigs, from pig herds naturally infected with Ap and from herds declared free of Ap serotypc 12 infection......The objective was to develop a blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Actinobacillus pleuropneumoniae (Ap) serotype 12 in pig serum. Lipopolysaccharide (LPS) from Ap serotype 12 was purified and used as antigen in the assay. Antibodies to the LPS antigen...

  9. Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies. (United States)

    Winkelmann, D A; Bourdieu, L; Kinose, F; Libchaber, A


    We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement.

  10. Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis. (United States)

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


    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.

  11. Development of an in Vitro Potency Assay for Anti-anthrax Lethal Toxin Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Sjoerd Rijpkema


    Full Text Available Lethal toxin (LT of Bacillus anthracis reduces the production of a number of inflammatory mediators, including transcription factors, chemokines and cytokines in various human cell lines, leading to down-regulation of the host inflammatory response. Previously we showed that the reduction of interleukin-8 (IL-8 is a sensitive marker of LT-mediated intoxication in human neutrophil-like NB-4 cells and that IL-8 levels are restored to normality when therapeutic monoclonal antibodies (mAb with toxin-neutralising (TN activity are added. We used this information to develop cell-based assays that examine the effects of TN therapeutic mAbs designed to treat LT intoxication and here we extend these findings. We present an in vitro assay based on human endothelial cell line HUVEC jr2, which measures the TN activity of therapeutic anti-LT mAbs using IL-8 as a marker for intoxication. HUVEC jr2 cells have the advantage over NB-4 cells that they are adherent, do not require a differentiation step and can be used in a microtitre plate format and therefore can facilitate high throughput analysis. This human cell-based assay provides a valid alternative to the mouse macrophage assay as it is a more biologically relevant model of the effects of toxin-neutralising antibodies in human infection.

  12. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

    DEFF Research Database (Denmark)

    Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper;


    relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means......A cell-based assay has been developed for the quantification of the activity of TNFa antagonists based on human erythroleukemic K562 cells transfected with a NF¿B regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NF¿B. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFa-induced firefly luciferase activity to be normalized...

  13. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

    DEFF Research Database (Denmark)

    Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper;


    relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means......A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized...

  14. Comparison of a Multiplex Flow Cytometric Assay with Enzyme-Linked Immunosorbent Assay for Quantitation of Antibodies to Tetanus, Diphtheria, and Haemophilus influenzae Type b


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


    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 ...

  15. An extended set of yeast-based functional assays accurately identifies human disease mutations (United States)

    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.


    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

  16. An improved method for utilization of peptide substrates for antibody characterization and enzymatic assays. (United States)

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


    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.

  17. Rapid Enzyme-Linked Immunosorbent Assay for the Detection of Hantavirus-Specific Antibodies in Divergent Small Mammals

    Directory of Open Access Journals (Sweden)

    Karla Cautivo


    Full Text Available We assessed the utility of an enzyme-linked immunosorbent assay (ELISA for the detection of hantavirus-specific antibodies from sera of Oligoryzomys longicaudatus, the principal reservoir of Andes virus (ANDV, using an antigen previously developed for detection of antibodies to Sin Nombre virus (SNV in sera from Peromyscus maniculatus. The assay uses a protein A/G horseradish peroxidase conjugate and can be performed in as little as 1.5 hours. Serum samples from Oligoryzomys longicaudatus collected in central-south Chile were used and the assay identified several that were antibody positive. This assay can be used for the rapid detection of antibodies to divergent hantaviruses from geographically and phylogenetically distant rodent species.

  18. Rapid enzyme-linked immunosorbent assay for the detection of hantavirus-specific antibodies in divergent small mammals. (United States)

    Cautivo, Karla; Schountz, Tony; Acuña-Retamar, Mariana; Ferrés, Marcela; Torres-Pérez, Fernando


    We assessed the utility of an enzyme-linked immunosorbent assay (ELISA) for the detection of hantavirus-specific antibodies from sera of Oligoryzomys longicaudatus, the principal reservoir of Andes virus (ANDV), using an antigen previously developed for detection of antibodies to Sin Nombre virus (SNV) in sera from Peromyscus maniculatus. The assay uses a protein A/G horseradish peroxidase conjugate and can be performed in as little as 1.5 hours. Serum samples from Oligoryzomys longicaudatus collected in central-south Chile were used and the assay identified several that were antibody positive. This assay can be used for the rapid detection of antibodies to divergent hantaviruses from geographically and phylogenetically distant rodent species.

  19. Antibodies with specificity for native and denatured forms of ovalbumin differ in reactivity between enzyme-linked immunosorbent assays

    DEFF Research Database (Denmark)

    Holm, B. E.; Bergmann, Ann Christina; Hansen, Paul Robert


    In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme......-linked immunosorbent assay (ELISA) employing surface-bound OVA and streptavidin-capture ELISA to determine whether effects of different coating influence antibody specificity and with respect to epitope specificity by peptide ELISA, using overlapping peptides, covering the complete OVA sequence. Polyclonal antibodies...... to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially...

  20. Development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein. (United States)

    Fan, Jing-Hui; Zuo, Yu-Zhu; Shen, Xiao-Qiang; Gu, Wen-Yuan; Di, Jing-Mei


    The recent dramatic increase in reported cases of porcine epidemic diarrhea (PED) in pig farms is a potential threat to the global swine industry. Therefore, the accurate diagnosis, serological monitoring, and surveillance of specific antibodies in pigs resulting from porcine epidemic diarrhea virus (PEDV) infection or vaccination would be essential in helping to control the spread of PED. We developed and validated an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant membrane (M) protein of PEDV. To detect PEDV antibodies in eight herds, 382 serum samples were collected from sows that had been immunized with a PED vaccine, and screened using the developed ELISA in parallel with a serum neutralization (SN) assay. Of the tested samples, 276 were positive for the presence of PEDV antibodies according to both assays, while 98 were negative. An excellent agreement between the ELISA and the SN assay was observed (kappa=0.947; 95% confidence interval=0.910-0.984; McNemar's test, P=0.727). No cross-reaction was detected for the developed ELISA with other coronaviruses or other common pig pathogens. The developed ELISA could be used for serological evaluation and indirect diagnosis of PED infection.

  1. Diagnostic accuracy and comparison of two assays for Borrelia-specific IgG and IgM antibodies

    DEFF Research Database (Denmark)

    Dessau, Ram


    Two assays (Liaison, Diasorin; IDEIA, Oxoid) for detection of Borrelia-specific antibodies were compared. A case-control design using patients with neuroborreliosis (n = 48), laboratory defined by a positive Borrelia-specific antibody index in the spinal fluid, was available and was intended...

  2. Development of a high-throughput opsonophagocytic assay for the determination of functional antibody activity against Streptococcus pyogenes using bioluminescence. (United States)

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


    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.

  3. Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum

    DEFF Research Database (Denmark)

    Klausen, Joan; Andresen, Lars Ole; Barfod, Kristen


    A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6...

  4. Development of 316v antibody enzyme-linked immunosorbent assay for detection of paratuberculosis in sheep. (United States)

    Gurung, R B; Begg, D J; Purdie, A C; Eamens, G J; Whittington, R J


    An enzyme-linked immunosorbent assay (ELISA) was developed and optimised using a Mycobacterium avium subspecies paratuberculosis (MAP) antigen prepared from a C strain (316v) passed through a French press. The optimised assay was evaluated with a panel of sera from MAP infected (n = 66) and uninfected (n = 1,092) sheep. Animals in the MAP infected category were positive on either tissue culture or histopathology but were of unknown serum antibody status. The diagnostic performance and cost of the assay were compared with those of a commercial ELISA (IDEXX). At 99.8% diagnostic specificity the assay showed a diagnostic sensitivity of 23% (95% CI: 15.1-35.8) compared with 36.4% (95% CI: 25.8-48.4) for the commercial ELISA (McNemar's test: chi-square 5.82, p ELISA for an equivalent number of tests, the multiple depending on the number of plates processed per run. For flock-level surveillance, to account for the lower sensitivity of the 316v ELISA compared with the commercial ELISA, sample sizes would be increased but the test cost would still be lower. The 316v assay will be useful for diagnosis of Johne's disease in sheep flocks, particularly in developing countries where labour costs are low relative to the cost of consumables.

  5. Detection of TGEV Antibody by Enzyme-Linked Immunosorbent Assay Using Recombinant Nucleocapsid Proteins

    Institute of Scientific and Technical Information of China (English)

    YU Li-yun; HOU Xi-lin


    An enzyme linked immunosorbent assays (ELISA) based on recombinant nucleocapsid (N) protein generated in Escherichia coli was evaluated for its sensitivity and specificity for diagnosis of transmissible gastroenteritis virus (TGEV) infection.The N gene encoding the N protein was cloned and expressed as a fusion protein with His tag protein in E. coli. The recombinant N protein migrated at 42 kDa and reacted with His6 tag specific monoclonal antibody by immunoblotting.Recombinant N protein ELISA (rnELISA) demonstrated 97.5% specificity among 80 TGEV-free individuals, and 97.3%sensitivity ranging among 110 clinical samples with TGEV. Taken together, these results indicated that nucleocapsid may be a useful antigen for the sera-diagnosis of TGEV and it was also suggested that the ELISA is a highly sensitive and specific test for detecting antibodies against TGEV.

  6. Peroxidase-linked assay for detection of antibodies against bovine leukosis virus. (United States)

    de Castro, Clarissa C; Nunes, Cristina F; Finger, Paula F; Siedler, Bianca S; Dummer, Luana; de Lima, Marcelo; Leite, Fábio P L; Fischer, Geferson; Vargas, Gilberto D'A; Hübner, Silvia de O


    A peroxidase linked assay (PLA) was designed to screen bovine sera for the presence of specific antibodies against bovine leukosis virus (BLV). Out of 201 samples of bovine sera analyzed, 52.2% were considered positive by PLA, 26.4% by AGID, and 38.9% by ELISA. Western blotting analyses excluded 27 samples found to be positive by PLA. PLA showed 100% of sensitivity when compared with AGID and ELISA. Specificity was 64.8% and 78%, respectively (kappa coefficients were 0.70 and 0.83). These findings indicate that PLA can be used as an alternative method for the diagnosis of BLV infection in cattle.

  7. Enzyme-linked immunosorbent Assay for detecting of antibody to canine distemper virus

    Directory of Open Access Journals (Sweden)



    Full Text Available Serum neutralisation test (SNT has been established for evaluating canine distemper vaccination, but until now SNT was rarely used due to the need for continuous tissue culture facilities and requires 3 days to perform. For detecting antibody to canine distemper virus, an enzyme-linked immunosorbent assay (ELISA is relatively simple and rapid seroassay. ELISA for canine immunoglobulin (Ig G antibodies to canine distemper virus (CDV was developed by using Onderstepoort strain of canine distemper virus as coating antigen. Rabbit anti canine IgG labelled with horse radish peroxidase was used as the conjugate, while phenylenediamine dihydrochloride (OPD was used as the substrate. The ELISA results were then compared with the results of the SNT, using the sera of 312 random-source dogs from West Java. The two test-results had a high degree of correlation. Very few discrepancies occurred and most of these were at the lower limits of each test. When the sera were tested at 1 : 100 dilutions, there was a 95.5% agreement between the ELISA and SNT. Their sensitivity and spesificity were 83.9 and 98.4%. Titrated SNT and ELISA also were performed on sera from 7 dogs whose lifetime medical histories were known. The antibodies were inclining up after two months of post vaccination, where the titre was not in zero/lower position at the day of vaccination. However, antibody zero or low position were found at 28 days post vaccination. All of the results indicated that ELISA can be used for evaluating antibody to canine distemper virus response, replacing the SNT.

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

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


    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.

  9. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains. (United States)

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki


    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  10. Evaluation of the Determine Syphilis TP assay for the detection of antibodies against Treponema pallidum for the serodiagnosis of syphilis. (United States)

    Zhuang, Y-H; Tian, Y; Chen, Y; Tang, J; Wang, J-Q; Li, P; Li, Q; Jiang, Y-Q


    Currently, infectious syphilis has been resurgent in China and has become a significant public health problem. The rapid expansion of syphilis screening programs is urgently required. In the present study, the performance of the Determine Syphilis TP assay (Determine TP assay) for the detection of antibodies against Treponema pallidum (T. pallidum) for syphilis serodiagnosis was evaluated. In total, 300 serum samples were tested for the presence of treponemal-specific antibodies using the Treponema pallidum particle agglutination (TPPA) assay, the Determine TP assay, and the InTec immunochromatography assay (InTec assay). The Determine TP assay detected 99, 11, and 5 positive results, whereas the InTec assay detected 97, 3, and 3 positive samples from group I (100 TPPA-positive sera), group II (13 TPPA 1:80 +/- sera), and group III (187 TPPA-negative sera), respectively. The sensitivity, specificity, and the rate concordant with TPPA for the Determine TP assay were 97.35, 98.91, and 97.33%, respectively. In comparison to the TPPA, the Determine TP assay is simple to perform and time-saving, making it a favorable alternative for the detection of T. pallidum-specific antibodies where other T. pallidum-specific confirmatory tests are not available. In addition, this rapid treponemal test promotes prompt treatment for syphilis by providing early laboratory diagnosis.

  11. Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum. (United States)

    Jin, Jun; Ma, Hongxia; Xu, Lin; An, Dong; Sun, Shiyang; Huang, Xueyong; Kong, Wei; Jiang, Chunlai


    Serum neutralizing antibody titers are indicative of protective immunity against Coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV71), the two main etiological agents of hand, foot and mouth disease (HFMD), and provide the basis for evaluating vaccine efficacy. The current CV-A16 neutralization assay based on inhibition of cytopathic effects requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay was developed by employing CV-A16 pseudoviruses expressing luciferase for detecting infectivity in rhabdomyosarcoma (RD) cells and measuring serum viral neutralizing antibodies. Without the need to use infectious CV-A16 strains, the neutralizing antibody titer against CV-A16 could be determined within 15h by measuring luciferase signals by this assay. The pseudovirus CV-A16 neutralization assay (pCNA) was validated by comparison with a conventional CV-A16 neutralization assay (cCNA) in testing 174 human serum samples collected from children (age <5 years). The neutralizing antibody titers determined by these two assays were well correlated (R(2)=0.7689). These results suggest that the pCNA can serve as a rapid and objective procedure for the measurement of neutralizing antibodies against CV-A16.

  12. Comparison of five commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis. (United States)

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


    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.

  13. Evaluation of the LIAISON ANA screen assay for antinuclear antibody testing in autoimmune diseases. (United States)

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


    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.

  14. Accurate and High-Coverage Immune Repertoire Sequencing Reveals Characteristics of Antibody Repertoire Diversification in Young Children with Malaria (United States)

    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.

  15. Label free checkerboard assay to determine overlapping epitopes of Ebola virus VP-40 antibodies using surface plasmon resonance. (United States)

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


    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.

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

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


    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

  17. Fluorescent competitive assay for melamine using dummy molecularly imprinted polymers as antibody mimics

    Institute of Scientific and Technical Information of China (English)

    DU Xin-wei; JIN Mao-jun; ZHENG Lu-fei; ZHANG Yan-xin; SHE Yong-xin; LIU Guang-yang; ZHAO Feng-nian; WANG Jing; WANG Shan-shan; JIN Fen; SHAO Hua


    A lfuorescent competitive assay for melamine was ifrst developed utilizing dummy molecularly imprinted polymers (DMIPs) as artiifcial antibodies. This method is based on the competition between lfuorescent substances and the unlabeled analyte for binding sites in synthesized DMIPs and the decreased binding of lfuorescent substances to DMIPs due to increased concentrations of melamine in the solutions. DMIPs for melamine were synthesized under a hot water bath in the pres-ence of the initiator azobisisobutyronitrile (AIBN) using 2,4-diamino-6-methyl-1,3,5-triazine (DAMT) as a dummy template, methacrylic acid (MAA) as a functional monomer, and ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent. The adsorption capacity and selectivity of DMIPs for melamine were evaluated by the isothermal adsorption curve and Scatchard analysis. The evaluation results showed that the synthesized DMIPs had speciifc recognition sites for melamine and the maximum adsorption amount was 1066.33 μg g–1. Later, 5-(4,6-dichlorotriazinyl) amino lfuorescein (DTAF) with a triazine ring, which slightly resembles melamine, was selected as the lfuorescent substance. The lfuorescent competitive assay using DMIPs as the antibody mimics was ifnaly established by selecting and optimizing the reaction solvents, DMIPs amount, DTAF concentration, and incubation time. The optimal detection system showed a linear response within range of 0.05–40 mg L–1 and the limit of detection (LOD) was 1.23 μg L–1. It was successfuly applied to the detection of melamine in spiked milk samples with satisfactory recoveries (71.9 to 86.3%). According to the comparative analysis, the result of optimized lfuorescent competitive assay revealed excelent agreement with the HPLC-MS/MS result for melamine.

  18. A comparison of a solid phase IRC assay and the PSIFT for detection of antibodies to platelets. (United States)

    Häcker-Shahin, B; Giannitsis, D J


    A rapid solid phase indicator red cells assay (IRCA) for detection of platelet antibodies was developed and its sensitivity compared with PSIFT. Platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After serum incubation bound platelet-specific antibodies were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an anti-platelet antibody was indicated by red cell adherence over the reaction surface. In the absence of serum antibodies to platelets the indicator red cells formed a pellet. The IRCA showed a high sensitivity; the anti-platelet antibody Thrombocyte was detectable until a dilution of 1:1,600 whereas the same antibody in the PSIFT could only be detected until a dilution of 1:400.

  19. Antiphospholipid antibody syndrome: the flow cytometric annexin A5 competition assay as a diagnostic tool. (United States)

    Tomer, A; Bar-Lev, S; Fleisher, S; Shenkman, B; Friger, M; Abu-Shakra, M


    The mechanism underlying hypercoagulability in antiphospholipid antibody syndrome (APS) is uncertain. Here, we present a flow-cytometric assay (FCA) based on the hypothesis that anti-platelet-anionic-phospholipid autoantibodies (aPL) interfere with the activity of the natural anticoagulant protein annexin A5, thereby accelerating platelet procoagulant activity. This study assessed the clinical utility of the feasible FCA, which demonstrates the competition of the patient's aPL with the binding of annexin A5 to the platelet-anionic-phospholipids, in the diagnosis of APS. Sixty-two (94%) of 66 APS patients, 20 (51%) of 39 patients with systemic lupus erythematosus and two (4%) of 49 healthy individuals were positive by FCA. Compared with the anticardiolipin (aCL) assay, the relative sensitivity was 82% and the specificity 73.3%. However, 19 (25%) aCL-negative patients were positive by FCA; 12 were positive for lupus-anticoagulant (LA). Compared with LA assay, the relative sensitivity was 85% and the specificity 72.2%. However, 21 (26%) LA-negative patients were FCA-positive, 12 were positive for aCL. The FCA was particularly sensitive for APS patients with arterial (97.0%) and gestational vascular complications (100%) with overall sensitivity of 95% and specificity of 97%. Our findings suggest that the FCA is practical, sensitive and specific for the detection of clinically relevant aPL in the diagnosis of APS.

  20. Sensitive measurement of thrombopoietin by a monoclonal antibody based sandwich enzyme-linked immunosorbent assay. (United States)

    Folman, C C; von dem Borne, A E; Rensink, I H; Gerritsen, W; van der Schoot, C E; de Haas, M; Aarden, L


    In this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs). The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 +/- 0.8 A.U./ml, mean +/- sd) was lower than the concentration found in controls (11 +/- 8 A.U./ml, mean +/- sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma. We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels. In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.

  1. Analysis of cross-reactive neutralizing antibodies in human HFMD serum with an EV71 pseudovirus-based assay. (United States)

    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


    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 ( 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.

  2. A Comparison of Anti-Nuclear Antibody Quantification Using Automated Enzyme Immunoassays and Immunofluorescence Assays

    DEFF Research Database (Denmark)

    Baronaite, Renata; Engelhart, Merete; Mørk Hansen, Troels;


    Anti-nuclear antibodies (ANA) have traditionally been evaluated using indirect fluorescence assays (IFA) with HEp-2 cells. Quantitative immunoassays (EIA) have replaced the use of HEp-2 cells in some laboratories. Here, we evaluated ANA in 400 consecutive and unselected routinely referred patients...... patients were compared. The majority of the results were the same between the two methods (n = 325, 84%); however, 8% (n = 30) yielded equivocal results (equivocal-negative and equivocal-positive) and 8% (n = 31) yielded divergent results (positive-negative). The results showed fairly good agreement......, with Cohen's kappa value of 0.30 (95% confidence interval (CI) = 0.14-0.46), which decreased to 0.23 (95% CI = 0.06-0.40) when the results for dsDNA were omitted. The EIA method was less reliable for assessing nuclear and speckled reactivity patterns, whereas the IFA method presented difficulties detecting...

  3. Monoclonal Antibody-Based Enzyme Linked Immunosorbent Assay for the Analysis of Jasmonates in Plants

    Institute of Scientific and Technical Information of China (English)

    Aixing Deng; Weiming Tan; Suping He; Wei Liu; Tiegui Nan; Zhaohu Li; Baomin Wang; Qing X.Li


    Methyl jasmonate (MeJA) and its free-acid form,jasmonic acid (JA) are naturally occurring plant growth regulators widely distributed in higher plants.In order to improve the sensitivity for the analysis of MeJA at low levels in small amounts of plant samples,a monoclonal antibody (MAb) (designated as MAb 3E5D7C4B6) against MeJA was derived from a JAbovine serum albumin (BSA) conjugate as an immunogen.The antibody belongs to the IgG1 subclass with a κ type light chain and has a dissociation constant of approximately 6.07 x 10-9 M.MAb3E5D7C4B6 is very specific to MeJA.It was used to develop a direct competitive enzyme-linked immunosorbent assay (dcELISA),conventional and simplified indirect competitive ELISAs (icELISA).JA was derivatized into MeJA for the ELISA analysis.The IC50 value and detection range for MeJA were,respectively,34 and 4-257 ng/mL by the conventional icELISA,21 and 3-226 ng/mL by the simplified icELISA and 5.0 and 0.7-97.0 ng/mL by the dcELISA.The dcELISA was more sensitive than either the conventional or simplified icELISA.The assays were used to measure the content of jasmonates as MeJA in tobacco leaves under drought stress or inoculated with tobacco mosaic virus and tomato leaves inoculated with tomato mosaic virus or Lirioinyza sativae Blanchard as compared with the corresponding healthy leaves.The increased jasmonates content indicated its role in response to the drought stress and pathogens.

  4. Evaluation of a pan-serotype point-of-care rapid diagnostic assay for accurate detection of acute dengue infection. (United States)

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


    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.

  5. Modification of solid phase red cell adherence assay for the detection of platelet antibodies in patients with thrombocytopenia. (United States)

    Vongchan, Preeyanat; Nawarawong, Weerasak; Linhardt, Robert J


    Platelet refractoriness is caused by HLA antibodies and platelet-specific antibodies. Current methods used to detect antiplatelet antibodies have limitations. Solid phase red cell adherence (SPRCA) lacks sensitivity and requires a second assay using chloroquine-treated intact platelets to specify the response due to anti-HLA. We modified SPRCA by using 2 types of antihuman platelet antibodies with different specificities toward platelet lysate and tested samples from 361 patients (69 with unexplained thrombocytopenia and 292 with poor response to platelet transfusions not explicable by alloimmunization or the clinical situation) and 50 from healthy volunteers. Our method compared favorably with platelet suspension direct immunofluorescence. All samples from healthy volunteers were negative; of the samples from the patient population, 240 were positive (147 samples had only antiplatelet and 3 samples had only anti-HLA antibodies). This modified technique had a sensitivity of 98% and a specificity of 91%.

  6. Comparison of competitive ligand-binding assay and bioassay formats for the measurement of neutralizing antibodies to protein therapeutics. (United States)

    Finco, Deborah; Baltrukonis, Daniel; Clements-Egan, Adrienne; Delaria, Kathy; Gunn, George R; Lowe, John; Maia, Mauricio; Wong, Teresa


    Administration of biological therapeutic proteins can lead to unwanted immunogenicity in recipients of these products. The assessment and characterization of such immune reactions can be helpful to better understand their clinical relevance and how they relate to patient safety and therefore, have become an integral part of a product development program for biological therapeutics. Testing for anti-drug antibodies (ADA) to biological/biotechnology-derived therapeutic proteins generally follows a tiered approach. Samples are initially screened for binding antibodies; presumptive positives are then confirmed in a confirmatory assay; subsequently, confirmed-positive samples may be further characterized by titration and with a neutralizing antibody (NAb) assay. Regulatory guidances on immunogenicity state that assessing the neutralizing capacity of antibodies should preferably be done using functional bioassays, while recognizing that competitive ligand-binding (CLB) assays may be substituted when neutralizing bioassays are inadequate or not feasible. This manuscript describes case studies from four companies in which CLB assays and functional bioassays were compared for their ability to detect neutralizing ADA against a variety of biotechnology-derived therapeutic proteins. Our findings indicate that CLB assays are comparable to bioassays for the detection of NAbs, in some cases offering better detection sensitivity, lower variability, and less matrix interference.

  7. A rugged, precise and accurate new gravimetry method for the determination of gold: an alternative to fire assay method. (United States)

    Singh, Nahar


    A new, rugged, precise, accurate and fast primary method of measurement has been proposed for the determination of gold in various gold articles. Precise and accurate measurement of gold is the primary requirement for hall marking and to trade gold internationally, as billions of dollars of gold are trading world wide for the various applications. At present Fire Assay ASTM E 1335-08 is the only Standard Test Method for the determination of Gold, which is accepted internationally. But the method is time consuming, cumbersome and required expertise to perform the test. In the present investigation, a method has been developed gravimetry based on direct determination of gold after reducing gold in zero-valent state by hydroxylamine hydrochloride. Gravimetry is the most reliable technique and having highest metrological qualities in comparison to titremetry and instrumental method and the results of gravimetry are directly traceable to SI unit. The results of gravimetric method are accepted without reference to a standard of the same quantity. Several experiments were carried out with and without impurities and it has been concluded that gold can be determined accurately and precisely in presence of several impurities. Five replicates of approximate 0.2 g gold samples were analyzed following method proposed and percentage purity were found to be 99.993 ± 0.0056 with 95% confidence level (k = 2). The combined uncertainty in gold measurement has also been evaluated using potential sources of the method according to the EURACHEM/GUM guidelines.

  8. Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human Myeloperoxidase. (United States)

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


    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 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.

  9. Enzyme-linked immunosorbent assay for total isoflavonoids in Pueraria candollei using anti-puerarin and anti-daidzin polyclonal antibodies. (United States)

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


    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.

  10. Validation of 2 commercial Neospora caninum antibody enzyme linked immunosorbent assays. (United States)

    Wu, John T Y; Dreger, Sally; Chow, Eva Y W; Bowlby, Evelyn E


    This is a validation study of 2 commercially available enzyme linked immunosorbent assays (ELISA) for the detection of antibodies against Neospora caninum in bovine serum. The results of the reference sera (n = 30) and field sera from an infected beef herd (n = 150) were tested by both ELISAs and the results were compared statistically. When the immunoblotting results of the reference bovine sera were compared to the ELISA results, the same identity score (96.67%) and kappa values (K) (0.93) were obtained for both ELISAs. The sensitivity and specificity values for the IDEXX test were 100% and 93.33% respectively. For the Biovet test 93.33% and 100% were obtained. The corresponding positive (PV+) and negative predictive (PV-) values for the 2 assays were 93.75% and 100% (IDEXX), and 100% and 93.75% (Biovet). In the 2nd study, competitive inhibition ELISA (c-ELISA) results on bovine sera from an infected herd were compared to the 2 sets of ELISA results. The identity scores of the 2 ELISAs were 98% (IDEXX) and 97.33% (Biovet). The K values calculated were 0.96 (IDEXX) and 0.95 (Biovet). For the IDEXX test the sensitivity and specificity were 97.56% and 98.53%, whereas for the Biovet assay 95.12% and 100% were recorded, respectively. The corresponding PV+ and PV- values were 98.77% and 97.1% (IDEXX), and 100% and 94.44% (Biovet). Our validation results showed that the 2 ELISAs worked equally well and there was no statistically significant difference between the performance of the 2 tests. Both tests showed high reproducibility, repeatability and substantial agreement with results from 2 other laboratories. A quality assurance based on the requirement of the ISO/IEC 17025 standards has been adopted throughout this project for test validation procedures.

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

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


    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.

  12. Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus (United States)

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


    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.

  13. A combination of serological assays to detect human antibodies to the avian influenza A H7N9 virus.

    Directory of Open Access Journals (Sweden)

    Libo Dong

    Full Text Available 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.

  14. Comparison of a commercial ELISA and an immunoperoxidase monolayer assay to detect antibodies directed against porcine respiratory and reproductive syndrome virus

    NARCIS (Netherlands)

    Nodelijk, G.; Wensvoort, G.; Kroese, B.; Leengoed, van L.A.M.G.; Colijn, E.; Verheijden, J.H.M.


    A commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against porcine respiratory and reproductive syndrome virus (PRRSV) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used were collected from pigs experimentally infected with

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

    Energy Technology Data Exchange (ETDEWEB)

    Hovi, T.; Roivainen, M.


    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.

  16. Detection by radioimmunoassay and enzyme-linked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions. (United States)

    Rodak, L; Babiuk, L A; Acres, S D


    The sensitivity of a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), and a serum neutralization assay (SN) for detecting antibodies to bovine coronavirus in serum and colostrum were compared. Although there proved to be a good correlation among all three assays (r = 0.915 and 0.964 for RIA with SN and ELISA, respectively), RIA and ELISA proved to be at least 10 times more sensitive than neutralization tests. By using these techniques, it was possible to detect a time-dependent decrease in antibody levels in bovine colostrum after parturition. Using ELISA, we demonstrated that 12 of 12 herds in Saskatchewan, and 109 of 110 animals tested, and antibody to bovine coronavirus. There was no elevated antibody response in serum or lacteal secretions of cows vaccinated once or twice with a commercially available modified live rota-coronavirus vaccine. In addition to being more sensitive than SN, ELISA and RIA proved to have other advantages for measuring antibody levels to bovine coronavirus and therefore warrant wider use as tools in diagnostic virology.

  17. Antibody performance in ChIP-sequencing assays: From quality scores of public data sets to quantitative certification (United States)

    Mendoza-Parra, Marco-Antonio; Saravaki, Vincent; Cholley, Pierre-Etienne; Blum, Matthias; Billoré, Benjamin; Gronemeyer, Hinrich


    We have established a certification system for antibodies to be used in chromatin immunoprecipitation assays coupled to massive parallel sequencing (ChIP-seq). This certification comprises a standardized ChIP procedure and the attribution of a numerical quality control indicator (QCi) to biological replicate experiments. The QCi computation is based on a universally applicable quality assessment that quantitates the global deviation of randomly sampled subsets of ChIP-seq dataset with the original genome-aligned sequence reads. Comparison with a QCi database for >28,000 ChIP-seq assays were used to attribute quality grades (ranging from ‘AAA’ to ‘DDD’) to a given dataset. In the present report we used the numerical QC system to assess the factors influencing the quality of ChIP-seq assays, including the nature of the target, the sequencing depth and the commercial source of the antibody.  We have used this approach specifically to certify mono and polyclonal antibodies obtained from Active Motif directed against the histone modification marks H3K4me3, H3K27ac and H3K9ac for ChIP-seq. The antibodies received the grades AAA to BBC ( We propose to attribute such quantitative grading of all antibodies attributed with the label “ChIP-seq grade”. PMID:27335635

  18. Antibody performance in ChIP-sequencing assays: From quality scores of public data sets to quantitative certification. (United States)

    Mendoza-Parra, Marco-Antonio; Saravaki, Vincent; Cholley, Pierre-Etienne; Blum, Matthias; Billoré, Benjamin; Gronemeyer, Hinrich


    We have established a certification system for antibodies to be used in chromatin immunoprecipitation assays coupled to massive parallel sequencing (ChIP-seq). This certification comprises a standardized ChIP procedure and the attribution of a numerical quality control indicator (QCi) to biological replicate experiments. The QCi computation is based on a universally applicable quality assessment that quantitates the global deviation of randomly sampled subsets of ChIP-seq dataset with the original genome-aligned sequence reads. Comparison with a QCi database for >28,000 ChIP-seq assays were used to attribute quality grades (ranging from 'AAA' to 'DDD') to a given dataset. In the present report we used the numerical QC system to assess the factors influencing the quality of ChIP-seq assays, including the nature of the target, the sequencing depth and the commercial source of the antibody.  We have used this approach specifically to certify mono and polyclonal antibodies obtained from Active Motif directed against the histone modification marks H3K4me3, H3K27ac and H3K9ac for ChIP-seq. The antibodies received the grades AAA to BBC ( We propose to attribute such quantitative grading of all antibodies attributed with the label "ChIP-seq grade".

  19. Gold nanoparticle-based enzyme-linked antibody-aptamer sandwich assay for detection of Salmonella Typhimurium. (United States)

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


    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.

  20. Detection of Lewis, P1, and some MNS blood group system antibodies by a solid phase assay. (United States)

    Rolih, S; Thomas, R; Sinor, L


    Some solid phase red cell adherence (SPRCA) assays are designed to detect IgG antibodies to red blood cell (RBC) antigens. These assays use anti-IgG-coated red cells as the indicator. It is reported that most antibodies to Lea, Leb, P1, M, and N fail to react by solid phase (SP), presumably because they are IgM antibodies. Those detected are assumed to be IgG. In one year, during routine testing using SPRCA to screen patients for intended RBC transfusion, 28 of 59 such examples were found to react: anti-Lea(9), -Leb(1), -M(14), -N(1), and -P1(3). A study was undertaken to determine if reactivity was due to crosslinking by IgM antibodies of antigen-positive indicator RBCs to antigen-positive reagent RBC monolayers, or due to detection of IgG antibodies. Antibodies were tested according to standard SP protocols, except where IgG-neutralized indicator RBCs were substituted for anti-IgG-active indicator cells. The 59 samples were retested with antigen-positive and antigen-negative indicator RBCs. Only 5 of 59 reacted optimally when antigen-positive indicator cells were used: anti-Lea(2), -Leb(1), -M(1), and -N(1). The reactions of all antibodies were abolished when the anti-IgG component of the indicator was neutralized by soluble IgG. These findings show that detection of most Lewis, P1, M, and N antibodies by SPRCA is dependent on the presence of an IgG antibody in the serum.

  1. An enzyme-linked immunosorbent assay for the detection of mouse polyomavirus-specific antibodies in laboratory mice.

    NARCIS (Netherlands)

    H.W.J. Broeders; J. Groen (Jan); A.D.M.E. Osterhaus (Albert); G. van Steenis (Bert)


    textabstractAn enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of IgM and IgG serum antibodies to mouse polyomavirus (MPV). To evaluate the potential of this ELISA for the screening of laboratory rodents, serum samples from specific pathogen free (SPF) BA

  2. Analysis of Liquid Bead Microarray Antibody Assay Data for Epidemiologic Studies of Pathogen-Cancer Associations (United States)

    Colombara, Danny V.; Hughes, James P.; Burnett-Hartman, Andrea N.; Hawes, Stephen E.; Galloway, Denise A.; Schwartz, Stephen M.; Bostick, Roberd M.; Potter, John D.; Manhart, Lisa E.


    Background Liquid bead microarray antibody (LBMA) assays are used to assess pathogen-cancer associations. However, studies analyze LBMA data differently, limiting comparability. Methods We generated 10,000 Monte Carlo-type simulations of log-normal antibody distributions (exposure) with 200 cases and 200 controls (outcome). We estimated type I error rates, statistical power, and bias associated with t-tests, logistic regression with a linear exposure and with the exposure dichotomized at 200 units, 400 units, the mean among controls plus two standard deviations, and the value corresponding to the optimal sensitivity and specificity. We also applied these models, and data visualizations (kernel density plots, receiver operating characteristic (ROC) curves, predicted probability plots, and Q-Q plots), to two empirical datasets to assess the consistency of the exposure-outcome relationship. Results All strategies had acceptable type I error rates (0.03≤P≤0.048), except for the dichotomization according to optimal sensitivity and specificity, which had a type I error rate of 0.27. Among the remaining methods, logistic regression with a linear predictor (Power=1.00) and t-tests (Power=1.00) had the highest power to detect a mean difference of 1.0 MFI (median fluorescence intensity) on the log scale and were unbiased. Dichotomization methods upwardly biased the risk estimates. Conclusion These results indicate that logistic regression with linear predictors and unpaired t-tests are superior to logistic regression with dichotomized predictors for assessing disease associations with LBMA data. Logistic regression with continuous linear predictors and t-tests are preferable to commonly used LBMA dichotomization methods. PMID:26071614

  3. Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans. (United States)

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


    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.

  4. Automated immunohistochemical assay for estrogen receptor status in breast cancer using monoclonal antibody CC4-5 on the Ventana ES. (United States)

    Nichols, G E; Frierson, H F; Boyd, J C; Hanigan, M H


    Determination of breast cancer estrogen receptor (ER) status as a predictor of tumor response to adjuvant endocrine therapy remains a mainstay of breast cancer management. Recent second generation anti-ER antibodies and new epitope retrieval methods have produced paraffin-based immunohistochemical results that correlate closely with the dextran-coated charcoal (DCC) assay and appear to represent a superior method of ER assay. The authors determined the ER status of 103 invasive breast cancers by paraffin-based, automated immunohistochemistry on the Ventana ES using a new monoclonal antibody, CC4-5, and compared the results to those of parallel DCC biochemical analysis and manual immunohistochemical analysis using anti-ER monoclonal antibody ER1D5. The specificity of the CC4-5 antibody for ER protein was confirmed by Western blot analysis. Sixty of 103 cases were positive for ER by CC4-5 automated immunohistochemistry. With a ligand binding assay threshold value of 20 fmol/mg protein, there were 50 positive cases by biochemical assay. The biochemical results corresponded to an 88% rate of agreement with automated CC4-5 staining. Analysis of discordant cases revealed that the majority of CC4-5 immunopositive only cases (8 of 11) were strongly positive, stroma rich tumors, suggesting that corresponding biochemical measurements were diluted by non representative stromal tissue. There was only one immunonegative, biochemically positive case (27 fmol/mg protein). Semiquantitation of CC4-5 staining using percent positive tumor cells or weighted average staining intensity (HSCORE) showed moderate to good correlation with quantitative DCC results (r = 0.64 and 0.62, P Ventana ES, most likely due to temperature constraints of the instrument. By manual ER1D5 staining, 40 of 79 examined cases were positive corresponding to a 99% rate of agreement with automated CC4-5 staining. Semiquantitation of ER1D5 staining by percent positive tumor cells and weighted average staining

  5. The effects of affinity-purified anti-DNA antibodies from patients with systemic lupus erythematosus on the fluorescent antinuclear antibody assay using HEp-2 cells. (United States)

    Suzuki, Kimihiro; Kawamura, Masahide; Mineo, Midori; Shinohara, Tadashi; Kataharada, Koji; Okada, Makoto; Takada, Kunio; Miyawaki, Shoji; Ohsuzu, Fumitaka


    The aim of this study was to clarify the effects of anti-dsDNA antibodies on the titer and the nuclear staining pattern(s) in a fluorescent antinuclear antibody (FANA) assay using HEp-2 cells. Anti-dsDNA derived from 14 patients with systemic lupus erythematosus (SLE) was individually affinity-purified. The anti-dsDNA titer of the purified anti-dsDNA solution was measured by radioimmunoassay (RIA) or by enzyme-linked immunosorbent assay (ELISA). In the FANA assay, the anti-dsDNA solution was diluted in a stepwise manner and its titer was expressed by the endpoint dilution. The nuclear staining pattern in the anti-dsDNA solution was examined at the 1:5 and 1:20 dilutions and at the endpoint dilution. The anti-dsDNA titers of the affinity-purified anti-dsDNA solution were high enough (13 to 126 IU/ml) to be measured by RIA. However, the antinuclear antibody (ANA) titers of this solution were relatively low: 1:20 to 1:320. In the study of nuclear staining the peripheral pattern was observed in nine of the 14 cases at a 1:5 dilution. However, at the endpoint dilution, all cases exhibited the homogeneous pattern. These findings indicate that in the FANA assay using HEp-2 cells, 1) although serum samples show high anti-dsDNA titers by RIA or by ELISA, the antibodies' direct contribution to ANA titers is limited, and 2) when samples reveal a homogeneous staining pattern at the endpoint dilution, this suggests the presence of anti-dsDNA.

  6. Best practice recommendations for the transfer of cell-based assays for the measurement of neutralizing anti-drug antibodies. (United States)

    Belouski, Shelley S; Born, Danika; Jacques, Susan; Harder, Brandon; Reynhardt, Kai; Kaliyaperumal, Arunan; Gupta, Shalini


    We recommend the application of a strategically designed step-wise approach to transfer cell-based assays that includes assessing analytical performance (through a fit for purpose validation and/or design of experiment robustness characterization), clinical performance (i.e., concordance) and performance or proficiency testing for long-term method monitoring. Here we focus on the application of this strategy to cell-based assays for the measurement of neutralizing anti-drug antibodies. This application is unique in that it requires a custom cell-based assay to be used over a long period of time (potentially phase 1a through the life of a marketed product) with the confidence of consistent method performance and result reporting. But, the process is adaptable to a variety of assay types and applications. We present lessons learned from two cell-based assay transfers that met relevant challenges while implementing alternative permutations of the recommended method transfer process.

  7. Ebolavirus nucleoprotein C-termini potently attract single domain antibodies enabling monoclonal affinity reagent sandwich assay (MARSA formulation.

    Directory of Open Access Journals (Sweden)

    Laura J Sherwood

    Full Text Available 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

  8. An optimized assay of specific IgE antibodies to reactive dyes and studies of immunologic responses in exposed workers. (United States)

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


    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.

  9. A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies. (United States)

    Chiang, S; Dar, A M; Goyal, S M; Sheikh, M A; Pedersen, J C; Panigrahy, B; Senne, D; Halvorson, D A; Nagaraja, K V; Kapur, V


    Avian pneumovirus (APV) infection of turkeys in Minnesota was first confirmed in March 1997. Serum samples (n = 5,194) from 539 submissions to Minnesota Veterinary Diagnostic Laboratory were tested by a modified enzyme-linked immunosorbent assay (ELISA). Of these, 2,528 (48.7%) samples from 269 submissions were positive and 2,666 (51.3%) samples from 270 submissions were negative for APV antibodies. Most positive samples were from Kandiyohi, Stearns, Morrison, and Meeker counties in Minnesota. In addition, 10 samples from South Dakota were positive. The sensitivity and specificity of the ELISA test with anti-chicken and anti-turkey conjugates were compared by testing field and experimental sera. The ELISA test with anti-turkey conjugate was more sensitive than that with anti-chicken conjugate. The ELISA tests with antigens prepared with APV strains isolated from Colorado and Minnesota were also compared. No difference was detectable. Currently, the Minnesota Veterinary Diagnostic Laboratory uses an antigen prepared from the Colorado isolate of APV and a goat anti-turkey conjugate in the ELISA test.

  10. An enzyme-linked immunosorbent assay to detect antibodies against glycoprotein gE of bovine herpesvirus 1 allows differentiation between infected and vaccinated cattle

    NARCIS (Netherlands)

    Oirschot, van J.T.; Kaashoek, M.J.; Maris-Veldhuis, M.A.; Weerdmeester, K.; Rijsewijk, F.A.M.


    A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against glycoprotein gE (gE) of bovine herpesvirus 1 (BHV1). The assay is based on the use of two monoclonal antibodies directed against different antigenic domains on gE. Sera from uninfected cattle and catt

  11. Extraction of Ku antigen and anti-Ku antibody assay in various autoimmune connective tissue diseases

    Institute of Scientific and Technical Information of China (English)


    Objective: To extract Ku antigen and to detect anti-Ku antibody in connective tissue diseases (CTDs). Methods: Ku antigen was prepared from rabbit thymus acetone powder. Anti-Ku antibodies were tested in 50 normal controls and 438 patients

  12. Detection of BRAF mutation in Chinese tumor patients using a highly sensitive antibody immunohistochemistry assay (United States)

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


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Parida, S; Clavijo, A


    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.

  14. Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay. (United States)

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio; Tanabayashi, Kiyoshi


    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

  15. Detection of Anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of Kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    J.J.N. Ngeranwa


    Full Text Available The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra and domestic animal (cattle, sheep and goat populations was studied in wildlife / livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA, using a recombinant antigen (MSP-5 from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was used as the primary antibody, while anti-mouse conjugated to horseradish peroxidase was used as the secondary antibody. The results indicate a high seroprevalence in both wildlife and livestock populations, in contrast to earlier reports from Kenya, which indicated a low seroprevalence. The differences are attributed to the accurate analytical method used (CI-ELISA, as compared with agglutination techniques, clinical signs and microscopy employed by the earlier workers.

  16. Analysis of antibody responses to Hymenolepis nana infection in mice by the enzyme-linked immunosorbent assay and immunoprecipitation. (United States)

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


    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.

  17. Detection of homocytotropic antibody in lambs infested with the louse, Bovicola ovis, using a basophil histamine-release assay. (United States)

    Pfeffer, A; Phegan, M D; Bany, J


    The utility of a basophil histamine-release assay using washed whole blood cells was examined in lambs and was used to determine if homocytotropic antibody with specificity for Bovicola ovis was produced in response to infestation with the louse. Maximal histamine release in the assay in response to Concanavalin A, anti-ovine IgE monoclonal antibody and, in sensitized lambs, to B. ovis antigen ranged from 18 to 48%. Histamine release from blood cells in response to B. ovis antigen was significantly higher in louse-infested lambs than in louse-naive lambs and was significantly correlated with louse and cockle scores. Passive cutaneous anaphylaxis (PCA) tests were negative with sera obtained from the lambs at the same time as blood for the basophil histamine-release assay. Serum histamine levels also were significantly higher in the louse-infested lambs than in louse-naive lambs and were significantly correlated with louse and cockle scores. The present results support a role for B. ovis-specific homocytotropic antibody in the development of cockle and indicate that the basophil histamine-release assay is more sensitive than the PCA test.

  18. High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses.

    Directory of Open Access Journals (Sweden)

    Peter Sehr

    Full Text Available A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2 = 0.7 was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.

  19. Improved performance of Brucella melitensis native hapten over Brucella abortus OPS tracer on goat antibody detection by the fluorescence polarization assay. (United States)

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


    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 (pgoat 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.

  20. 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. (United States)

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


    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 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.

  1. Quality control and evaluation of human immunodeficiency virus antibody assays used for screening donated blood in China

    Institute of Scientific and Technical Information of China (English)



    During 2004, a total of 124 batches of HIV antibody ELISAs from domestic and overseas manufacturers, comprising approximately 60 million tests, were tested for quality and released for screening blood in China. The inter- and intra-batch variation, specificity, and sensitivity were evaluated using a laboratory panel and clinical samples. The inter-batch variation was less than 15% and only 2 of 12 assays had intra-batch variation of less than 20% for 4 dilutions of a control specimen.257 samples confirmed positive for HIV antibody and 4826 negative samples from different regions in China were used to evaluate the sensitivity and specificity of the assays. The results showed that the sensitivity is in the range from 93.7% to 100% for assays sampled directly from the manufacturers,and 91.4%-99.6% for those retrieved from the consumers; the specificity was in the range from 97.88% to 99.97%. The testing environment may vary in different regions of China. Therefore, manufacturers should provide robust assays to satisfy the requirements of these diverse environments, and especially reduce the intra-assay variation and improve the stability of the kits.

  2. Evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus

    Institute of Scientific and Technical Information of China (English)

    MO Hong-ying; XU Jun; REN Xiao-lan; ZENG Guang-qiao; TAN Ya-xia; CHEN Rong-chang; Moira Chan-Yeung; ZHONG Nan-shan


    Background Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerging virus that gives rise to SARS patients with high rates of infectivity and fatality. To study the humoral immune responses to SARS-CoV, the authors evaluated IgG and IgM specific antibodies in patients' sera.Methods Two methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent assay (IFA), were used to detect specific serum IgG and IgM against SARS-CoV in 98 SARS patients and 250 controls consisting of patients with pneumonia, health-care professionals and healthy subjects. The serum antibody profiles were investigated at different times over one and a half years in 18 of the SARS patients. Results The sensitivity and specificity of ELISA for detecting IgG against SARS-CoV were 100.0% and 97.2% and for IgM 89.8% and 97.6% respectively; the figures using IFA for IgG were 100.0% and 100.0% and for IgM 81.8% and 100.0% respectively. During the first seven days of the antibodies trace test, no IgG and IgM were detected, but on day 15, IgG response increased dramatically, reaching a peak on day 60, remaining high up to day 180 and decreasing gradually until day 540. On day 15, IgM was detected, rapidly reached a peak, then declined gradually until day 180 when IgM was undetectable. Conclusion The detection of antibodies against SARS virus is helpful in the clinical diagnosis of SARS.

  3. Modular, Antibody-free Time-Resolved LRET Kinase Assay Enabled by Quantum Dots and Tb3+-sensitizing Peptides (United States)

    Cui, Wei; Parker, Laurie L.


    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.

  4. EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies. (United States)

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


    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.

  5. A rapid, accurate and robust particle-based assay for the simultaneous screening of plasma samples for the presence of five different anti-cytokine autoantibodies

    DEFF Research Database (Denmark)

    Guldager, Daniel Kring Rasmussen; von Stemann, Jakob Hjorth; Larsen, Rune


    PURPOSE: To establish and validate a rapid, cost-effective and accurate screening assay for the simultaneous testing of human naturally occurring anti-cytokine autoantibodies (c-aAb) targeting interleukin-1α (IL-1α), interleukin-6 (IL-6), interleukin-10 (IL-10), granulocyte-macrophage colony...... plasma samples and pooled normal immunoglobulin preparations were used to validate the assay. Plasma samples from 98 transfusion recipients, half of whom presented with febrile reactions, were tested by the assay. RESULTS: The assay detected specific and saturable immunoglobulin G (IgG) binding to each...... cytokine autoantibodies quantities in the negative plasma samples ranged between 80% and 125%. The analytical intra- and inter-assay variations were 4% and 11%, respectively. Varying c-aAb levels were detectable in the transfusion recipients. There was no difference in c-aAb frequency between the patients...

  6. Relation between enzyme-linked immunosorbent assay and radioimmunoassay for detection of antibodies to the capsular polysaccharide of Haemophilus influenzae type b

    Energy Technology Data Exchange (ETDEWEB)

    Kristensen, K. (Streptococcus Department, Statens Seruminstitut, Copenhagen (Denmark)); Weis Bentzon, M. (Department of Biostatistics, Statens Seruminstitut, Copenhagen (Denmark))


    The measurement of antibodies to the capsular polysaccharide (PRP) of Haemophilus influenzae type b (Hib) is important because vaccines inducing such antibodies are now available. We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for detection of these antibodies based on direct coating of the plates with tyraminated PRP. The assay fulfilled the requirements for parallel line assays; it was sensitive, specific, and reproducible with a coefficient of variation between days of 19%. Results from the ELISA were compared with results from radioimmunoassay and a correlation coefficient of 0.93 was found. Results obtained by the two methods were proportional and the relation was indepenedent of the antibody level. The relation between them was also unaffected by the contribution of different antibody isotypes, indicating that these were measured to the same extent by both methods. ELISA employing direct coating of the plates with tyraminated PRP represents a useful alternative for detection of antibodies when studying immunogenicity of Hib vaccines. (au).

  7. Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus. (United States)

    Abdelwahab, Mohamed; Loa, Chien Chang; Wu, Ching Ching; Lin, Tsang Long


    Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks.

  8. Development of monoclonal antibodies and quantitative sandwich enzyme linked immunosorbent assay for the characteristic sialoglycoprotein of edible bird's nest. (United States)

    Zhang, Shiwei; Lai, Xintian; Liu, Xiaoqing; Li, Yun; Li, Bifang; Huang, Xiuli; Zhang, Qinlei; Chen, Wei; Lin, Lin; Yang, Guowu


    The article presents a sandwich enzyme linked immunosorbent assay (ELISA) for identification of edible bird's nest. The characteristic sialoglycoproteins were found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified by liquid-phase isoelectric focusing (LIEF). According to the analysis, the molecular weight was 106-128 kDa and the isoelectric point was ≤pH 3.0. Two anti-characteristic sialoglycoprotein monoclonal antibodies were produced. The monoclonal antibodies were examined by western-blot assay. One of the monoclonal antibody was used as coating and the other as the enzyme-labeled antibody after being coupled to horseradish peroxidase (HRP). Based on the optimized ELISA condition, the method was established with IC(50) of 1.5 ng/mL, and low cross-reactivity with various fake materials (ELISA provided a suitable means for screening of a large number of samples. The coefficients of variation were between 2.9% and 5.8%.

  9. Auto-antibodies to double-stranded DNA as biomarker in systemic lupus erythematosus : comparison of different assays during quiescent and active disease

    NARCIS (Netherlands)

    de Leeuw, Karina; Bungener, Laura; Roozendaal, Caroline; Bootsma, Hendrika; Stegeman, Coen A


    OBJECTIVE: Auto-antibodies directed to dsDNA (anti-dsDNA) are used in diagnosis and follow-up for SLE. However, multiple assays are used. The objective of this study was to determine the best-performing assays, especially in prediction of exacerbations. METHODS: Seven assays were compared during LN

  10. A Novel IgM-capture enzyme-linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection. (United States)

    Otsuka, Nao; Gotoh, Kensei; Nishimura, Naoko; Ozaki, Takao; Nakamura, Yukitsugu; Haga, Kiyohito; Yamazaki, Makoto; Gondaira, Fumio; Okada, Kenji; Miyaji, Yusuke; Toyoizumi-Ajisaka, Hiromi; Shibayama, Keigo; Arakawa, Yoshichika; Kamachi, Kazunari


    An ELISA that measures anti-PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG-based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti-PT IgG antibodies. To solve this problem, we developed a novel IgM-capture ELISA that measures serum anti-Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti-Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti-Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti-Vag8 IgM-capture ELISA. The results revealed that the anti-Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti-Vag8 IgM-capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti-PT IgG ELISA kit. Moreover, it was shown that anti-Vag8 IgM antibodies were induced earlier than anti-PT IgG antibodies on sequential patients' sera. These data indicate that our novel anti-Vag8 IgM-capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.

  11. Antibody (United States)

    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 ...

  12. A pseudovirus-based hemagglutination-inhibition assay as a rapid, highly sensitive, and specific assay for detecting avian influenza A (H7N9 antibodies

    Directory of Open Access Journals (Sweden)

    Anli Zhang


    Full Text Available Background Increased surveillance of avian-origin influenza A (H7N9 virus infection is critical to assess the risk of new outbreaks in China. A high-throughput assay with a good safety profile, sensitivity, and specificity is urgently needed. Methods We used a hemagglutination-inhibition (HI assay based on an H7N9-enveloped pseudovirus to assess serum neutralization antibodies level in 40 H7N9 positive sera and 40 H7N9 negative sera and compared the efficacy of the assay with traditional HI test and micro-neutralization (MN test. Results Spearman’s rank correlation coefficient analysis showed pseudovirus HI (PHI titers correlated well with both HI titers and MN titers. Receiver operating characteristic (ROC curves test revealed using a PHI cut-off titer of 10, the sensitivity and specificity reached 1.0. Conclusions PHI can be used in H7N9-related serological studies. This assay is high-throughput, very sensitive and specific, and cost effective.

  13. Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate. (United States)

    Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred


    Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.

  14. Profiling of multiple signal pathway activities by multiplexing antibody and GFP-based translocation assays. (United States)

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


    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.

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

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


    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.

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

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


    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.

  17. Combined Hepatitis C Virus (HCV) Antigen-Antibody Detection Assay Does Not Improve Diagnosis for Seronegative Individuals with Occult HCV Infection▿


    Quiroga, Juan A.; Castillo, Inmaculada; Pardo, Margarita; Rodríguez-Iñigo, Elena; CARREÑO, VICENTE


    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.

  18. Preparation of Monoclonal Antibody and Development of Enzyme-linked Immunosorbent Assay Specific for Escherichia coli O157 in Foods

    Institute of Scientific and Technical Information of China (English)


    Objective To prepare monoclonal antibodies (Mab) and antisera specific for Escherichia coli (E.coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect E.coli O157 in foods. Methods Spleen cells from BALB/c mice immunized with the somatic antigen of E.coli O157:H7 were fused with murine Sp2/0 myeloma cells. The hybridoma cell line specific for E.coli O157 was established after having been subcloned. Antisera specific for E.coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E.coli O157:H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the Mab 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized milk were tested to confirm efficiency of the method. Results Mab 3A5 specific for E.coli O157 and O113:H21 belonged to subtype IgM. The ascetic titers of the antibody was 1:1×106. No cross-reactivity of the Mab was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1:1×105 with E.coli O157. The detection limit of this sandwich ELISA was 103-104 cfu E.coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E.coli O157:H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. Conclusion Mab 3A5 specific for E.coli O157 and O113:H21 can be produced by immunizing BALB/c mice with a strain of E.coli O157:H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the Mab 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E.coli O157 in food.

  19. Establishment and preliminary application of Dengue virus envelope domain Ⅲ IgG antibody capture enzyme-linked immuno-absorbent assay

    Institute of Scientific and Technical Information of China (English)



    Objective To establish a highly sensitive and specific assay to detect Dengue virus(DENV) envelope protein domainⅢ(EDⅢ) IgG antibody,and to explore its value in the diagnosis and seroepidemiological survey of dengue

  20. LabKey Server NAb: A tool for analyzing, visualizing and sharing results from neutralizing antibody assays

    Directory of Open Access Journals (Sweden)

    Gao Hongmei


    Full Text Available Abstract Background Multiple types of assays allow sensitive detection of virus-specific neutralizing antibodies. For example, the extent of antibody neutralization of HIV-1, SIV and SHIV can be measured in the TZM-bl cell line through the degree of luciferase reporter gene expression after infection. In the past, neutralization curves and titers for this standard assay have been calculated using an Excel macro. Updating all instances of such a macro with new techniques can be unwieldy and introduce non-uniformity across multi-lab teams. Using Excel also poses challenges in centrally storing, sharing and associating raw data files and results. Results We present LabKey Server's NAb tool for organizing, analyzing and securely sharing data, files and results for neutralizing antibody (NAb assays, including the luciferase-based TZM-bl NAb assay. The customizable tool supports high-throughput experiments and includes a graphical plate template designer, allowing researchers to quickly adapt calculations to new plate layouts. The tool calculates the percent neutralization for each serum dilution based on luminescence measurements, fits a range of neutralization curves to titration results and uses these curves to estimate the neutralizing antibody titers for benchmark dilutions. Results, curve visualizations and raw data files are stored in a database and shared through a secure, web-based interface. NAb results can be integrated with other data sources based on sample identifiers. It is simple to make results public after publication by updating folder security settings. Conclusions Standardized tools for analyzing, archiving and sharing assay results can improve the reproducibility, comparability and reliability of results obtained across many labs. LabKey Server and its NAb tool are freely available as open source software at under the Apache 2.0 license. Many members of the HIV research community can also access the Lab

  1. Comparison of enzyme-linked immunosorbent assays and virus neutralization test for detection of antibodies to avian pneumovirus. (United States)

    Alkahalaf, A N; Halvorson, D A; Saif, Y M


    Two different whole-virus enzyme-linked immunosorbent assays (ELISAs), developed in Ohio (OH) with APV/Minnesota/turkey/2a/97 and in Minnesota (MN) with APV/Colorado/turkey/97, and the virus neutralization (VN) test were used to test 270 turkey serum samples from 27 Minnesota turkey flocks for avian pneumovirus (APV) antibodies. In addition, 77 turkey serum samples and 128 ostrich serum samples from Ohio were tested. None of the turkey samples from Ohio had antibodies to APV by the VN test and OH ELISA. The ostrich samples were only tested with the VN test and were all negative for antibodies to APV. For the Minnesota serum samples, 107, 115, and 120 were positive by the VN test, the OH ELISA, and the MN ELISA, respectively. The Kappa values of 0.938 and 0.825 showed excellent agreement between the VN test and the OH ELISA and the MN ELISA, respectively, for detection of antibodies to the APV. The OH ELISA and MN ELISA had sensitivities of 1.0 and 0.953, specificities of 0.950 and 0.889, and accuracies of 0.970 and 0.914, respectively. Our results indicate that the 3 methods are sensitive and specific for diagnosis of the APV infection.

  2. 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. (United States)

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


    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.

  3. 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▿


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


    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 assa...

  4. Seq4SNPs: new software for retrieval of multiple, accurately annotated DNA sequences, ready formatted for SNP assay design

    Directory of Open Access Journals (Sweden)

    Dunning Alison M


    Full Text Available Abstract Background In moderate-throughput SNP genotyping there was a gap in the workflow, between choosing a set of SNPs and submitting their sequences to proprietary assay design software, which was not met by existing software. Retrieval and formatting of sequences flanking each SNP, prior to assay design, becomes rate-limiting for more than about ten SNPs, especially if annotated for repetitive regions and adjacent variations. We routinely process up to 50 SNPs at once. Implementation We created Seq4SNPs, a web-based, walk-away software that can process one to several hundred SNPs given rs numbers as input. It outputs a file of fully annotated sequences formatted for one of three proprietary design softwares: TaqMan's Primer-By-Design FileBuilder, Sequenom's iPLEX or SNPstream's Autoprimer, as well as unannotated fasta sequences. We found genotyping assays to be inhibited by repetitive sequences or the presence of additional variations flanking the SNP under test, and in multiplexes, repetitive sequence flanking one SNP adversely affects multiple assays. Assay design software programs avoid such regions if the input sequences are appropriately annotated, so we used Seq4SNPs to provide suitably annotated input sequences, and improved our genotyping success rate. Adjacent SNPs can also be avoided, by annotating sequences used as input for primer design. Conclusion The accuracy of annotation by Seq4SNPs is significantly better than manual annotation (P Using Seq4SNPs to incorporate all annotation for additional SNPs and repetitive elements into sequences, for genotyping assay designer software, minimizes assay failure at the design stage, reducing the cost of genotyping. Seq4SNPs provides a rapid route for replacement of poor test SNP sequences. We routinely use this software for assay sequence preparation. Seq4SNPs is available as a service at and

  5. A Simple and Accurate Way to Assay the Concentration of Nitric Acid——Back-titration Method

    Institute of Scientific and Technical Information of China (English)


    Using 1mol/L (NH4)2SO4-aturated (NH4)2C2O4 as a masking reagent, V-△V/△pH, V-pH and back-titration methods are used to assay the concentration of nitric acid under various conditions and theresults are compared with that from phenolphthalein indicator method. It is shown that the data treatment

  6. Establishment of an Enzyme-linked Immunosorbent Assay for Thyroglobulin Antibody

    Institute of Scientific and Technical Information of China (English)


    Thyroglobulin is coated on the microtiter plate and labeled with horseradish peroxidase(HRP). Thetwo-step assay is established based on enzyme-linked immunosorbent assay(ELISA). TMB-H2O2 solution

  7. Antibodies to immunoglobulin-G in dog sera, synovial fluids and aqueous humor : a comparative study of rheumatoid factor assays, suitable for routine application

    NARCIS (Netherlands)

    Bernadina, W.E.; Kol, P.J. van; Willemse, A.


    The incidence of anti-IgG antibodies (rheumatoid factors, RF) in body fluids (sera, synovial fluids and aqueous humor) selected from 62 normal and 275 diseased dogs was studied. Fluids were assayed by canine versions of standard agglutinating and/or precipitating RF assays with routine application i

  8. Indirect enzyme-linked immunosorbent assay for detection of Brucella melitensis-specific antibodies in goat milk. (United States)

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


    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 melitensis-specific antibodies in goat milk.

  9. Detection of drug-dependent, platelet-reactive antibodies by solid-phase red cell adherence assays. (United States)

    Leach, M F; Cooper, L K; AuBuchon, J P


    We developed a simple modification of the solid-phase red cell adherence (SPRCA) assay system that can be used to identify drug-dependent platelet antibodies (DDPAs) reactive by either the hapten or immune complex reaction mechanisms. Between January 1994 and August 1996 we tested sera from 173 patients [123 (71%) with unexplained thrombocytopenia and 50 (29%) because of poor responses to platelet transfusions not explicable by alloimmunization or the clinical situation] for DDPAs possibly associated with the receipt of 61 different drugs. We correlated positive results with patients' clinical courses. DDPAs were identified in samples from 138 (80%) of the patients tested. Antibodies reactive only by the hapten mechanism were identified in 51 (37%) of those sera exhibiting positive reactions. The clinical courses of 108 (78%) patients were evaluable. Discontinuation of the implicated drug(s) resulted in prompt (<5 d) resolution of the thrombocytopenia or improvement in response to transfusion in all of these patients. In four cases thrombocytopenia returned upon re-exposure to the implicated drug. This adaptation of SPRCA provides a simple means of investigating the possibility of DDPAs and documents a higher frequency of these antibodies than has previously been suspected.

  10. High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays


    Hill, Danika L.; Eriksson, Emily M.; Schofield, Louis


    Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibito...

  11. A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma

    DEFF Research Database (Denmark)

    Cham, Gerald K K; Kurtis, Jonathan; Lusingu, John;


    BACKGROUND: The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample...... of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1......-based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both...

  12. An automated packed protein G micro-pipette tip assay for rapid quantification of polyclonal antibodies in ovine serum. (United States)

    Chhatre, Sunil; Francis, Richard; Bracewell, Daniel G; Titchener-Hooker, Nigel J


    The demands on the biopharmaceutical sector to expedite process development have instigated the deployment of micro-biochemical engineering techniques to acquire manufacturing insight with extremely small sample volumes. In conjunction with automated liquid handlers, this permits the simultaneous evaluation of multiple operating conditions and reduces manual intervention. For these benefits to be sustained, novel ways are now required to accelerate analysis and so prevent this becoming a throughput bottleneck. For example, although Protein G HPLC is used to quantify antibody titres in bioprocess feedstocks, it can be time-consuming owing to the serial nature of its application. Although commercial options are available that can process many samples simultaneously, these require separate, potentially expensive instruments. A more integrated approach is desirable wherein the assay is implemented directly on a robot. This article describes a high-throughput alternative to antibody HPLC analysis which uses an eight-channel liquid handler to control pipette tips packed with 40 μL of Protein G affinity matrix. The linearity, range, limit of detection, specificity and precision of the method were established, with results showing that antibody was detected reliably and specifically between 0.10 and 1.00 mg/mL. Subsequently, the technique was used to quantify the antibody titre in ovine serum, which is used as feed material by BTG PLC for manufacturing FDA-approved polyclonal bio-therapeutics. The mean concentration determined by the tips was comparable to that found by HPLC, but the tip method delivered its results in less than 40% of the time and with the potential for further, substantial time-savings possible by using higher capacity robots.

  13. A feasible enzyme-linked immunosorbent assay system using monoclonal and polyclonal antibodies against glucosyltransferase-B from Streptococcus mutans. (United States)

    Shinozaki-Kuwahara, Noriko; Hashizume-Takizawa, Tomomi; Hirasawa, Masatomo; Takada, Kazuko


    Streptococcus mutans has been considered the principal etiological agent of dental caries in humans. S. mutans can secrete three kinds of glucosyltransferases (GTFs). One of these, GTF-B, which synthesizes water-insoluble glucans from sucrose, has been considered to be one of the most important factors of cariogenic dental plaque formation. Therefore, determination of whether GTF-B is present in plaque and saliva samples may contribute to the evaluation of individual virulence potential (caries risk). The aim of this study was to develop a feasible enzyme-linked immunosorbent assay (ELISA) for the routine quantification of GTF-B in plaque-derived cultures and clinical samples, and to apply this assay to an epidemiological study. To determine the presence of GTF-B in plaque samples, a sandwich-ELISA was devised, consisting of mouse monoclonal and rabbit polyclonal antibodies against GTF-B and a horseradish peroxidase-conjugated anti-rabbit antibody. The developed ELISA allowed for quantification of the amounts of purified GTF-B with satisfactory sensitivity and specificity; this method was not affected by other components such as plaque and saliva. Plaque samples from healthy volunteers were examined using this ELISA method and microbial analysis to apply the assay to an epidemiological study. A correlation was observed between the amount of extracted GTF-B and S. mutans levels as determined by ELISA and cultivated with Mitis Salivarius Bacitracin agar plates derived from plaque samples, although there were some exceptions. In this regard, this ELISA system has the advantage of estimating both the individual numbers of S. mutans and the productivity of GTF-B, namely, the cariogenic potential of S. mutans simultaneously. These results indicate that this ELISA method is a useful tool for the diagnosis of caries risk.

  14. Feasibility studies for assaying alpha-fetoprotein using antibody-activated magnetic nanoparticles

    Directory of Open Access Journals (Sweden)

    Huang KW


    Full Text Available Kai-Wen Huang1, Shieh-Yueh Yang2,3, Yu-Wei Hong3, Jen-Jie Chieh3, Che-Chuan Yang3, Herng-Er Horng3, Chau-Chung Wu4, Chin-Yih Hong5, Hong-Chang Yang61Department of Surgery and Hepatitis Research Center, National Taiwan University Hospital, National Taiwan University, Taipei, 2MagQu Co, Ltd, Sindian Dist, New Taipei City, 3Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei, 4Departments of Internal Medicine and Primary Care Medicine, College of Medicine, National Taiwan University, Taipei, 5Graduate Institute of Bio-medical Engineering, National Chung Hsing University, Taichung, 6Department of Physics, National Taiwan University, Taipei, TaiwanAbstract: Some previous reports have already shown the characterizations of immunomagnetic reduction (IMR. The assay technology involves the utilities of biofunctionalized magnetic nanoparticles to label target biomolecules. However, the detection threshold and interference tests for IMR have not been investigated in detail. In this study, alpha-fetoprotein (AFP was used as a target biomolecule. The signals for AFP solutions of various concentrations, or with interfering materials, were detected via IMR. These samples were also used for characterizing the detection threshold and interference with enzyme-linked immunosorbent assay (ELISA. The results of assaying AFP level with IMR and ELISA were compared. The detection threshold for assaying AFP with IMR was found to be 3 ng/mL, which is 15 times lower than that of ELISA, and definitely suppresses false negative. For the interfering materials noted commonly in serum such as hemoglobin, bilirubin, triglyceride, and vascular endothelial growth factor, there was no detectable interfering effect when assaying AFP with IMR. Several serum samples from normal people and liver-tumor-bearing patients were used for the detections of AFP concentration via IMR. These results reveal the feasibilities of assaying AFP in blood

  15. An Innovative Pseudotypes-Based Enzyme-Linked Lectin Assay for the Measurement of Functional Anti-Neuraminidase Antibodies.

    Directory of Open Access Journals (Sweden)

    Marua Prevato

    Full Text Available Antibodies (Ab to neuraminidase (NA play a role in limiting influenza infection and might help reduce the disease impact. The most widely used serological assay to measure functional anti-NA immune responses is the Enzyme-Linked Lectin Assay (ELLA which relies on hemagglutinin (HA mismatched virus reassortants, or detergent treated viruses as the NA source to overcome interference associated with steric hindrance of anti-HA Ab present in sera. The difficulty in producing and handling these reagents, which are not easily adapted for screening large numbers of samples, limits the routine analysis of functional anti-NA Ab in clinical trials. In this study, we produced influenza lentiviral pseudoparticles (PPs containing only the NA antigen (NA-PPs with a simple two-plasmid co-transfection system. NA-PPs were characterized and tested as an innovative source of NA in the NA inhibition (NI assay. Both swine A/California/07/2009 (H1N1 and avian A/turkey/Turkey/01/2005 (H5N1 N1s within NA-PPs retained their sialidase activity and were specifically inhibited by homologous and N1 subtype-specific, heterologous sheep sera. Moreover, A/California/07/2009 N1-PPs were a better source of NA compared to whole live and detergent treated H1N1 viruses in ELLA, likely due to lack of interference by anti-HA Ab, and absence of possible structural modifications caused by treatment with detergent. This innovative assay is safer and applicable to all NAs. Taken together, these results highlight the potential of NA-PPs-based NI assays to be developed as sensitive, flexible, easy to handle and scalable serological tests for routine NA immune response analysis.

  16. Development of a Rapid Qualitative Assay for Determining Elevated Antibody Levels to Periodontopathic Organisms (United States)


    another report, showed no elevation in IgM or IgA in these patients, with some-elevation of total~lgG (Rahney et’aL. 1981)’. Murray & Genco (1980) found...leukotoxic activity compared to 24% of normal, 39%of adult periodontitis,.,and 38% of ANUG sera. Genco et at. (1980b) found that.89% of LJP patients...Aactinomycetemcomitans have been notedin patients with rapidly progressive disease ( Genco et al. 1985). Theserum antibody response in adult periodontitis is more

  17. Novel Monoclonal Antibody-Based Immunodiagnostic Assay for Rapid Detection of Deamidated Gluten Residues. (United States)

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


    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.

  18. A hybrid stochastic-deterministic computational model accurately describes spatial dynamics and virus diffusion in HIV-1 growth competition assay. (United States)

    Immonen, Taina; Gibson, Richard; Leitner, Thomas; Miller, Melanie A; Arts, Eric J; Somersalo, Erkki; Calvetti, Daniela


    We present a new hybrid stochastic-deterministic, spatially distributed computational model to simulate growth competition assays on a relatively immobile monolayer of peripheral blood mononuclear cells (PBMCs), commonly used for determining ex vivo fitness of human immunodeficiency virus type-1 (HIV-1). The novel features of our approach include incorporation of viral diffusion through a deterministic diffusion model while simulating cellular dynamics via a stochastic Markov chain model. The model accounts for multiple infections of target cells, CD4-downregulation, and the delay between the infection of a cell and the production of new virus particles. The minimum threshold level of infection induced by a virus inoculum is determined via a series of dilution experiments, and is used to determine the probability of infection of a susceptible cell as a function of local virus density. We illustrate how this model can be used for estimating the distribution of cells infected by either a single virus type or two competing viruses. Our model captures experimentally observed variation in the fitness difference between two virus strains, and suggests a way to minimize variation and dual infection in experiments.

  19. Isothermal microcalorimetry accurately detects bacteria, tumorous microtissues, and parasitic worms in a label-free well-plate assay. (United States)

    Braissant, Olivier; Keiser, Jennifer; Meister, Isabel; Bachmann, Alexander; Wirz, Dieter; Göpfert, Beat; Bonkat, Gernot; Wadsö, Ingemar


    Isothermal microcalorimetry is a label-free assay that allows monitoring of enzymatic and metabolic activities. The technique has strengths, but most instruments have a low throughput, which has limited their use for bioassays. Here, an isothermal microcalorimeter, equipped with a vessel holder similar to a 48-well plate, was used. The increased throughput of this microcalorimeter makes it valuable for biomedical and pharmaceutical applications. Our results show that the sensitivity of the instrument allows the detection of 3 × 10(4) bacteria per vial. Growth of P. mirabilis in Luria Broth medium was detected between 2 and 9 h with decreasing inoculum. The culture released 2.1J with a maximum thermal power of 76 μW. The growth rate calculated using calorimetric and spectrophotometric data were 0.60 and 0.57 h(-1) , respectively. Additional insight on protease activities of P. mirabilis matching the last peak in heat production could be gathered as well. Growth of tumor microtissues releasing a maximum thermal power of 2.1 μW was also monitored and corresponds to a diameter increase of the microtissues from ca. 100 to 428 μm. This opens new research avenues in cancer research, diagnostics, and development of new antitumor drugs. For parasitic worms, the technique allows assessment of parasite survival using motor and metabolic activities even with a single worm.

  20. Significance of enzyme linked immunosorbent assay (ELISA) for antibodies to double stranded and single stranded DNA in patients with lupus nephritis: correlation with severity of renal histology. (United States)

    Okamura, M; Kanayama, Y; Amastu, K; Negoro, N; Kohda, S; Takeda, T; Inoue, T


    The correlation between renal histology and class specific (IgG and IgM) antibodies to double stranded DNA (dsDNA) and single stranded DNA (ssDNA) was studied by enzyme linked immunosorbent assay (ELISA) in 40 untreated patients with systemic lupus erythematosus (SLE). The levels of IgG antibodies to dsDNA were significantly higher in patients with World Health Organisation class IV nephritis than in those with class I, class II, or class III nephritis. IgG antibodies to ssDNA were higher in patients with class IV than in those with class II nephritis. IgG antibodies to dsDNA showed a close correlation with the histological activity score and the amount of electron dense deposit. IgG antibodies to ssDNA showed only a weak correlation with the renal histological activity score. IgM antibodies to dsDNA and IgM antibodies to ssDNA were not correlated with renal histological features. Patients with moderate to severe nephritis had a lower ratio of IgM antibodies to dsDNA to IgG antibodies to dsDNA than those with mild nephritis. These results indicate that the measurement of IgG antibodies to dsDNA is predictive in evaluating renal histological activity in patients with SLE.

  1. Comparison of antibody assays for detection of autoantibodies to Ro 52, Ro 60 and La associated with primary Sjögren's syndrome. (United States)

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


    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.

  2. Evaluation of an egg yolk enzyme-linked immunosorbent assay antibody test and its use to assess the prevalence of Mycoplasma gallisepticum infection in laying hens in Italy

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    Marco Tamba


    Full Text Available The prevalence of Mycoplasma gallisepticum infection in commercial layers was established by the presence of antibodies in eggs. Saline-extracted yolks were used with a commercial enzyme-linked immunosorbent assay kit. For the prevalence study, yolks from 30 eggs were obtained from each of 66 flocks coming from 36 layer farms. The prevalence of egg antibodies to Mycoplasma gallisepticum was 33.3% in single-age farms and 77.8% in multi-age farms. In 27 flocks, antibody titers were compared with results obtained from blood samples taken in the same flock and in the same period and analyzed with the same kit. This study has confirmed that egg yolk enzyme-linked immunosorbent assay antibody test is a suitable and practical approach for assessing the flock prevalence of Mycoplasma gallisepticum infection in layer hens.

  3. Preparation of a specific monoclonal antibody to asiaticoside for the development of an enzyme-linked immunosorbent assay. (United States)

    Juengwatanatrakul, Thaweesak; Sritularak, Boonchoo; Amornnopparattanakul, Paveena; Tassanawat, Patcharin; Putalun, Waraporn; Tanaka, Hiroyuki; Morimoto, Satoshi


    Asiaticoside (AS), the major active component of Centella asiatica (L.) Urban, is used as a memory enhancer and for wound healing. We have successfully prepared monoclonal antibodies (MAbs) against AS, and developed an enzyme-linked immunosorbent assay (ELISA) system for its determination. AS was conjugated to the carrier protein bovine serum albumin (BSA), which acted as an immunogen. In order to confirm its immunogenicity, the ratio of hapten in the AS-BSA conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization, hybridomas secreting MAbs against AS were produced by fusing splenocytes with the mouse myeloma cell line, SP2/0-Ag14. After the screening, anti-asiaticoside MAb 2B4 was obtained. Weak cross-reactivities occurred with madecassoside (7.08%), but no cross-reactivities were observed with other related triterpenoid glycosides (<0.01%). The assay was suitable for quantitating AS in the range of 0.78 to 50 µg mL(-1). A good correlation of AS concentrations in crude extracts of C. asiatica between ELISA and HPLC methods was obtained (r(2) = 0.999). The contents of AS in various cultivated C. asiatica samples were assayed by the newly established ELISA. The recovery rates of AS in the samples were in the range of 95-103% with coefficients of variation of <10%. The intra- and inter-assay variations were 3.9 and 4.5%, respectively. The ELISA method described should prove useful as an analytical tool for quality control and standardization of medicinal plants and pharmaceutical products containing AS.

  4. Rapid and accurate detection of Mycobacterium tuberculosis in sputum samples by Cepheid Xpert MTB/RIF assay--a clinical validation study.

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    Andrea Rachow

    Full Text Available BACKGROUND: A crucial impediment to global tuberculosis control is the lack of an accurate, rapid diagnostic test for detection of patients with active TB. A new, rapid diagnostic method, (Cepheid Xpert MTB/RIF Assay, is an automated sample preparation and real-time PCR instrument, which was shown to have good potential as an alternative to current reference standard sputum microscopy and culture. METHODS: We performed a clinical validation study on diagnostic accuracy of the Xpert MTB/RIF Assay in a TB and HIV endemic setting. Sputum samples from 292 consecutively enrolled adults from Mbeya, Tanzania, with suspected TB were subject to analysis by the Xpert MTB/RIF Assay. The diagnostic performance of Xpert MTB/RIF Assay was compared to standard sputum smear microscopy and culture. Confirmed Mycobacterium tuberculosis in a positive culture was used as a reference standard for TB diagnosis. RESULTS: Xpert MTB/RIF Assay achieved 88.4% (95%CI = 78.4% to 94.9% sensitivity among patients with a positive culture and 99% (95%CI = 94.7% to 100.0% specificity in patients who had no TB. HIV status did not affect test performance in 172 HIV-infected patients (58.9% of all participants. Seven additional cases (9.1% of 77 were detected by Xpert MTB/RIF Assay among the group of patients with clinical TB who were culture negative. Within 45 sputum samples which grew non-tuberculous mycobacteria the assay's specificity was 97.8% (95%CI = 88.2% to 99.9%. CONCLUSIONS: The Xpert MTB/RIF Assay is a highly sensitive, specific and rapid method for diagnosing TB which has potential to complement the current reference standard of TB diagnostics and increase its overall sensitivity. Its usefulness in detecting sputum smear and culture negative patients needs further study. Further evaluation in high burden TB and HIV areas under programmatic health care settings to ascertain applicability, cost-effectiveness, robustness and local acceptance are required.

  5. Performance of three microimmunofluorescence assays for detection of Chlamydia pneumoniae immunoglobulin M, G, and A antibodies

    DEFF Research Database (Denmark)

    Bennedsen, Mette; Berthelsen, Lene; Lind, Inga


    Labsystems (LAB) and MRL Diagnostics (MRL) by investigation of sera from three groups of patients: group I, 83 sera from 28 patients with atypical pneumonia; group II, 37 sera from 16 patients with acute C. pneumoniae or Chlamydia psittaci respiratory tract infection confirmed by PCR or culture; group III......The microimmunofluorescence (MIF) test is considered the "gold standard" for laboratory diagnosis of acute and chronic Chlamydia pneumoniae infection. The performance of a MIF test based on C. pneumoniae antigen from Washington Research Foundation (WRF) was compared with those of assays from...

  6. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. (United States)

    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


    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.

  7. Comparison of seven commercial enzyme-linked immunosorbent assays for the detection of anti-diphtheria toxin antibodies. (United States)

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


    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.

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

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


    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

  9. Comparison of enzyme linked immunosorbent assay (ELISA with indirect immunofluorescence for detection of anti-nuclear antibody

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    G.L.S. Sumanth Kumar


    Full Text Available Background: Detection of antinuclear antibody (ANA is used as one of the diagnostic criteria for autoimmune rheumatic diseases (ARD. Both indirect immunofluorescence (IIF and enzyme linked immunosorbant assay (ELISA methods are used for this purpose. However, there are lack of data comparing these two tests from India. Methods: We prospectively studed 294 patients clinically suspected to be having ARD between April 2012 and September 2013. They were tested for ANA by IIF and ELISA methods. Representative samples positive by both the tests were processed again by a line immunoassay test to detect the specific antinuclear antibodies. Considering the IIF results as the ‘gold standard’, the utility of ELISA for ANA detection was analyzed. Results: Of the 294 samples processed, 181 (61.5% were from female patients. By IIF 30% of samples in males and 40.3% sample in females tested positive. We found ELISA to have a poor sensitivity (45.8% but good specificity (99.5%. The positive predictive value for ELISA were 98% and negative predictive value 76.2% respectively. Forty four samples positive by both IIF and ELISA were tested by Western blot to detect individual autoantibodies. Of these, only 24 samples showed the presence of one or more bands, while the remaining 20 (45.4% were negative by line immunoassay. In our study anti-nuclear ribonucleoprotein/Smith was the most common ANA detected. Conclusions: The poor sensitivity raises concerns regarding the practice of initial screening for ANA by ELISA

  10. 抗核抗体筛查试验与特异性抗体确认试验的相关性研究%Correlation between screening assay of antinuclear antibody and confirmatory assay of specific antibody

    Institute of Scientific and Technical Information of China (English)

    周仁芳; 胡朝军; 张蜀澜; 白伊娜; 宋宁; 李丽君; 李永哲


    Objective To investigate the correlation between screening assay of antinuclear antibody ( ANA ) and confirmatory assay of specific antibody. Methods Five hundred clinical patients' serum samples were detected by indirect immunofluorescence ( I1F) for screening assay of ANAs and line immunoassay ( LIA) for confirmatory assay of specific ANAs. According to the results of IIF-ANA and LIA-ANAs, 500 samples were divided into 4 groups; IIF-ANA ~+/LIA-ANAs~+ group, IIF-ANA~+/LIA-ANAs~-group, IIF-ANA~-/LIA-ANAs~+ group and IIF-ANA~-LIA-ANAs~- group. The samples in IIF-ANA~-VLIA-ANAs~+ group were collected and rechecked by ELISA and DID for detection of specific ANAs. Results Five hundred clinical samples were identified including 247 cases of IIF-ANA~+/LJA-ANAs~+86 cases of IIF-ANA ~+/LIA-ANAs~- , 152 cases of IIF-ANA~-/LIA-ANAs~- and 15 cases of IIF-ANA~- /LIA-ANAs~+, In IIF-ANA ~+/LIA-ANAs~+ group, 48. 2% ,23. 9% and 27. 9% patients had the titre of IIF-ANA 1:1 280, 1: 80-1:160 and 1:320-1:640. Fifteen cases of LJA-ANAs~+ were identified in 167 cases of HF-ANA(9. 0% ). Among 15 cases of IIF-ANA~-/LIA-ANAs~+ 11 positive cases were identified as anti-SSA and anti-Ro52 positive, which were rechecked by ELISA and DID, and the results were consistent with the prior detection. Other positive cases were identified including 1 case of anti-Histone, 1 case of anti-Scl-70, 1 case of anti-Jo-1 and 1 case of anti-AMA-M2. The positive consistent rates between LJA-ANAs and ELJSA, DID were 100% , 69% respectively. And 8 cases of IIF-ANA~-/LJA-ANAs~+ patients were diagnosed with autoimmune disease after clinical data analysis. Among 86 cases of IIF-ANA ~+ /LJA-ANAs~- , 50.0% and 36.1% patients had the titre of 1: 80-1: 160 and homogeneous fluorescence pattern respectively. There was a significant difference in IIF-ANA~+/LIA-ANAs~- group and IIF-ANA ~+/LIA-ANAs~+ (χ~2 =20.47,12.42, P<0. 05). Conclusions It is recommend that specific antinuclear antibodies can be used once screening

  11. 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. (United States)

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


    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.

  12. An integrated microfluidic system for measurement of glycated hemoglobin levels by using an aptamer-antibody assay on magnetic beads. (United States)

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


    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.

  13. Comparison of a Monoclonal Antibody-Blocking Enzyme-Linked Immunoassay and a Strip Immunoblot Assay for Identifying Type-Specific Herpes Simplex Virus Type 2 Serological Responses


    van Doornum, G. J. J.; Slomka, M.J.; Buimer, M; Groen, J.; van den Hoek, J.A.R.; Cairo, I.; Vyse, A.; Brown, D. W G


    Detection of herpes simplex virus type 2 (HSV-2)-specific antibodies by a monoclonal antibody (MAb)-blocking enzyme-linked immunoassay (EIA) was compared with detection by a strip immunoblot assay (SIA) in a sexually transmitted disease (STD) clinic population. The study population consisted of 1,683 genitourinary medicine clinic attendees (582 women and 1,101 men). Sera were tested for the presence of HSV-2 antibody by use of the blocking EIA, in which binding of the MAb AP-1 to HSV-2 glycop...

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

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


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Jaszczak, R.J.


    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.

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

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


    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

  17. Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen. (United States)

    Quinn, Conrad P; Semenova, Vera A; Elie, Cheryl M; Romero-Steiner, Sandra; Greene, Carolyn; Li, Han; Stamey, Karen; Steward-Clark, Evelene; Schmidt, Daniel S; Mothershed, Elizabeth; Pruckler, Janet; Schwartz, Stephanie; Benson, Robert F; Helsel, Leta O; Holder, Patricia F; Johnson, Scott E; Kellum, Molly; Messmer, Trudy; Thacker, W Lanier; Besser, Lilah; Plikaytis, Brian D; Taylor, Thomas H; Freeman, Alison E; Wallace, Kelly J; Dull, Peter; Sejvar, Jim; Bruce, Erica; Moreno, Rosa; Schuchat, Anne; Lingappa, Jairam R; Martin, Sandra K; Walls, John; Bronsdon, Melinda; Carlone, George M; Bajani-Ari, Mary; Ashford, David A; Stephens, David S; Perkins, Bradley A


    The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 micro g/mL, a reliable lower limit of detection of 0.09 micro g/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 micro g/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 97.6%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.

  18. Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Anaplasma phagocytophilum in sheep. (United States)

    Woldehiwet, Z; Yavari, C


    An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Anaplasma phagocytophilum in ovine serum samples was evaluated. The assay used purified A. phagocytophilum grown in tick cell cultures as antigen. Serum samples were diluted 1 in 200 and binding was detected with anti-sheep IgG conjugated to horseradish peroxidase. All tests were carried out in the presence of positive and negative control samples. Optical density (OD) values obtained for each test sample at 490 nm were used to calculate percentage positivity (PP) of each sample based on the ratio of the OD of the test sample that of the positive reference sample. Known negative samples (n=69) obtained from uninfected sheep bred and maintained in a tick-free environment and subsequently shown to be susceptible to A. phagocytophilum were used to establish the cut-off point between negative and positive samples and to establish the specificity of the test. Serum samples obtained from 92 animals 14-21 days after infection were used to establish the sensitivity of the test. Using a cut-off point of 20PP (mean+2 standard deviations of the PP of 69 control samples) the test was shown to have a sensitivity of 84.8% and a specificity of 95.7%. Lowering the cut-off point to 15PP increased the sensitivity to 94.6%, but reduced the specificity to 92.8%.

  19. Limits of diagnostic accuracy of anti-hepatitis C virus antibodies detection by ELISA and immunoblot assay. (United States)

    Suslov, Anatoly P; Kuzin, Stanislav N; Golosova, Tatiana V; Shalunova, Nina V; Malyshev, Nikolai A; Sadikova, Natalia V; Vavilova, Lubov M; Somova, Anna V; Musina, Elena E; Ivanova, Maria V; Kipor, Tatiana T; Timonin, Igor M; Kuzina, Lubov E; Godkov, Mihail A; Bajenov, Alexei I; Nesterenko, Vladimir G


    When human sera samples are tested for anti-hepatitis C virus (HCV) antibodies using different ELISA kits as well as immunoblot assay kits discrepant results often occur. As a result the diagnostics of HCV infection in such sera remains unclear. The purpose of this investigation is to define the limits of HCV serodiagnostics. Overall 7 different test kits of domestic and foreign manufacturers were used for the sampled sera testing. Preliminary comparative study, using seroconversion panels PHV905, PHV907, PHV908 was performed and reference kit was chosen (Murex anti-HCV version 4) as the most sensitive kit on the base of this study results. Overall 1640 sera samples have been screened using different anti-HCV ELISA kits and 667 of them gave discrepant results in at least two kits. These sera were then tested using three anti-HCV ELISA kits (first set of 377 samples) or four anti-HCV ELISA kits (second set of 290 samples) at the conditions of reference laboratory. In the first set 17.2% samples remained discrepant and in the second set - 13.4%. "Discrepant" sera were further tested in RIBA 3.0 and INNO-LIA immunoblot confirmatory assays, but approximately 5-7% of them remained undetermined after all the tests. For the samples with signal-to-cutoff ratio higher than 3.0 high rate of result consistency by reference, ELISA routing and INNO-LIA immunoblot assay was observed. On the other hand the results of tests 27 "problematic" sera in RIBA 3.0 and INNO-LIA were consistent only in 55.5% cases. Analysis of the antigen spectrum reactive with antibodies in "problematic" sera, demonstrated predominance of Core, NS3 and NS4 antigens for sera, positive in RIBA 3.0 and Core and NS3 antigens for sera, positive in INNO-LIA. To overcome the problem of undetermined sera, methods based on other principles, as well as alternative criteria of HCV infection diagnostics are discussed.

  20. Surface plasmon resonance-based competition assay to assess the sera reactivity of variants of humanized antibodies. (United States)

    Gonzales, Noreen R; Schuck, Peter; Schlom, Jeffrey; Kashmiri, Syed V S


    While clinical trials are the only way to evaluate the immunogenicity, in patients, of murine or genetically engineered humanized variants of a potentially therapeutic or diagnostic monoclonal antibody (MAb), ethical and logistical considerations of clinical trials do not permit the evaluation of variants of a given MAb that are generated to minimize its immunogenicity. The most promising variant could be identified by comparing the reactivities of the parental antibody (Ab) and its variants to the sera of patients containing anti-variable region (anti-VR) Abs to the administered parental Ab. We have developed a surface plasmon resonance (SPR) biosensor-based assay to monitor the binding of the sera anti-VR Abs to the parental Ab and the inhibition of this binding by the variants. SPR biosensors allow the real-time detection and monitoring of the binding between an immobilized protein and its soluble ligand without the need for prior purification and labeling of the mobile analyte. This new assay requires no radiolabeling, is relatively less time-consuming, and uses only small amounts of serum (5-20 microl of diluted serum) through a new microfluidic sample handling technique. To validate the assay, we have tested the relative reactivities of the CDR-grafted anti-carcinoma Ab, HuCC49, and its two variants, designated V5 and V10, to the sera of patients who were earlier administered radiolabeled murine CC49 in a clinical trial. A comparison of IC(50)s (the concentrations of the competitor Abs required for 50% inhibition of the binding of sera to immobilized HuCC49) showed that V5 and V10 were less reactive than HuCC49 to the three patients' sera tested. We have also demonstrated, for the first time, the specific detection and comparison of relative amounts of anti-VR Abs present in the sera of different patients without prior removal of anti-murine Fc Abs and/or circulating antigen. This may facilitate the rapid screening, for the presence of anti-VR Abs, of the

  1. In vivo neutralization assays by monoclonal antibodies against white spot syndrome virus in crayfish (Cambarus proclarkii)

    Institute of Scientific and Technical Information of China (English)

    WANG Yinan; ZHAN Wenbin; XING Jing; JIANG Yousheng


    The neutralizing activities of eight monoclonal antibodies (MAbs) against white spot syndrome virus (WSSV) (2D2,2B2,1D2,1DS,1C2,4A1,6A4 and 6B4) were analyzed by in vivo experiments.Gills from WSSV-infected shrimp were homogenized and ten-fold serially diluted by PBS,and then incubated with MAbs (hybridoma culture supernatant),respectively.The mixture of WSSV and MAbs were injected into crayfish (Procambarus clarkii).Mter challenge,the death rates of crayfish were counted to determine the neutralizing activities of MAbs.At the same time,the mixture of myeloma culture supernatant and WSSV or PBS was served as positive or negative control,respectively.The results showed that,at each virus dilution,the mean time to death of the crayfish injected with MAb-treated virus was significantly longer than that in the positive control,though they all showed 100% mortality within 25 d,and meanwhile,few crayfish died in the negative control.Among the eight MAbs,2D2,2B2,1D2 and 1DS,especially the former two,delayed the mortality significantly,and 1C2,4Al and 6A4 delayed the mortality as well but not so efficiently,while MAb 6B4 was efficient only when the virus concentration increased.The results indicated that the anti-WSSV MAbs can neutralize WSSV in different virus dilutions.

  2. Confirmation of positive antibody screens by solid-phase red cell adherence assay using a tube technique method with polyethylene glycol enhancement. (United States)

    Gammon, R R; Lake, M; Velasquez, N; Prichard, A


    Our blood bank routinely screens donors for antibodies using a solid-phase red cell adherence (SPRCA) assay. Positive results are then confirmed using a tube technique with polyethylene glycol (PEG) enhancement due to reported higher specificity than with SPRCA. Over a 5-month period, 49,084 donor serum or plasma samples were tested using the SPRCA assay. Further identification of positive samples was performed using a PEG enhancement method. Testing was performed with strict adherence to the manufacturers' inserts. Of 49,084 samples, 313 (0.64%) were positive by the SPRCA assay. Of these, 99 (31.6%) samples remained positive when tested with PEG enhancement. The remaining 214 (68.4%) were negative, giving specificity for the SPRCA assay of 99.6 percent (48,985/ 49,199). We report a high specificity for antibody screening using the SPRCA assay. However, it is cost effective to perform a confirmatory tube test with PEG enhancement because 214 SPRCA assay samples were interpreted as having a negative antibody screen, thus allowing the release of valuable blood components for transfusion.

  3. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay to quantify soluble beta-glucans in oats and barley. (United States)

    Rampitsch, Christof; Ames, Nancy; Storsley, Joanne; Marien, Lindsay


    A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.

  4. Comparison of the inhibition of urokinase-type plasminogen activator (u-PA) activity by monoclonal antibodies specific for u-PA as assessed by different assays

    NARCIS (Netherlands)

    Boheemen, P.A. van; Hoogen, N.M. van den; Koolwijk, N.


    Six murine monoclonal antibodies (MAbs) specific for urokinase-type plasminogen activator (u-PA) were tested for their ability to inhibit u-PA activity in three different assays with respect to amidolytic activity, plasminogen activation and fibrinolytic activity. Two of the MAbs were able to inhibi

  5. Development and validation of an indirect Enzyme-linked Immunosorbent Assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants

    NARCIS (Netherlands)

    Heijden, van der H.M.J.F.; Bouwstra, R.J.; Mars, M.H.; Poel, van der W.H.M.; Wellenberg, G.J.; Maanen, van C.


    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 sample

  6. Evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); N. Juntti; B. Klingeborn; J. Groen (Jan); F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)


    textabstractAn enzyme-linked immunosorbent assay (ELISA) system was developed for the detection of canine parvovirus (CPV) or CPV antigen in dog faeces and two other ELISA systems were developed for the detection of CPV-specific antibodies in dog sera. The ELISA's were based on the use of CPV-specif

  7. Clinical relevance of anti-HLA antibodies detected by flow-cytometry bead-based assays--single-center experience. (United States)

    Mihaylova, Anastassia; Baltadjieva, Daniela; Boneva, Petia; Ivanova, Milena; Penkova, Kalina; Marinova, Daniela; Mihailova, Snejina; Paskalev, Emil; Simeonov, Petar; Naumova, Elissaveta


    The purpose of this study was to define the incidence, dynamics, and profiles of anti-human leukocyte antigen antibodies (HLA-Abs) produced after kidney transplantation and their impact on graft outcome. A total of 72 first cadaver donor kidney recipients were prospectively monitored for the development of HLA-Abs using bead-based flow-cytometry assays (One Lambda FlowPRA tests). Sixteen recipients (22.2%) developed HLA-Abs after transplantation (class I, n = 7; class I+II, n = 6; class II, n = 3), in most cases (81.25%) within the first 2 weeks posttransplantation. A strong association between alloantibody presence and delayed graft function (Chi-square = 7.659, p < 0.01), acute rejection (Chi-square = 14.504, p < 0.001), chronic rejection (Chi-square = 12.84, p < 0.001), and graft loss (Chi-square = 20.283, p < 0.001) was found. Patients with higher alloantibody titers experienced acute rejections and even early graft loss, compared with those with lower titers for whom chronic rejections were more common. Immunologic complications occurred in recipients with both donor-specific and cross-reacting groups or non-donor-specific antibodies alone. A positive correlation (Pearson correlation, 0.245; p < 0.05) between HLA class I amino acid triplet incompatibility and alloantibody production was observed, mainly resulting from immunogenic triplotypes. Given the results obtained in this study, an alloantibody testing algorithm has been designed and implemented for routine monitoring and to define optimally the alloantibody reactivity in kidney transplant recipients.

  8. [Comparative study on the assay for IgE antibodies specific for Chamaecyparis obtusa pollen between AlaSTAT and CAP-RAST]. (United States)

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


    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.

  9. Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta

    DEFF Research Database (Denmark)

    Hermanrud, Christina; Ryner, Malin; Luft, Thomas


    Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach...... was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated...

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

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


    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.

  11. A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice. (United States)

    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


    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.

  12. 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. (United States)

    Shang, Yuqin; Mernaugh, Ray; Zeng, Xiangqun


    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.

  13. Evaluation of five different antigens in enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies. (United States)

    Maherchandani, Sunil; Patnayak, Devi P; Muñoz-Zanzi, Claudia A; Lauer, Dale; Goyal, Sagar M


    Five different antigens were evaluated in enzyme-linked immunosorbent assay (ELISA) tests for the detection of avian pneumovirus (APV) antibodies. Two of the 5 antigens were prepared from recent APV isolates from Minnesota. The 2 older isolates were passage 63 of a strain currently used as a live, attenuated vaccine and a Colorado strain isolated for the first time in the United States and currently used in an ELISA test. The fifth antigen is based on an APV recombinant N-protein. Basic parameters and positive-negative threshold of the assays were established for all 5 antigens on the basis of data obtained by testing 46 known negative and 46 known positive serum samples. Subsequently, 449 field samples were tested by all 5 ELISAs. The optical density difference (ODD) was calculated by subtracting optical density of the sample in the negative antigen well from that in the positive antigen well. In the current ELISA test based on the Colorado strain, an ODD of 0.2 is considered to be the cutoff value to classify samples as negative or positive. In this study, however, use of different cutoffs, based on ODD of negative control plus 3 SD or values estimated from Receiver operating characteristic analysis, was considered to be more appropriate for the various antigens used. Overall person-to-person and day-to-day variability was found to be large for all tests using either ODD or sample to positive ratio to report results. In addition, results suggest that antigenicity of the APV isolates in the United States has not changed between 1997 and 2000.

  14. Determination of anti-endomysium IgA antibodies in the diagnosis of celiac disease: Comparison of a novel ELISA-based assay with conventional immunofluorescence

    Institute of Scientific and Technical Information of China (English)

    Dennis CW Poland; Huib Ceelie; Rob B Dinkelaar; Cornelis Beijer


    AIM: To evaluate the novel anti-endomysium (anti-EMA)detection based on ELISA.METHODS: Anti-EMA IgA was measured by a novel ELISA in 196 patients with gastrointestinal symptoms and suspected mal-absorption. Data were compared with those obtained by the conventional IF test.RESULTS: A good concordance of 98% was found between these two assays. In sera of 161 patients (82%)both assays tested negative whereas in sera of 31 patients (16%) both assays tested positive for the presence of anti-EMA antibodies. Discrepancies between EMAELISA and EMA-immunofluorescence (IF) were found in only 4 patients (2%).CONCLUSION: This ELISA can replace IF for the detection of anti-EMA antibodies and provide clinicians with an excellent tool to screen for celiac disease in patients with gastrointestinal complaints.

  15. 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. (United States)

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


    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

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

    Directory of Open Access Journals (Sweden)

    Gortázar Christian


    Full Text Available Abstract 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 (

  17. The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

    Directory of Open Access Journals (Sweden)

    Claudio R Madruga


    Full Text Available There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

  18. Sexing murine embryos with an indirect immunofluorescence assay using phage antibody B9-Fab against SDM antigen. (United States)

    Wang, Naidong; Yuan, Anwen; Ma, Jun; Deng, Zhibang; Xue, Liqun


    The use of serologically detectable male (SDM; also called H-Y) antigens to identify male embryos may be limited by the source of anti-SDM antibody. In the present study, novel anti-SDM B9-Fab recombinant clones (obtained by chain shuffling of an A8 original clone) were used to detect SDM antigens on murine embryos. Murine morulae and blastocysts (n=138) were flushed from the oviducts of Kunming mice and incubated with anti-SDM B9-Fab for 30 min at 37°C. With an indirect immunofluorescence assay, the membrane and inner cell mass had bright green fluorescence (presumptive males). Overall, 43.5% (60/138) were classified as presumptive males and 56.5% (78/138) as presumptive females, with 85.0 and 88.5% of these, respectively, confirmed as correct predictions (based on PCR analysis of a male-specific [Sry] sequence). We concluded that the anti-SDM B9-Fab molecule had potential for non-invasive, technically simple immunological sexing of mammalian embryos.

  19. Evaluation of assay interference and interpretation of CXCR4 receptor occupancy results in a preclinical study with MEDI3185, a fully human antibody to CXCR4 (United States)

    Chavez, Carlos; Henderson, Simon; Vainshtein, Inna; Standifer, Nathan; DelNagro, Christopher; Mehrzai, Freshta; Schneider, Amy; Roskos, Lorin; Liang, Meina


    Background Receptor occupancy (RO) assays provide a means to measure the direct interaction of therapeutics with their cell surface targets. Free receptor assays quantify cell‐surface receptors not bound by a therapeutic while total receptor assays quantify the amount of target on the cell surface. Methods We developed both a flow cytometry‐based free RO assay to detect free surface CXCR4, and a total surface CXCR4 assay. In an effort to evaluate potential displacement interference, we performed in vitro experiments to compare on‐cell affinity with the IC50 values from in vitro and in vivo from the free CXCR4 assay. We determined free and total surface CXCR4 on circulating blood cells in cynomolgus monkeys dosed with MEDI3185, a fully human monoclonal antibody to CXCR4. Results We devised an approach to evaluate displacement interference during assay development and showed that our free assay demonstrated little to no displacement interference. After dosing cynomolgus monkeys with MEDI3185, we observed dose‐dependence in the magnitude and duration of receptor occupancy and found CXCR4 to increase on lymphocytes, monocytes, and granulocytes. In a multiple dose study, we observed time points where surface CXCR4 appeared fully occupied but MEDI3185 was not detectable in serum. These paradoxical results represented a type of assay interference, and by comparing pharmacokinetic, ADA and total CXCR4 results, the most likely reason for the free CXCR4 results was the emergence of neutralizing anti‐drug antibodies (ADA). The total CXCR4 assay was unaffected by ADA and provided a reliable marker of target modulation in both in vivo studies. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published byWiley Periodicals, Inc. PMID:26384735

  20. 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

    Directory of Open Access Journals (Sweden)

    Anju Mohan


    Full Text Available 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.

  1. 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 (United States)

    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


    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

  2. 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

    Directory of Open Access Journals (Sweden)

    Miriam Lizette Díaz-Toscano


    Full Text Available 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.

  3. Establishment of the Method of Immunohistochemistry Assay for the Detection of Scrapie in Chinese Short-Tailed Han Sheep by Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)


    The method of immunohistochemistry assay for the detection of scrapie in Chinese Short-tailed Han sheep was established using monoclonal antibody. Genomic DNA was isolated from Chinese Short-tailed Han sheep blood. Using the polymerase chain reaction technique, PrP27-30 gene sequence was amplified from Chinese Short-tailed Han sheep genomic DNA. By recombinant DNA technology, the recombinant protein of Chinese Short-tailed Han sheep PrP27-30 was obtained. Then, using standard methodology of myeloma cell fusion, a panel of monoclonal antibodies was generated. With mAbs, scrapie in Chinese Short-tailed Han sheep was detected by immunohistochemistry assay. The recombinant protein of Chinese Short-tailed Han sheep PrP27-30 was obtained and a panel of six hybridoma cell lines secreting specific antibodies to Chinese Short-tailed Han sheep PrP27-30 related to scrapie was obtained with one fusion between myeloma Sp2/0 and spleen cells from mice immunized with the purified recombinant protein. Four hybridoma cell lines can be used in immunohistochemistry assay for the detection of scrapie in Chinese Short-tailed Han sheep. So that the special monoclonal antibody developed in author's institute can be used to detect PrPsc of scrapie in Chinese Short-tailed Han sheep by immunohistochemistry in China.

  4. Murine monoclonal antibody to platelet factor 4/heparin complexes as a potential reference standard for platelet activation assays in heparin-induced thrombocytopenia. (United States)

    Asada, Reiko; Wanaka, Keiko; Walenga, Jeanine; Prechel, Margaret; Miyashita, Kumiko; Escalante, Vicki; Kaneko, Chieko; Hoshino, Nobuhiro; Oosawa, Mitsuru; Matsuo, Miyako


    Quality control of the platelet activation assays to diagnose heparin-induced thrombocytopenia (HIT), (14)C-serotonin release assay (SRA) and platelet aggregation test (PAT) has yet to be established due to lack of reference standards and the difficulty of obtaining significant amounts of HIT antibodies from patients with HIT. We prepared a murine monoclonal antibody to human platelet factor 4 (hPF4)/heparin complexes (HIT-MoAb) and investigated the platelet activating action of HIT-MoAb by using SRA and PAT. The HIT-MoAb activated human platelets at low heparin concentration and the platelet activations were inhibited at high heparin concentration in both SRA and PAT. The HIT-MoAb produced a concentration-dependent effect. Moreover, the platelet activation at low heparin concentration was inhibited by anti-FcγRIIa antibody. These results indicated that HIT-MoAb has characteristics similar to human HIT antibodies regarding heparin-dependent platelet activation. Therefore, it is suggested that HIT-MoAb has the potential to be a positive control or reference standard in platelet activation assays.

  5. A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry. (United States)

    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


    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.

  6. 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. (United States)

    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


    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.

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

    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


    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.

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

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


    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.

  9. 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. (United States)

    Lyng, J; Heron, I


    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.

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

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


    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.

  11. A novel bead-based assay to detect specific antibody responses against Toxoplasma gondii and Trichinella spiralis simultaneously in sera of experimentally infected swine

    Directory of Open Access Journals (Sweden)

    Bokken Gertie CAM


    Full Text Available Abstract Background A novel, bead-based flow cytometric assay was developed for simultaneous determination of antibody responses against Toxoplasma gondii and Trichinella spiralis in pig serum. This high throughput screening assay could be an alternative for well known indirect tests like ELISA. One of the advantages of a bead-based assay over ELISA is the possibility to determine multiple specific antibody responses per single sample run facilitated by a series of antigens coupled to identifiable bead-levels. Furthermore, inclusion of a non-coupled bead-level in the same run facilitates the determination of, and correction for non-specific binding. The performance of this bead-based assay was compared to one T. spiralis and three T. gondii ELISAs. For this purpose, sera from T. gondii and T. spiralis experimentally infected pigs were used. With the experimental infection status as gold standard, the area under the curve, Youden Index, sensitivity and specificity were determined through receiver operator curve analysis. Marginal homogeneity and inter-rater agreement between bead-based assay and ELISAs were evaluated using McNemar's Test and Cohen's kappa, respectively. Results Results indicated that the areas under the curve of the bead-based assay were 0.911 and 0.885 for T. gondii and T. spiralis, respectively, while that of the T. gondii ELISAs ranged between 0.837 and 0.930 and the T. spiralis ELISA was 0.879. Bead-based T. gondii assay had a sensitivity of 86% and specificity of 96%, while the ELISAs ranged between 64-84% and 93-99%, respectively. The bead-based T. spiralis assay had a sensitivity of 68% and specificity of 100% while the ELISA scored 72% and 95%, respectively. Marginal homogeneity was found between the T. gondii bead-based test and one of the T. gondii ELISAs. Moreover, in this test combination and between T. spiralis bead-based assay and respective ELISA, an excellent inter-rater agreement was found. When results of

  12. Rheumatoid arthritis and its association with HLA-DR antigens. II. Antibodies to native connective tissue antigens detected by enzyme linked immunosorbent assay. (United States)

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


    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).

  13. Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

    Directory of Open Access Journals (Sweden)

    Virgínia MG Silva


    Full Text Available Indirect enzyme-linked immunosorbent assays (ELISAs based on recombinant major surface protein 5 (rMSP5 and initial body (IB antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2% and specificities (100% for rMSP5 and 93.8% for IB ELISA which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA to 15% (IB ELISA of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

  14. Particle counting assay for anti-toxoplasma IgG antibodies. Comparison with four automated commercial enzyme-linked immunoassays. (United States)

    Galanti, L M; Dell'Omo, J; Wanet, B; Guarin, J L; Jamart, J; Garrino, M G; Masson, P L; Cambiaso, C L


    An assay for anti-toxoplasma IgG antibodies based on agglutination of latex particles was set up and compared with commercial immunoassays. The reaction was measured by instrumental counting of particles remaining unagglutinated. The running time was 45 min. This test (PaC) was compared using 243 serum samples with four automated commercial immunoassays: the Enzymum test Toxo IgG (ES300, Boehringer), the Vidas Toxo IgG (Biomérieux), the IMX Toxo IgG (Abbott), the Magia Toxoplasma gondii IgG (Merck). The mean values (+/- SD) obtained by IMX (25 IU +/- 68) and ES300 (45 IU +/- 142) were significantly lower than the values obtained by Vidas (73 IU +/- 237, p Magia (80 IU +/- 300, p < 10(-4) and p = 0.0005) and by PaC (70 IU +/- 260, p < 10(-4) and p = 0.0126). The correlations between PaC and Toxo IgG Boehringer, Biomérieux, Abbott, Merck were r = 0.97, r = 0.98, r = 0.94, r = 0.98, respectively. The correlation coefficients between the enzyme-immunoassays ranged from 0.96 to 0.99. All positive samples by PaC were found to be positive by enzyme-immunoassays except for eight sera which were doubtful positives by the Enzymum test ToxoIgG from Boehringer. No negative sample by PaC was found positive by any of the enzyme-immunoassays. In PaC, when two latex preparations coated with different antigen were compared, the correlation was rather weak (r = 0.93) suggesting that the selection of the antigen can be critical. In conclusion, the four automated commercial immunoassays now available gave similar results. However, the discrepancies observed in this study underlined the importance of clinical and biological follow-up of the patients and the necessity to confirm the result. The introduction of a new technique such as PaC, which is now available for a large variety of assays in Clinical Chemistry and Microbiology, is justified by its intrinsic advantage of homogeneity. Therefore, automation is easy as well as the control of possible interference.

  15. Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori. (United States)

    Xuan, Shi-Hai; Zhou, Yu-Gui; Shao, Bo; Cui, Ya-Lin; Li, Jian; Yin, Hong-Bo; Song, Xiao-Ping; Cong, Hui; Jing, Feng-Xiang; Jin, Qing-Hui; Wang, Hui-Min; Zhou, Jie


    Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

  16. Antibodies to immunoglobulin-G in dog sera, synovial fluids and aqueous humor : a comparative study of rheumatoid factor assays, suitable for routine application


    Bernadina, W.E.; Kol, P.J. van; Willemse, A.


    The incidence of anti-IgG antibodies (rheumatoid factors, RF) in body fluids (sera, synovial fluids and aqueous humor) selected from 62 normal and 275 diseased dogs was studied. Fluids were assayed by canine versions of standard agglutinating and/or precipitating RF assays with routine application in human practice. The number of RF detected by dog IgG-coated particles was substantially higher by latex fixation test (LFT) than by modified Rose-Waaler (RW) test (61/144 vs. 14/144). This did no...

  17. Platelet antibody screening by flow cytometry is more sensitive than solid phase red cell adherence assay and lymphocytotoxicity technique: a comparative study in Thai patients. (United States)

    Buakaew, Jarin; Promwong, Charuporn


    The objective of this study was to compare the sensitivity and specificity of lymphocytotoxicity test (LCT), solid phase red cell adherence assay (SPRCA) and flow cytometry in detecting platelet reactive antibodies against human leukocyte antigens (HLA) class I and human platelet antigens (HPA). Sera from 38 thrombocytopenic patients and 5 mothers of thrombocytopenic newborns were screened for platelet reactive antibodies by these three methods using screening platelets and/or lymphocytes panels derived from six subjects. The sensitivity and specificity of each method and levels of agreement were analysed. HLA antibodies were found in 18, 17 and 19 out of 43 patients' sera tested by LCT, SPRCA and flow cytometry, respectively. Four out of 43 patients' sera were reactive against HPA by flow cytometry, but were reactive to only 2 sera by SPRCA. Using flow cytometry as the reference method, the sensitivities/specificities of SPRCA and LCT in HLA antibody detection were 84.21/95.83% and 94.73/100%, respectively, with a good strength of agreement. SPRCA had 50% sensitivity and 100% specificity in HPA antibody detection compare to flow cytometry. Flow cytometry appeared to be the most sensitive technique compared with SPRCA and LCT for both HPA and HLA antibody screening. SPRCA sensitivity was too low for HPA antibody detection, but this might be because of the small number of samples. There was one serum from the mother of a baby suffering neonatal alloimmune thrombocytopenia (NAIT), in whom SPRCA could not detect HPA antibodies, while flow cytometry came out positive. Therefore, SPRCA should not be used in NAIT investigation and flow cytometry should be employed instead.

  18. An Improved Ultrasensitive Enzyme-Linked Immunosorbent Assay Using Hydrangea-Like Antibody-Enzyme-Inorganic Three-in-One Nanocomposites. (United States)

    Wei, Tianxiang; Du, Dan; Zhu, Mei-Jun; Lin, Yuehe; Dai, Zhihui


    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.

  19. Laboratory Evaluation of a Point-of-Care Downward-Flow Assay for Simultaneous Detection of Antibodies to Treponema pallidum and Human Immunodeficiency Virus


    Herbst de Cortina, S.; Bristow, C. C.; Vargas, S. K.; Perez, D. G.; Konda, K. A.; Caceres, C. F.; Klausner, J. D.


    Combining the detection of syphilis and HIV antibodies into one point-of-care test integrates syphilis screening into already existing HIV screening programs, which may be particularly beneficial in settings such as antenatal care. Using the INSTI Multiplex downward-flow immunoassay, we tested 200 stored serum samples from high-risk patients enrolled in a longitudinal study on HIV infection and syphilis in Peruvian men who have sex with men and transgender women. This rapid assay detected HIV...

  20. Multiplex assay (Mikrogen recomBead) for detection of serum IgG and IgM antibodies to 13 recombinant antigens of Borrelia burgdorferi sensu lato in patients with neuroborreliosis

    DEFF Research Database (Denmark)

    Dessau, Ram Benny; Møller, Jens K.; Kolmos, Birte


    A multiplex-bead-based assay for the detection of serum antibodies to Borrelia burgdorferi sensu lato was evaluated. The assay contained 13 different antigens in both the IgG and the IgM assay; thus, a total of 26 measurement results were available from each sample. A total of 49 Danish patients...

  1. A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding.

    Directory of Open Access Journals (Sweden)

    Hadassah Sade

    Full Text Available We have adapted an in vitro model of the human blood-brain barrier, the immortalized human cerebral microvascular endothelial cells (hCMEC/D3, to quantitatively measure protein transcytosis. After validating the receptor-mediated transport using transferrin, the system was used to measure transcytosis rates of antibodies directed against potential brain shuttle receptors. While an antibody to the insulin-like growth factor 1 receptor (IGF1R was exclusively recycled to the apical compartment, the fate of antibodies to the transferrin receptor (TfR was determined by their relative affinities at extracellular and endosomal pH. An antibody with reduced affinity at pH5.5 showed significant transcytosis, while pH-independent antibodies of comparable affinities at pH 7.4 remained associated with intracellular vesicular compartments and were finally targeted for degradation.

  2. Optimization of fixed-permeabilized cell monolayers for high throughput micro-neutralizing antibody assays: application to the zebrafish/viral hemorrhagic septicemia virus (vhsv) model. (United States)

    Chinchilla, Blanca; Encinas, Paloma; Estepa, Amparo; Coll, Julio; Gomez-Casado, Eduardo


    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.

  3. Studies to assess the biological relevance of anti-Tamm-Horsfall protein antibodies detected by direct-binding enzyme-linked immunosorbent assay. (United States)

    Hunt, J S; Groufsky, A; Lynn, K L


    1. A role has been suggested for anti-Tamm-Horsfall protein (THP) antibodies in renal disease based on the results of immunoassays of pathological sera. The putative autoantibodies have not been isolated from such sera nor have definitive inhibition studies of their binding been carried out. We have carried out such studies using rabbit anti-THP antibodies as control reagents. 2. Urinary THP prepared by salt precipitation was used to prepare four immunoabsorbent columns by covalent coupling to CNBr-activated Sepharose 4B. After washing with a variety of dissociating agents to remove any non-covalently bound subunit THP, each column was incubated with normal and immune rabbit serum. Fractions washed and eluted from columns were tested for anti-THP antibodies by enzyme-linked immunosorbent assay (ELISA) and THP antigen by radioimmunoassay, and showed NH4SCN (3 mol/l) and guanidine hydrochloride (GuHCl) (6 mol/l) equivalent and sodium dodecyl sulphate (20 g/l) to be inferior in their capacity to produce immunoabsorbent THP capable of isolating specific antibodies from immune rabbit serum. 3. The column treated with GuHCl (6 mol/l) was used further in attempts to isolate putative anti-THP antibodies from five patients, who had a history of urinary tract infections and whose sera showed strong binding by ELISA. 4. Results from direct and inhibition ELISA experiments on fractions collected after washing and elution with all sera suggested that the putative human anti-THP antibodies were of very low affinity and/or directed against non-subunit THP. 5. The pathological relevance of human anti-THP antibodies measured by ELISA remains to be established.

  4. Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.; Marks, James D.; Varnum, Susan M.


    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.

  5. Wide dynamic range of surface-plasmon-resonance-based assay for Hepatitis-B-surface-antigen-antibody optimal detection in comparison with ELISA. (United States)

    Tam, Yew Joon; Zeenathul, Nazariah Allaudin; Rezaei, Morvarid Akhavan; Mustafa, Nor Hidayah; Azmi, Mohd Lila Mohd; Bahaman, Abdul Rani; Lo, Sewn Cen; Tan, Joo Shun; Hani, Homayoun; Rasedee, Abdullah


    Limit of detection (LOD), limit of quantification (LOQ) and the dynamic range of detection of hepatitis B surface antigen antibody (anti-HBs) using surface plasmon resonance (SPR) chip-based approach with Pichia pastoris derived recombinant hepatitis B surface antigen (HBsAg) as recognition element were established through the scouting for optimal conditions for the improvement of immobilization efficiency and in the use of optimal regeneration buffer. Recombinant HBsAg was immobilized onto the sensor surface of CM5 chip at a concentration of 150 mg/L, in sodium acetate buffer at pH 4 with added 0.6% Triton X-100. Regeneration solution of 20 mM HCl was optimally found to effectively unbind analytes from the ligand, thus allowing for multiple screening cycles. A dynamic range of detection of ∼0.00098 to 0.25 mg/L was obtained and a 7-fold higher LOD, as well as a 2-fold increase in coefficient of variance (CV) of the replicated results, were shown as compared to enzyme-linked immunosorbent assay (ELISA). Evaluation of the assay for specificity showed no cross-reactivity with other antibodies tested. The ability of SPR chip-based assay and ELISA to detect anti-HBs in human serum was comparable, indicating that the SPR chip-based assay with its multiple screening capacity has greater advantage over ELISA. This article is protected by copyright. All rights reserved.

  6. Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay. (United States)

    Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K


    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 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.

  7. Serum antibodies from a subset of horses positive for Babesia caballi by competitive enzyme-linked immunosorbent assay demonstrate a protein recognition pattern that is not consistent with infection. (United States)

    Awinda, Peter O; Mealey, Robert H; Williams, Laura B A; Conrad, Patricia A; Packham, Andrea E; Reif, Kathryn E; Grause, Juanita F; Pelzel-McCluskey, Angela M; Chung, Chungwon; Bastos, Reginaldo G; Kappmeyer, Lowell S; Howe, Daniel K; Ness, SallyAnne L; Knowles, Donald P; Ueti, Massaro W


    Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence of Theileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi-seropositive horses using rhoptry-associated protein 1 (RAP-1)-competitive enzyme-linked immunosorbent assay (cELISA), despite B. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1-cELISA as a single serological test to determine the infection status of B. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted with B. caballi lysate and purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions where B. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected with B. caballi and from areas where B. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence of B. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis of B. caballi.

  8. Clinical implications of a new TSH-receptor-antibody-assay (DYNOtest {sup trademark} TRAKhuman) in autoimmune thyroid diseases; Klinische Implikationen eines neuen TSH-Rezeptor-Antikoerper-Assays (DYNOtest {sup trademark} TRAKhuman) bei autoimmunen Schilddruesenerkrankungen

    Energy Technology Data Exchange (ETDEWEB)

    Meller, J.; Schreivogel, I.; Becker, W. [Goettingen Univ. (Germany). Abt. fuer Nuklearmedizin; Bergmann, A.; Morgenthaler, N. [B.R.A.H.M.S Diagnostica, Berlin (Germany); Huefner, M. [Goettingen Univ. (Germany). Abt. Innere Medizin


    Aim: Conventional radioreceptor-antibody-assays (RAAs) fail in the detection of TSH-receptor antibodies (TRAKs) in 10-30% of patients with Graves' disease (GD). The aim of this study was the evaluation of the diagnostic and clinical impact of a new RRA (DYNOtest {sup trademark} TRAKhuman) which uses the human recombinant TSH-Receptor in the diagnosis of autoimmune thyroid disease. Methods: Sera from 142 consecutive patients (GD: n=50, autoimmune thyroiditis/AIT: n=92) and from 55 controls (31 patients without any thyroid disease and 14 with euthyroid goiter) were evaluated both with the DYNOtest {sup trademark} TRAKhuman-assay and a conventional RRA (TRAK-Assay {sup trademark}). Thyroid in vitro parameters and thyroid sonography were performed in all patients. Results: The DYNOtest {sup trademark} TRAK-assay was significantly superior to the conventional RRA in the diagnosis of GD (p<0,00012), especially in those who were treated by thionamides (p<0,003) and in the diagnosis of TRAK-positive patients with AIT (p<0,003). The majority of TRAK-positive AIT-patients suffered from hypothyroidism. One false positive result in patients with euthyroid goiter was found in the TRAK-Assay {sup trademark} as well as in the DYNOtest {sup trademark} TRAKhuman-Assay. Therefore the specifity of the DYNOtest {sup trademark} TRAKhuman was not inferior compared with the conventional assay. Conclusion: The DYNOtest {sup trademark} TRAK-assay is superior in the diagnostic work up of Graves' disease compared with a conventional TRAK-assay and offers an equal specifity. (orig.) [German] Ziel: Bei konventionellen Radiorezeptor-Antikoerper-Assays (RRAs) misslingt der Nachweis von TSH-Rezeptor Antikoerpern (TRAKS) bei 10-30% der immunogenen Hyperthyreosen (IH). Ziel der Studie war es, den diagnostischen und klinischen Stellenwertes eines neuen RRA (DYNOtest {sup trademark} TRAKhuman) bei autoimmunen Schilddruesenerkrankungen zu evaluieren. Methoden: Serumproben von 142

  9. Evaluation of fully automated assays for the detection of Rubella IgM and IgG antibodies by the Elecsys(®) immunoassay system. (United States)

    van Helden, Josef; Grangeot-Keros, Liliane; Vauloup-Fellous, Christelle; Vleminckx, Renaud; Masset, Frédéric; Revello, Maria-Grazia


    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 (prubella 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.

  10. Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

    Directory of Open Access Journals (Sweden)

    Yanil R Parma


    Full Text Available 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-Stx2 B 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 400 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 2 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.

  11. Human anti-animal antibody and the interference in immunological assays%人抗动物抗体及其对免疫分析的干扰

    Institute of Scientific and Technical Information of China (English)



    人抗动物抗体可引起临床不良反应,干扰动物源性抗体制剂治疗或成像,干扰免疫分析而引起假性结果。临床上可通过应用免疫抑制剂治疗和人源性抗体、聚乙二醇处理的抗体或抗体Fab段制剂等方法防止人抗动物抗体应答形成;样品预处理或分析技术的改进可消除人抗动物抗体对免疫分析的干扰。目前已设计出了多种分析技术检测人抗动物抗体,并形成了检测试剂盒,甚至床边检验试剂。%Human anti-animal antibody response may result in some adversereaction interfere with antibodies from animal therapy or imaging,and cause both false-positive and false-negative test results in immunoassays Strategies to prevent the development of human anti-animal antibody responses include immunosuppressant therapy and the use of humanized,polyethylene glycolytated antibodies,or Fab fragment of antibody agents.Sample pretreatments or assay redesign can eliminate immunoassay interference caused by human anti-animal antibodies.Currently,several assays have been designed to detect human anti-animal antibodies.There are the assays available in kit form,even a simple point-of care test.

  12. Monoclonal antibody-based dipstick assay: a reliable field applicable technique for diagnosis of Schistosoma mansoni infection using human serum and urine samples. (United States)

    Demerdash, Zeinab; Mohamed, Salwa; Hendawy, Mohamed; Rabia, Ibrahim; Attia, Mohy; Shaker, Zeinab; Diab, Tarek M


    A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.

  13. Detection of antibodies to hepatitis B core antigen using the Abbott ARCHITECT anti-HBc assay: analysis of borderline reactive sera. (United States)

    Ollier, Laurence; Laffont, Catherine; Kechkekian, Aurore; Doglio, Alain; Giordanengo, Valérie


    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.

  14. Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

    DEFF Research Database (Denmark)

    Foddai, Alessandro; Enøe, Claes; Stockmarr, Anders


    successfully been used to eradicate BVD in Sweden. Data (2003–2010) on changes in median herd size and milk production levels, occurrence of viremic animals and bulk milk surveillance were analysed. Additionally, the Danish blocking ELISA and the SVANOVIR ELISA were compared analyzing milk and serum samples....... The prevalence of antibody positive milking cows that could be detected by each test was estimated, by diluting positive individual milk samples and making artificial milk pools. Results: During the study period, the median herd size increased from 74 (2003) to 127 cows (2010), while the prevalence of BVDV...... infected herds decreased from 0.51 to 0.02 %. The daily milk yield contribution of a single seropositive cow to the entire daily bulk milk was reduced from 1.61 % in 2003 to 0.95 % in 2010 due to the increased herd size. It was observed that antibody levels in bulk milk decreased at national level...

  15. Development of an enzyme-linked immunosorbent assay for the detection of isomiroestrol, an identical marker, in White Kwao Krua using a monoclonal antibody. (United States)

    Kitisripanya, Tharita; Jutathis, Kamonthip; Inyai, Chadathorn; Komaikul, Jukrapun; Udomsin, Orapin; Tanaka, Hiroyuki; Putalun, Waraporn


    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.

  16. Highly sensitive enzyme-free immunosorbent assay for porcine circovirus type 2 antibody using Au-Pt/SiO2 nanocomposites as labels. (United States)

    Wu, Long; Yin, Wenmin; Tang, Kun; Shao, Kang; Li, Qin; Wang, Pan; Zuo, Yunpeng; Lei, Xiaomin; Lu, Zhicheng; Han, Heyou


    Improving the performance of conventional enzyme-linked immunosorbent assay (ELISA) is of great importance to meet the demand of early clinical diagnosis of various diseases. Herein, we report a feasible enzyme-free immunosorbent assay (EFISA) system using antibody conjugated Au-Pt/SiO2 nanocomposites (APS NCs) as labels. In this system, Au-Pt/SiO2 nanospheres (APS NPs) were first synthesized by wet chemical method and exhibited intrinsic peroxidase and catalase-like activity with excellent water-solubility. Then APS NCs were utilized as labels to replace HRP conjugated antibody, and Fe3O4 magnetic beads (MBs) to entrap the analyte. To discuss the performance of EFISA system, Human IgG was served as a model analyte, and porcine circovirus type 2 (PCV2) serums as real samples. The system boosted the detection limit of HIgG to 75pgmL(-1) with a RSD below 5%, a 264-fold improvement as compared with conventional ELISA. This is the first time that APS NCs have been used and successfully optimized for the sensitive dilution detection of PCV2 antibody (5:10(7)) in ELISA. Besides, APS NCs have advantages related to low cost, easy preparation, good stability and tunable catalytic activity, which make them a potent enzyme mimetic candidate and may find potential applications in bioassays and clinical diagnostics.

  17. Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographic strip. (United States)

    Liu, Biing-Hui; Hung, Chun-Tse; Lu, Chuan-Chen; Chou, Hong-Non; Yu, Feng-Yih


    Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and causes diarrhetic shellfish poisoning (DSP). This study generated a monoclonal antibody (mAb) specific to OA from a hybridoma cell line, 6B1A3, which was obtained by fusion of myeloma cells (P3/NS1/1-AG4-1) with spleen cells isolated from a BALB/c mouse immunized with OA-γ-globulin. The 6B1A3 mAb belongs to the immunoglobulin G1 (κ chain) isotype. Both competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) were established for characterization of the antibody. The concentrations causing 50% inhibition of binding of OA-horseradish peroxidase to the antibody by OA were calculated to be 0.077 ng/mL in the cdELISA. A rapid and sensitive mAb-based gold nanoparticle immunochromatographic strip was also established. This proposed strip has a detection limit of 5 ng/mL for OA and can be finished in 10 min. Extensive analyses of 20 seafood samples with ELISA revealed that 10 were slightly contaminated with OA, with a mean concentration of 0.892 ng/g. Analysis of OA in shellfish samples showed that data acquired by the immunochromatographic strip agreed well with those acquired by the ELISA. The mAb-based ELISA and immunochromatographic strip assay developed in this study have adequate sensitivity and accuracy for rapid screening of OA in shellfish samples.

  18. The effect on kaolin adsorption of serum on the virus neutralization and enzyme-linked immunosorbent assays of antibody to bovine herpesvirus 1. (United States)

    Darcel, C L; Kozub, G C


    Two procedures have been used for measuring antibody titres to bovine herpes virus 1 (BHV1): the serum neutralization (SN) test and enzyme-linked immunosorbent assay (ELISA). One hundred and thirty-two sera selected for their low SN titres were tested both unadsorbed and after adsorption with kaolin to determine the effect of kaolin on the titres. With ELISA, the titres of unadsorbed and kaolin adsorbed were not significantly different but with the SN test many treated sera, originally with weak positive titres, became negative after kaolin adsorption. Thus, if the ELISA results are specific for BHV1 antibody then the SN test findings suggest that treatment of sera with kaolin, rather than removing a viral inhibitor, removes a substance from the serum which potentiates SN antibody. This in turn indicates that low SN titres (reciprocal of titre less than or equal to 4, for instance) are probably specific for BHV1 SN antibody whether or not they are abolished by kaolin treatment of the serum.

  19. Development of an antigen-capture enzyme-linked immunosorbent assay using monoclonal antibodies for detecting H6 avian influenza viruses. (United States)

    Chen, Yi-Tung; Tsao, Zak; Chang, Shu-Ting; Juang, Ron-Huay; Wang, Lih-Chiann; Chang, Chung-Ming; Wang, Ching-Ho


    The H6 subtype of avian influenza virus (AIV) infection occurs frequently in wild and domestic birds. AIV antigen detection is preferred for controlling AIV as birds are infected before they produce antibodies. The purpose of this study was to develop an early diagnostic method for AIV detection. Six monoclonal antibodies (mAbs) developed from a field H6N1 AIV strain were tested for their ability to bind to viruses. The two that showed the greatest binding ability to AIVs were used for antigen detection. An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect H6 AIVs was developed using these mAbs. One mAb was coated onto an ELISA plate as the capture antibody. The other mAb was used as the detector antibody after labeling with horseradish peroxidase. The antigen-capture ELISA detected H6N1 AIVs but not H5 AIVs, human H1N1, H3N2 influenza or other viruses. This antigen-capture ELISA could be used to specifically detect H6N1 AIV.

  20. Specific Capture of Peptide-Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide-Binding Assay in Live Cells. (United States)

    Dirscherl, Cindy; Palankar, Raghavendra; Delcea, Mihaela; Kolesnikova, Tatiana A; Springer, Sebastian


    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.

  1. HIV中和抗体评价方法研究进展%Research progress evaluation assay in HIV neutralizing antibody

    Institute of Scientific and Technical Information of China (English)

    聂建辉; 王佑春


    诱导广谱有效的中和抗体被认为是预防性疫苗成功的关键,HIV中和抗体检测方法的建立和标准化为HIV疫苗评价和设计提供指导,也为不同HIV疫苗的比较提供了平台.本文从方法的建立、假病毒株的选择、评价标准的制定、评价策略的确定、该方法在HⅣ其它研究领域的应用等方面,对目前应用最广泛的以假病毒为基础的中和抗体评价方法进行了综述.%It is well -known that induction of effective broad -spectrum neutralizing antibodies is the key for successful prophylactic vaccines. Establishment and standardization of the HIV neutralizing antibody assays will assist the vaccine design and provide a platform to compare the vaccines from different laboratories. In this paper,we reviewed the most acceptable pseudovirus -based neutralization assay in following aspects,establishment of the assay,principles for pseudovirus selection,determination of the evaluation criteria and strategy,and applications in other fields.

  2. Development and application of a double-antigen sandwich enzyme-linked immunosorbent assay for detection of antibodies to porcine circovirus 2. (United States)

    Ge, Meng; Luo, Wei; Jiang, Daliang; Li, Runcheng; Zhao, Wenwei; Chen, Guoliang; Yang, Xingdong; Yu, Xinglong


    A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases.

  3. Application of the Ceditest FMDV type O and FMDV-NS enzyme-linked immunosorbent assays for detection of antibodies against Foot-and-mouth disease virus in selected livestock and wildlife species in Uganda

    DEFF Research Database (Denmark)

    Ayebazibwe, Chrisostom; Mwiine, Frank Norbert; Balinda, Sheila Nina


    Diagnosis and control of Foot-and-mouth disease virus (FMDV) requires rapid and sensitive diagnostic tests. Two antibody enzyme-linked immunosorbent assay (ELISA) kits, Ceditest FMDV-NS for the detection of antibodies against the nonstructural proteins of all FMDV serotypes and Ceditest FMDV type...

  4. An undetectable source of technical error that could lead to false negative results in enzyme linked immunosorbent assay of antibodies to HIV-1. (United States)

    Wiltbank, T B; McCarroll, D R; Wartick, M G


    Since the institution of routine testing for antibodies to Human Immunodeficiency Virus (HIV) using the enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of this assay system has received significant scrutiny. During previous use of this methodology, we have quantified rates of false biological positive results using commercial kit assays in a normal donor population. In this study, we have identified a potential source for false negative results. Using multiple lots of two different commercial ELISA kits, the absorbance readings at the test end point could not differentiate between normal non-reactive donor samples and blanks containing no sample. These results occur using normal donor samples, even though the assays could distinguish between blank wells and the manufacturers' "normal controls", provided with the assay. Our findings suggest that a technical pipetting error is presently undetectable, either visually or by statistical methods, and could permit an untested, potentially HIV-1 positive, unit to be released into the transfusable blood supply. A possible solution is suggested.

  5. A novel rabbit immunospot array assay on a chip allows for the rapid generation of rabbit monoclonal antibodies with high affinity.

    Directory of Open Access Journals (Sweden)

    Tatsuhiko Ozawa

    Full Text Available Antigen-specific rabbit monoclonal antibodies (RaMoAbs are useful due to their high specificity and high affinity, and the establishment of a comprehensive and rapid RaMoAb generation system has been highly anticipated. Here, we present a novel system using immunospot array assay on a chip (ISAAC technology in which we detect and retrieve antigen-specific antibody-secreting cells from the peripheral blood lymphocytes of antigen-immunized rabbits and produce antigen-specific RaMoAbs with 10(-12 M affinity within a time period of only 7 days. We have used this system to efficiently generate RaMoAbs that are specific to a phosphorylated signal-transducing molecule. Our system provides a new method for the comprehensive and rapid production of RaMoAbs, which may contribute to laboratory research and clinical applications.

  6. Performance of an automated solid-phase red cell adherence system compared with that of a manual gel microcolumn assay for the identification of antibodies eluted from red blood cells. (United States)

    Finck, R H; Davis, R J; Teng, S; Goldfinger, D; Ziman, A F; Lu, Q; Yuan, S


    IgG antibodies coating red blood cells (RBCs) can be removed by elution procedures and their specificity determined by antibody identification studies. Although such testing is traditionally performed using the tube agglutination assay, prior studies have shown that the gel microcolumn (GMC) assay may also be used with comparable results. The purpose of this study was to compare an automated solid-phase red cell adherence (SPRCA) system with a GMC assay for the detection of antibodies eluted from RBCs. Acid eluates from 51 peripheral blood (PB) and 7 cord blood (CB) samples were evaluated by both an automated SPRCA instrument and a manual GMC assay. The concordance rate between the two systems for peripheral RBC samples was 88.2 percent (45 of 51), including cases with alloantibodies (n = 8), warm autoantibodies (n = 12), antibodies with no identifiable specificity (n = 2), and negative results (n = 23). There were six discordant cases, of which four had alloantibodies (including anti-Jka, -E, and -e) demonstrable by the SPRCA system only. In the remaining 2 cases, anti-Fya and antibodies with no identifiable specificity were demonstrable by the GMC assay only. All seven CB specimens produced concordant results, showing anti-A (n = 3), -B (n = 1), maternal anti-Jka (n = 2), or a negative result (n = 1). Automated SPRCA technology has a performance that is comparable with that of a manual GMC assay for identifying antibodies eluted from PB and CB RBCs.

  7. Varicella Zoster Virus Myelitis in Two Elderly Patients: Diagnostic Value of Nested Polymerase Chain Reaction Assay and Antibody Index for Cerebrospinal Fluid Specimens

    Directory of Open Access Journals (Sweden)

    Teruyuki Takahashi


    Full Text Available Background: Myelitis is one of the rarest neurological complications of the varicella zoster virus (VZV infection. Focal muscle weakness with or without sensory disturbance occurs in approximately 5% of the cases after acute VZV infection, with complete recovery in 50-70%. Case Presentation: This report describes two rare cases of elderly patients with VZV myelitis secondary to dermatomal zoster rash. Patient 1 was a 79-year-old woman who developed paraplegia, numbness and decreased sensation in the left arm and below thoracic (Th-10 after sacral zoster. Spinal cord MRI showed a high-signal-intensity lesion at the cervical spinal nerve 2 on a T2-weighted image. Patient 2 was a 73-year-old man who developed right flaccid leg weakness and urinary retention after right dorsal Th 5-8 zoster. Spinal cord MRI showed a high-signal-intensity lesion at Th 3-4 on a T2-weighted image. In both cases, although the conventional single polymerase chain reaction (PCR assays all showed negative results, the original nested PCR assay detected VZV DNA in the cerebrospinal fluid (CSF specimen collected on admission. In addition, the anti-VZV IgG antibody by enzyme immunoassay and antibody index were elevated in the CSF specimens during the clinical courses of both patients. On the basis of these findings, both patients were diagnosed with VZV myelitis and were treated with high-dose acyclovir and corticosteroid. This combined treatment was appropriate and effective for the improvement of their functional outcomes. Conclusion: The detection of VZV DNA in CSF by nested PCR assay and the evaluation of the antibody index to VZV had significant diagnostic value.

  8. Downfall of the current antibody correlates of influenza vaccine response in yearly vaccinated subjects: Toward qualitative rather than quantitative assays. (United States)

    Cagigi, Alberto; Cotugno, Nicola; Rinaldi, Stefano; Santilli, Veronica; Rossi, Paolo; Palma, Paolo


    Response to seasonal influenza vaccination is currently evaluated by antibody correlates that estimate vaccine seroconversion as well as immune protection. These correlates rely on the general dogmas surrounding seasonal influenza vaccination; that is, that vaccine-induced antibodies would exclusively generate immunity to influenza vaccine strains and that protective immunity would wane before the next season. Here, we summarize recently reported data on immunity to seasonal influenza in healthy individuals and rediscuss results on yearly vaccinated pediatric immunocompromised patients that together highlight the need for revision of the current correlates of vaccine response to shift from quantitative to qualitative measurements.

  9. 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. (United States)

    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


    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.

  10. Development of an Antigen-DNAzyme Based Probe for a Direct Antibody-Antigen Assay Using the Intrinsic DNAzyme Activity of a Daunomycin Aptamer

    Directory of Open Access Journals (Sweden)

    Noorsharmimi Omar


    Full Text Available G-Quadruplex (G-4 structures are formed when G-rich DNA sequences fold into intra- or intermolecular four-stranded structures in the presence of metal ions. G-4-hemin complexes are often effective peroxidase-mimicking DNAzymes that are applied in many detection systems. This work reports the application of a G-rich daunomycin-specific aptamer for the development of an antibody-antigen detection assay. We investigated the ability of the daunomycin aptamer to efficiently catalyze the hemin-dependent peroxidase activity independent of daunomycin. A reporter probe consisting of biotinylated antigen and daunomycin aptamer coupled to streptavidin gold nanoparticles was successfully used to generate a colorimetric readout. In conclusion, the daunomycin aptamer can function as a robust alternative DNAzyme for the development of colorimetric assays.

  11. Clinical implications of measuring drug and anti-drug antibodies by different assays when optimizing infliximab treatment failure in Crohn's disease

    DEFF Research Database (Denmark)

    Steenholdt, Casper; Bendtzen, Klaus; Brynskov, Jørn;


    classifications. The four different assays did not differ in terms of the ability to predict response to interventions defined by the algorithm. CONCLUSIONS: Despite variable analytical properties, common assays result in similar classifications and interventions in patients with IFX treatment failure......OBJECTIVES: Cost-effective guidance of therapeutic strategy in Crohn's disease patients with secondary infliximab (IFX) treatment failure may be achieved by serum IFX and anti-IFX antibody (Ab) measurements by radioimmunoassay (RIA). This study investigated implications of using other techniques...... on classification of underlying mechanism for treatment failure in most cases (79-94%). The majority (74-88%) failed IFX owing to pharmacodynamic problems, or had noninflammatory pathophysiology for symptoms resembling relapse. Applied threshold for therapeutic vs. subtherapeutic IFX level influenced...

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

    DEFF Research Database (Denmark)

    Schroeder, S.; von Rosen, Tanya; Blome, S.


    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...

  13. Malignant monoblasts can function as effector cells in natural killer cell and antibody-dependent cellular cytotoxicity assays

    DEFF Research Database (Denmark)

    Hokland, P; Hokland, M; Ellegaard, J


    This is the first report describing natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) of malignant monoblasts. Pure acute monoblastic leukemia was diagnosed in bone marrow aspirations from two patients by use of conventional cytochemical methods as well as multiple immunologic...

  14. Evaluation of an in-house dot enzyme-linked immunosorbent assay to detect antibodies against feline panleukopenia virus. (United States)

    Mende, Katherina; Stuetzer, Bianca; Truyen, Uwe; Hartmann, Katrin


    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.

  15. Development of Enzyme Linked Immunosorbent Assay for Quantita-tive Determination of Agonistic DR5 Monoclonal Antibody%抗 DR5单克隆抗体 ELISA 检测新方法的建立

    Institute of Scientific and Technical Information of China (English)

    郭聪; 陈知航; 许先兴; 刘运龙; 车津晶; 董俊兴; 程远国


    Objective: To develop a specific, sensitive and rapid ELISA method for the quantification of agonistic death receptor 5(DR5) monoclonal antibody. Methods: An quantitative sandwich enzyme immunoassay was devel⁃oped in using goat anti-human IgG for capturing as well as detecting. Following that, color was developed by the substrate solution and the reaction was stopped by stop solution. Finally the plate was read at a wavelength of 450 nm using a microplate reader. Results: An ELISA assay was developed with a wide dynamic range of con⁃centrations from 12.5 to 800 ng/mL. The lowest quantification of this assay was 12.5 ng/mL, both accuracy of the intra- and inter-assay were less than 15%. Conclusion: The assay is highly sensitive, accurate, specific, and re⁃producibility over a wide dynamic range of concentrations, which was proven to be a feasible quantitative method for agonistic DR5 monoclonal antibody analysis.%  目的:建立一种灵敏、特异、快速的 ELISA 方法,用猕猴血清中抗死亡受体5(DR5)单克隆抗体的检测.方法:采用双抗夹心 ELISA 法,用猴血清吸附的羊抗人 IgG 包被96孔酶标板,加入待测样品,用 HRP 标记的猴血清吸附的羊抗人 IgG 进行检测,加底物显色,读取 D450nm值.结果:建立了检测抗 DR5单克隆抗体的 ELISA 方法并进行了确证,方法的线性范围为12.5~800 ng/mL,定量下限为12.5 ng/mL,板内和板间精密度均小15%,准确度为±15%,冻融稳定性和稀释稳定性良好.结论:方法学确证结果表明,本研究建立的抗 DR5单克隆抗体检测方法符合新生物制品临床前药代动力学研究指导则要求,可用抗 DR5单克隆抗体的检测.

  16. 结核抗体检测在住院患者结核病诊断中的临床意义%Clinical value of tuberculosis antibody assays in diagnosis of inpatients with tuberculosis

    Institute of Scientific and Technical Information of China (English)

    马秀清; 陈良安; 李春笋; 梁媛; 于玲


    目的 探讨结核抗体检测在住院患者结核病诊断中的临床价值.方法 对医院1911例住院患者,应用3种方法同时检测血清结核抗体的结果进行回顾性分析.结果 3种方法联合检测结核抗体的敏感性为79.8%,特异性为81.9%,结果优于单一检测,均P=0.000;与传统方法相比,3种方法联合检测结核抗体的阳性率为77.6%,优于抗酸染色法(33.6%,P=0.000),略高于结核菌素试验法(66.4%),差异无统计学意义.结论 3种方法联合检测结核抗体,敏感性高,操作简单、快捷,成本低,对住院患者结核病辅助诊断有重要的临床应用价值.%OBJECTIVE To investigate the clinical value of the tuberculosis antibody assays in the diagnosis of InPatients with tuberculosis.METHODS The results of serum Mycobacterium tuberculosis antibody (TB-Ab) tests of 1911 inpatients were retrospectively analyzed.RESULTS The sensitivity and specificity of combined measurement of immunochromatographic method, dot-immunogold filtration assay (DTBA) and immunoblotting method were 79.8% and 81.9%, respectively.They were obviously higher than that of single detection.TB-Ab assay, P=0.000.Compared with traditional methods, the positive rate of the combined measurement of three methods (77.6%) was significantly higher than sputum acid-fast bacillus (AFB) smear method (33.6%, p=0.000), and higher than PPD test (66.4 %), while there was no statistical difference, P= 0.068.CONCLUSION With high sensitivity, simple operation and low cost, the combined measurement of 3 TB-Ab assays helps to make an accurate clinical diagnosis of tuberculosis.

  17. Monoclonal antibody production and indirect competitive enzyme-linked immunosorbent assay development of 3-methyl-quinoxaline-2-carboxylic acid based on novel haptens. (United States)

    Li, Guopeng; Zhao, Liang; Zhou, Feng; Li, Jiaying; Xing, Yuan; Wang, Tiangang; Zhou, Xilong; Ji, Baoping; Ren, Wanpeng


    Two novel immunizing haptens of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) were synthesized and conjugated with cationized bovine serum albumin. Female BALB/c mice were immunized with above conjugates, splenocytes were fused with Sp2/0 cells to produce monoclonal antibody. Compared with previous studies, antibodies raised in this work showed higher sensitivity. Meantime, a novel heterologous coating hapten was also prepared. The indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the optimum condition showed an IC50 of 3.1μg/kg (ppb), and the linear range of 0.46-10.5ppb for MQCA. The limit of detect (LOD) of MQCA in swine muscle, swine liver and chicken was 0.32, 0.54, and 0.28ppb, respectively. The LOD of this assay can satisfy the minimum required performance levels (4ppb) for MQCA. These results indicated that the proposed ELISA, with high sensitivity and specificity, as well as good reproducibility and accuracy, is suitable for determination of MQCA residues in food samples.

  18. Development and field application of a competitive enzyme-linked immunosorbent assay for detection of Newcastle disease virus antibodies in chickens and ducks. (United States)

    Phan, L V; Park, M-J; Kye, S-J; Kim, J-Y; Lee, H-S; Choi, K-S


    A competitive enzyme-linked immunosorbent assay (C-ELISA) using a baculovirus-expressed recombinant nucleocapsid protein antigen (rNDV-N) and an rNDV-N-specific monoclonal antibody (5B3) was developed for the detection of Newcastle disease virus (NDV) antibodies, and its diagnostic performance was evaluated. The specificity and sensitivity of the C-ELISA was found to be 98.4 and 98.9%, respectively, for chickens, and 98.2 and 97.9% for ducks. However, the C-ELISA showed weak cross-reaction with hyperimmune antisera to some other avian paramyxovirus serotypes. In all experimentally vaccinated chickens, seroconversion rates at 7 d postinoculation were 100 and 40% when measured by C-ELISA and hemagglutination inhibition (HI), respectively. In field trials, the C-ELISA showed positive results in 98.9% of HI-positive sera and 40.8% of HI-negative sera from NDV-vaccinated chickens (n = 705). In domestic ducks (n = 158) from NDV-positive duck farms (n = 8), the positive rates according to C-ELISA were significantly higher than those according to the HI test. At the same time, 98.1% of ducks (n = 209) from NDV-negative duck farms (n = 11) were also negative by C-ELISA. Our results indicate that C-ELISA could be a useful alternative to HI testing for detecting NDV antibodies in different avian species such as chickens and ducks.

  19. Nucleoprotein-based indirect enzyme-linked immunosorbent assay(indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

    Institute of Scientific and Technical Information of China (English)

    Yi; Huang; Youjie; Zhu; Mengshi; Yang; Zhenqing; Zhang; Donglin; Song; Zhiming; Yuan


    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 antiEbola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G(Ig G) 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.

  20. In vitro killing assays of antisera antibody sheep post-infected with Fasciola gigantica with the presence of macrophages cells against homologous and heterologous liver flukes

    Directory of Open Access Journals (Sweden)

    S.E Estuningsih


    Full Text Available The previous artificial infection known that the Indonesian Thin Tail (ITT sheep was resistance against the liver fluke of Fasciola gigantica, the resistances occurred in the early infection. In order to observe the immune resistance, some in vitro studies were undertaken in the laboratory, to assay the ability of the antisera antibody of ITT sheep post-infected with F. gigantica, with the presence of macrophages cells in killing the homologous and heterologous liver flukes. The viability of liver flukes were observed within 24-72 hours of incubation period by observing their motility (motile flukes were designated live and non-motile once were death. The results showed that after 72 hours incubation, the motilities of the Newly Excysted Juvenile (NEJ of F. gigantica incubated with the presence of post-infected sera and macrophages cells solution were significantly lower (P0.05. It seems that the occurrence of homologous antibody to the antigens is very important in the development of killing mechanism. The absence of homologous antibody did not reduce the number of flukes or the ability of macrophages cells in killing F. hepatica was not apparent.

  1. Evaluation of an indirect enzyme-linked immunosorbent assay for routine screening of Theiler's murine encephalomyelitis virus antibodies in mice colonies. (United States)

    Laborde, Juan M; Carbone, Cecilia; Corva, Santiago G; Galosi, Cecilia M


    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.

  2. Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus. (United States)

    Huang, Yi; Zhu, Youjie; Yang, Mengshi; Zhang, Zhenqing; Song, Donglin; Yuan, Zhiming


    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.

  3. Development of polyclonal antibody-based indirect enzyme-linked immunosorbent assay for the detection of Alicyclobacillus strains in apple juice. (United States)

    Wang, Zhouli; Yue, Tianli; Yuan, Yahong; Cai, Rui; Guo, Caixia; Wang, Xin; Niu, Chen


    A sort of specific polyclonal anti-Alicyclobacillus antibody was generated by immunizing New Zealand white rabbits, and a sensitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for Alicyclobacillus detection in apple juice. A set of experimental parameters such as concentration of antigen, dilutions of the antibody and goat anti-rabbit IgG-horseradish peroxidase conjugate, selection of the blocking reagent, incubation time, and temperature was optimized. The cross-reactivity of the antibody was evaluated by ELISA and the result was consistent with Western blot analysis. The detection limit of the ELISA was about 10(5) colony forming units (CFU)/mL in apple juice samples. Samples were detected by ELISA and conventional culture method, and the ELISA results gave a good agreement with the results obtained by plating on Alicyclobacillus acidoterrestris medium agar. ELISA takes a total detection time of 6 to 7 h, which is less than the time of conventional techniques requiring more than 24 to 48 h. These results indicated that the established ELISA was a potential useful analytical method for detection of Alicyclobacillus in apple juice.

  4. Anti-VP1 and anti-VP2 antibodies detected by immunofluorescence assays in patients with acute human parvovirus B19 infection

    Directory of Open Access Journals (Sweden)

    Pereira Renata FA


    Full Text Available Acute human parvovirus B19 infection is followed by an antibody response to the structural proteins of the viral capsid (VP1 and VP2. We used 80 sera collected from 58 erythema infectiosum and 6 transient aplastic crisis patients to test IgM and IgG antibodies against these two proteins in an immunofluorescence assay (IFA using Sf9 cells infected with recombinant baculovirus expressing either VP1 or VP2 antigen. Although less sensitive than IgM capture enzyme immunoassay using native antigen (MACEIA, we could detect anti-VP1 or anti-VP2 IgM antibodies by IFA in 49 patients with acute infection (76.6%. Detection of IgG anti-VP1 and anti-VP2 by IFA, however, was as sensitive as IgG detection by indirect enzyme immunoassay. By applying IgG avidity IFA to sera of the 15 IgM IFA negative patients we were able to confirm acute infection in further 12 cases by IFA. Overall, acute infection was confirmed by IFA in 61 (95.3% of the 64 patients.

  5. Antibody Response to Plague Vaccination in Humans as Assayed by Staphylococcal Radioimmune Precipitation (St-RIP) Test (United States)


    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

  6. Fluorescent ligand binding and internalization assays to identify and profile anti Formyl Receptor 1 (FPR1) antibodies


    Peter Cariuk


    GPCR's are involved in a variety of diseases spanning a range of therapeutic areas and have historically been the domain of small molecule drugs. Recent technological advances have opened the possibility to develop therapeutic human monoclonal antibodies in multiple disease areas, conferring additional benefits such as specificity and longer duration of action. FPR1 is a Class A GPCR and is activated by N-formylated peptides (FPR), which can be released by bacterial colonization and lung tiss...

  7. An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software (United States)

    Shah, Pranav; Kerns, Edward; Nguyen, Dac-Trung; Obach, R. Scott; Wang, Amy Q.; Zakharov, Alexey; McKew, John; Simeonov, Anton; Hop, Cornelis E. C. A.


    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

  8. 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. (United States)

    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


    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.

  9. The Cobas AmpliPrep/Cobas TaqMan HCV Test, Version 2.0, Real-Time PCR Assay Accurately Quantifies Hepatitis C Virus Genotype 4 RNA


    Chevaliez, Stéphane; Bouvier-Alias, Magali; Rodriguez, Christophe; Soulier, Alexandre; Poveda, Jean-Dominique; Pawlotsky, Jean-Michel


    Accurate hepatitis C virus (HCV) RNA quantification is mandatory for the management of chronic hepatitis C therapy. The first-generation Cobas AmpliPrep/Cobas TaqMan HCV test (CAP/CTM HCV) underestimated HCV RNA levels by >1-log10 international units/ml in a number of patients infected with HCV genotype 4 and occasionally failed to detect it. The aim of this study was to evaluate the ability of the Cobas AmpliPrep/Cobas TaqMan HCV test, version 2.0 (CAP/CTM HCV v2.0), to accurately quantify H...

  10. Antithyroglobulin antibody (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  11. Antibody levels correlate with detection of Trypanosoma cruzi DNA by sensitive PCR assays in seropositive blood donors and possible resolution of infection over time (United States)

    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.


    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

  12. Evaluation of a covalent mix-enzyme linked immunosorbent assay for screening of Salmonella antibodies in pig serum

    DEFF Research Database (Denmark)

    Chow, E.Y.W.; Wu, J.T.Y.; Jauho, E.S.


    In this study, a commercial Salmonella covalent mix-enzyme linked immunosorbent assay (ELISA) for serological detection of Salmonella infection in swine was evaluated by comparing it with the conventional fecal culture method and inter-laboratory proficiency testing, using a panel of sera tested...

  13. Detection of Salmonella enterica serovar Enteritidis (SE) Antibodies in Serum Using A Polystyrene Bead/SE Flagella Agglutination Assay (United States)

    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...

  14. An ultra-sensitive monoclonal antibody-based enzyme-linked immunosobent assay for dibutyl phthalate in human urinary

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Lifang [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058 (China); Lei, Yajing [Hangzhou EPIE Bio-detection Technology Limited, Hangzhou 310051 (China); Zhang, Dai; Ahmed, Shabbir [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058 (China); Chen, Shuqing, E-mail: [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058 (China)


    Dibutyl phthalate (DBP) has been extensively used as a plasticizer in many daily products, which is highly toxic to human, notably affecting the reproductive and developmental function. As the previous method is expensive, time-consuming, low sensitivity and just focused on the environment. Present study was aimed to establish an ultra-sensitive and simple method based on good quality monoclonal antibody, applying to evaluate excretion level of DBP in urine samples of Chinese population directly. A monoclonal antibody was generated and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mouse. The mouse was previously immunized using a specially designed amino derivative of DBP conjugated with bovine serum albumin (BSA) as immunogen. Cross-reactivity values of the monoclonal antibody against DBP, di-isobutyl phthalate (DIBP) were observed 100% and 1.25%, while for dimethyl phthalate (DMP), butyl benzyl phthalate (BBP) and didecyl phthalate (DDP) the values were < 0.06%. The standard curve was constructed at 0–50 ng mL{sup −1} and good linearity (R{sup 2} = 0.994) was achieved. The observed IC{sub 50} (7.34 ng mL{sup −1}) and LOD (0.06 ng mL{sup −1}) values was improved 1000-fold to polyclonal antibody and 5-fold to other monoclonal antibodies. A total 1246 urine samples were analyzed and the detection frequency of DBP was observed 72.87% by ic-ELISA. The 95th percentile and mean concentration of DBP were 12.07 and 3.00 ng mL{sup −1}. Acceptable recovery rates of DBP were 97.8–114.3% and coefficients variation 5.93–11.09%. The concentrations of DBP in females were found significantly higher (p < 0.05) than males. Similarly, the DBP in middle aged and low educated individuals was found higher (p < 0.001) than the others. Considering the adverse health effects, DBP internal exposure in the Chinese population should be reduced. The ic-ELISA method has been proved as a cost effective, specific, and highly sensitive screening

  15. IgA anti-Actin antibodies in children with celiac disease: comparison of immunofluorescence with Elisa assay in predicting severe intestinal damage

    Directory of Open Access Journals (Sweden)

    Mora Stefano


    Full Text Available Abstract Background Previous studies have demonstrated that the presence of serum IgA antibodies against actin filaments (AAA in patients with celiac disease (CD is strongly associated with mucosal damage and severe degrees of villous atrophy. The aims of the present study were (1 to verify the effectiveness of IgA-AAA in newly diagnosed CD patients in a clinical setting (2 to compare the immunofluorescence assay with ELISA assay; (3 to compare the correlation of our IgA anti-tissue transglutaminase antibodies (tTG-Ab class with mucosal intestinal lesions. Methods 90 patients underwent endoscopy and multiple biopsies for suspected CD on the basis of symptoms, in presence of positive tTG-Ab tests. Twenty biopsied and 25 not-biopsied subjects with negative tTG-Ab were tested as control groups. IgA-AAA assays were performed by indirect immunofluorescence using rat epithelial intestinal cells, and by ELISA with a commercial kit. tTG-Ab assay was a radio-binding assay. Intestinal specimens were collected by upper endoscopy and the histological study was done according to the Marsh's classification modified by Oberhuber (M/O. Auto-antibodies assays and histological evaluation have been performed blindly by skilled operators. Results CD diagnosis was confirmed in 82 patients (type I M/O in 2 patients, IIIA in 18 patients, IIIB in 29 patients and IIIC in 33 patients. Two patients with type 1 lesion in presence of positive tTG-Ab and abdominal complaints, started a gluten free diet. The rate of IgA-AAA positivity (sensitivity by IFI and ELISA in histologically proven celiac disease patients, were 5.5% and 25% patients in IIIA, 27.5% and 34.4% patients in IIIB, 78.8% and 75% in IIIC patients, respectively. Patients with normal or nearly normal mucosa, regardless of tTG-Ab status, presented negative IgA-AAA IFI assay. On the other hand, 1 patient with normal mucosa but positive tTG-Ab, also presented positive IgA-AAA ELISA. All healthy non biopsied

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


    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


    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-ba...

  17. Detection of Lassa virus antinucleoprotein immunoglobulin G (IgG) and IgM antibodies by a simple recombinant immunoblot assay for field use. (United States)

    Ter Meulen, J; Koulemou, K; Wittekindt, T; Windisch, K; Strigl, S; Conde, S; Schmitz, H


    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

  18. An evaluation study of enzyme-linked immunosorbent assay (ELISA) using recombinant protein Pap31 for detection of antibody against Bartonella bacilliformis infection among the Peruvian population. (United States)

    Angkasekwinai, Nasikarn; Atkins, Erin H; Romero, Sofia; Grieco, John; Chao, Chien Chung; Ching, Wei Mei


    Reliable laboratory testing is of great importance to detect Bartonella bacilliformis infection. We evaluated the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) using recombinant protein Pap31 (rPap31) for the detection of antibodies against B. bacilliformis as compared with immunofluorescent assay (IFA). Of the 302 sera collected between 1997 and 2000 among an at-risk Peruvian population, 103 and 34 samples tested positive for IFA-immunoglobulin G (IgG) and IFA-IgM, respectively. By using Youden's index, the cutoff values of ELISA-IgG at 0.915 gave a sensitivity of 84.5% and specificity of 94%. The cutoff values of ELISA-IgM at 0.634 gave a sensitivity of 88.2% and specificity of 85.1%. Using latent class analysis, estimates of sensitivity and specificity of almost all the assays were slightly higher than those of a conventional method of calculation. The test is proved beneficial for discriminating between infected and non-infected individuals with the advantage of low-cost and high-throughput capability.

  19. An antibody induction method in mice for the potency assays of diphtheria and tetanus components in combined vaccines. (United States)

    Maheshwari, S C; Sharma, S B; Kumar, A; Sokhey, J


    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.

  20. Diagnostic accuracy of IgA anti-tissue transglutaminase antibody assays in celiac disease patients with selective IgA deficiency. (United States)

    Villalta, D; Alessio, M G; Tampoia, M; Tonutti, E; Brusca, I; Bagnasco, M; Pesce, G; Bizzaro, N


    Clinical studies have estimated a 10- to 20-fold increased risk for celiac disease (CD) in patients with selective IgA deficiency (SIgAD). For this reason, screening for CD is mandatory in SIgAD patients, but it represents a special challenge since the specific IgA class antibodies against gliadin (AGA), endomysium (EMA), and tissue-transglutaminase (tTG) are not produced in patients with CD. IgG class counterparts of these antibodies may be informative; in particular IgG EMA has been demonstrated to be a valid marker for diagnosing CD in SIgAD cases, but it is not used much in clinical laboratories, because it is cumbersome and involves some technical difficulties. Even if it was widely used in clinical laboratories, the measuring of IgG AGA has shown a less-than-optimum diagnostic accuracy, so that now it tends to be substituted by tests for anti-tTG IgG, for which the few available studies have shown diagnostic performances superior to AGA. Since it is not known whether various available methods for measuring IgG anti-tTG antibodies offer similar diagnostic performances, we have compared the results obtained from nine second-generation commercial methods (D-tek, Phadia, Immco, Orgentec, Radim, Euroimmun, Inova, Aesku, Generic Assays), measuring IgG anti-tTG antibodies in 20 patients with CD and SIgAD and in 113 controls (9 patients with SIgAD without CD, 54 patients with chronic liver disease, and 50 healthy individuals). Diagnostic sensitivity, calculated by means of ROC plot analysis, ranged between 75% and 95%, and specificity ranged from 94% to 100%. In the same population, the diagnostic sensitivity and specificity of AGA IgG were 40% and 87%, respectively. Even though they perform differently, all IgG anti-tTG methods evaluated are reliable serological assays for the diagnosis of CD in SIgAD patients, with diagnostic accuracy superior to the AGA IgG method. The methods that use a mix of tTG and gliadin peptides as the antigenic preparation have a

  1. Monitoring of Anti-Hepatitis E Virus Antibody Seroconversion in Asymptomatically Infected Blood Donors: Systematic Comparison of Nine Commercial Anti-HEV IgM and IgG Assays (United States)

    Vollmer, Tanja; Diekmann, Juergen; Eberhardt, Matthias; Knabbe, Cornelius; Dreier, Jens


    Diagnosis of hepatitis E virus (HEV) is usually determined serologically by detection of the presence of immunoglobulin (Ig)M antibodies or rising anti-HEV IgG titers. However, serological assays have demonstrated a significant variation in their sensitivities and specificities. In this study, we present the systematic comparison of different immunological anti-HEV assays using complete seroconversion panels of 10 virologically confirmed HEV genotype 3 infected individuals. Assay sensitivities were further evaluated by testing serially diluted World Health Organization (WHO) reference reagent for hepatitis E virus antibody and one patient sample infected with HEV genotype 3. Anti-HEV IgM and IgG antibody presence was determined using the immunological assays Wantai HEV IgM/IgG enzyme-linked immunosorbent assay (ELISA) (Sanbio, Uden, The Netherlands), recomWell HEV IgM/IgG (Mikrogen, Neuried, Germany), HEV IgM ELISA 3.0, HEV ELISA, HEV ELISA 4.0, Assure HEV IgM Rapid Test (all MP Biomedicals Europe, Illkirch Cedex, France) and Anti-HEV ELISA (IgM/IgG, Euroimmun, Lübeck, Germany). The assays showed differences regarding their analytical and diagnostic sensitivities, with anti-HEV IgM assays (n = 5) being more divergent compared to anti-HEV IgG (n = 4) assays in this study. Considerable variations were observed particularly for the detection period of IgM antibodies. This is the first study systematically characterizing serologic assays on the basis of seroconversion panels, providing sample conformity for a conclusive comparison. Future studies should include the assay comparison covering the four different genotypes. PMID:27556482

  2. Evaluation of modified Ziehl-Neelsen, direct fluorescent-antibody and PCR assay for detection of Cryptosporidium spp. in children faecal specimens. (United States)

    Aghamolaie, S; Rostami, A; Fallahi, Sh; Tahvildar Biderouni, F; Haghighi, A; Salehi, N


    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.

  3. Toxoplasma gondii: Recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay. (United States)

    Holec-Gasior, Lucyna; Kur, Józef


    In this study, for the first time, the evaluation of Toxoplasma gondii full-length recombinant GRA5 antigen for the serodiagnosis of human toxoplasmosis is shown. The recombinant GRA5 antigen as a fusion protein containing His-tag at both terminals was obtained using an Escherichia coli expression system. The usefulness of rGRA5 for the diagnosis of toxoplasmosis in an ELISA was tested on a total of 189 sera from patients with different stages of the infection and 31 sera from sero-negative individuals, obtained during routine diagnostic tests. Anti-GRA5 IgG antibodies were detected in 70.9% of all seropositive serum samples. This result was comparable to ELISA using a Toxoplasma lysate antigen (TLA) and six combinations of recombinant antigens. The sensitivity of IgG ELISA calculated from all positive serum samples was similar for TLA (94.2%), rMAG1+rSAG1+rGRA5 (92.6%), rGRA2+rSAG1+rGRA5 (93.1%) and rROP1+rSAG1+rGRA5 (94.2%) cocktails, whereas the sensitivity of cocktails without rGRA5 antigens was lower giving 82.0%, 86.2% and 87.8%, respectively. Thus, the present study showed that the full-length rGRA5 is suitable for use as a component of an antigen cocktail for the detection of anti-T. gondii IgG antibodies.

  4. Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

    Directory of Open Access Journals (Sweden)

    Tereza Cristina Cardoso


    Full Text Available The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN and mouse neutralization test (MNT to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125 as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5 cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100 and r = 0.9307 for cattle sera (n = 99, as well as good specificity (94.7%, sensitivity (87.5%, and agreement (96.6% were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

  5. Indirect Enzyme-Linked Immunosorbent Assay for Detection of Brucella melitensis-Specific Antibodies in Goat Milk


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


    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...

  6. Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv with a two-color fluorometric real-time PCR assay.

    Directory of Open Access Journals (Sweden)

    Lindsey R Pierce

    Full Text Available 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.

  7. Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay. (United States)

    Pierce, Lindsey R; Willey, James C; Palsule, Vrushalee V; Yeo, Jiyoun; Shepherd, Brian S; Crawford, Erin L; Stepien, Carol A


    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.

  8. Epidemiological, biological and histological characterization of patients with indeterminate third-generation recombinant immunoblot assay antibody results for hepatitis C virus. (United States)

    Ríos, M; Diago, M; Rivera, P; Tuset, C; Cors, R; García, V; Carbonel, P; Gonzalez, C


    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.

  9. Development and validation of an indirect Enzyme-linked immunosorbent assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants. (United States)

    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


    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.

  10. Analysis and solution of false-positives when testing CVA16 sera using an antibody assay against the EV71 virus. (United States)

    Wang, Changbing; You, Aiping; Tian, Xingui; Zhao, Mingqi; Chen, Yi; Lin, Tao; Zheng, Jianbin; Xiao, Misi; Zhang, Yingying; Kuang, Lu; Zhou, Zhenwen; Zhu, Bing


    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.

  11. Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay. (United States)

    Malgor, R; Nonaka, N; Basmadjian, I; Sakai, H; Carámbula, B; Oku, Y; Carmona, C; Kamiya, M


    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%.

  12. Magnetic beads-based enzymatic spectrofluorometric assay for rapid and sensitive detection of antibody against ApxIVA of Actinobacillus pleuropneumoniae. (United States)

    Wei, Bo; Li, Fang; Yang, Huicui; Yu, Lei; Zhao, Kaihong; Zhou, Rui; Hu, Yonggang


    In this paper, a simple, easily-operated and enzyme-amplified fluorescence immunoassay method using magnetic particles for the detection of antibody against Actinobacillus pleuropneumoniae (APP) has been presented. The A protein of APP Repeats-in-Toxin IV (ApxIVA) with high specificity to the APP species was immobilized onto the magnetic bead surfaces. Horseradish peroxidase (HRP), which can catalyze the substrate 4-hydroxyphenylacetic acid (p-HPA), generating fluorescent bi-p, p'-hydroxyphenylacetic acid (DBDA), was selected as an enzymatic-amplified tracer. The ApxIVA antibody was detected for the presence of APP infection by measuring the fluorescence intensity of DBDA. Under optimal conditions, the calibration plot obtained for standard positive serum was approximately linear within the dilution range 1:160-1:5120. The limit of detection (LOD) for the assay was 1:10240, considerably lower than that of ApxIVA-ELISA (1:320) (S/N=3). A series of repeatability measurements of using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation (RSD) of 4.8% (n=11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor yielded an efficiency of 89.7%, sensitivity of 90.9% and specificity of 89.3% compared with ApxIVA-ELISA.

  13. Identification of specific antinuclear antibodies in dogs using a line immunoassay and enzyme-linked immunosorbent assay. (United States)

    Bremer, Hanna D; Lattwein, Erik; Renneker, Stefanie; Lilliehöök, Inger; Rönnelid, Johan; Hansson-Hamlin, Helene


    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

  14. Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay. (United States)

    Zhang, Xian; Sun, Mengjiao; Kang, Yue; Xie, Hui; Wang, Xin; Song, Houhui; Li, Xiaoliang; Fang, Weihuan


    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.

  15. A multilaboratory evaluation of a commercial enzyme-linked immunosorbent assay test for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in cattle. (United States)

    Dargatz, David A; Byrum, Beverly A; Collins, Michael T; Goyal, Sagar M; Hietala, Sharon K; Jacobson, Richard H; Kopral, Christine A; Martin, Barbara M; McCluskey, Brian J; Tewari, Deepanker


    Five laboratories participated in a study to evaluate sources of variation in results from an enzyme-linked immunosorbent assay (ELISA) for antibodies against Mycobacterium avium subsp. paratuberculosis. Each laboratory repeatedly tested duplicates of a negative, positive (P), and high-positive (HP) serum sample, which were supplied by the United States Department of Agriculture: Animal and Plant Health Inspection Service: Veterinary Services, National Veterinary Services Laboratories, Ames, IA, on all 96-well microtiter plates when routinely testing other samples for M. avium subsp. paratuberculosis antibodies. These 3 sera were aliquoted and sent to the 5 participating laboratories. This study focused on variation in test results because of assay reagents and laboratory techniques and did not account for biologic variability associated with the time course of infection in cattle. Overall, results from 868 microtiter plates were used in the study. For each sample a sample-to-positive (S/P) ratio was calculated according to the manufacturer's directions. The S/ P ratio for the P sample ranged from 0.06 to 1.039 (mean = 0.466 and 0.484 for wells 1 and 2, respectively) and those for the HP sample ranged from 2.446 to 8.727 (mean = 4.027 and 3.980 for wells 1 and 2, respectively). The majority of the variation in S/P ratio for the P sample was attributed to kit lot (37.5%), followed by random (unexplained) error (27.0%), laboratory (18.3%), and kit lot by laboratory (11.9%). By eliminating plates in which the separation between negative and positive control ODs was less than 0.4, the proportion of variation attributed to laboratory was reduced markedly. These results confirm that there is variability in M. avium subsp. paratuberculosis ELISA results and that several sources contribute to the observed variability. The study gives a relative estimate of the contribution of various sources to the overall variability observed in the M. avium subsp. paratuberculosis ELISA

  16. Highly sensitive and reproducible immunoradiometric assay for total human renin using monoclonal antibodies, iodogen labelling and polystyrene star tubes

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, M.D.; Rasumussen, P.H.; Giese, J.


    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.

  17. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

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    Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)


    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

  18. The diagnostic sensitivity of dengue rapid test assays is significantly enhanced by using a combined antigen and antibody testing approach.

    Directory of Open Access Journals (Sweden)

    Scott R Fry


    Full Text Available BACKGROUND: Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1 has been identified as an early marker for acute dengue, and is typically present between days 1-9 post-onset of illness but following seroconversion it can be difficult to detect in serum. AIMS: To evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test. METHODOLOGY: The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples. KEY RESULTS: In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6% and 96% (95% CI: 92.2% to 99.8 respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1% and 96.7% specificity (95% CI: 82.8% to 99.9% compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers. CONCLUSIONS: This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

  19. Validation of a Poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations. (United States)

    Ramírez, Juan David; Herrera, Claudia; Bogotá, Yizeth; Duque, María Clara; Suárez-Rivillas, Alejandro; Guhl, Felipe


    Trypanosoma cruzi is the causative agent of American trypanosomiasis, a complex zoonotic disease that affects more than 10million people in the Americas. Strains of this parasite possess a significant amount of genetic variability and hence can be divided into at least six discrete typing units (DTUs). The life cycle of this protist suggests that multiclonal infections may emerge due to the likelihood of contact of triatomine insects with more than 100 mammal species. To date, there have been a few studies on but no consensus regarding standardised methodologies to identify multiclonal infections caused by this parasite. Hence, the aim of this study was to develop and validate a limiting dilution assay (LDA) to identify multiclonal infections in T. cruzi populations by comparing the feasibility and reliability of this method with the widely applied solid phase blood agar (SPBA) methodology. We cloned reference strains belonging to three independent genotypes (TcI, TcII, and TcIV) and mixed infections (TcI+TcII) using LDA and SPBA; the comparison was conducted by calculating the feasibility and reliability of the methods employed. Additionally, we implemented LDA in strains recently isolated from Homo sapiens, Rhodnius prolixus, Triatoma venosa, Panstrongylus geniculatus, Tamandua tetradactyla, Rattus rattus, Didelphis marsupialis and Dasypus novemcinctus, with the aim of resolving multiclonal infections using molecular characterization employing SL-IR (spliced leader intergenic region of mini-exon gene), the 24Sα rDNA gene and microsatellite loci. The results reported herein demonstrate that LDA is an optimal methodology to distinguish T. cruzi subpopulations based on microsatellite markers by showing the absence of multiple peaks within a single locus. Conversely, SPBA showed patterns of multiple peaks within a single locus suggesting multiclonal events. The biological consequences of these results and the debate between multiclonality and aneuploidy are

  20. Assessment of Neutralising Activity of Colostrum-Derived, Polyclonal, Bovine Antibodies: Use of the J774A.1 Anthrax Lethal Toxin Cytototoxity Assay (United States)


    Assessment of Neutralising Activity of Colostrum - Derived, Polyclonal, Bovine Antibodies: Use of the J774A.1 Anthrax Lethal Toxin...activity of colostrum -derived, polyclonal, bovine antibodies. Antibodies against lethal factor and protective antigen were found to protect...APPROVED FOR PUBLIC RELEASE Assessment of Neutralising Activity of Colostrum - Derived, Polyclonal, Bovine Antibodies: Use of the J774A.1

  1. An AS-PCR assay for accurate genotyping of FAD2A/FAD2B genes in peanuts (Arachis hypogaea L.

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    Yu, H. T.


    Full Text Available FAD2A and FAD2B are homoeologous genes from A and B genomes in cultivated peanuts (Arachis hypogaea L. encoding fatty acid desaturates which convert oleate to linoleate. To study the genetics of oleate and breed high oleate peanut cultivars, a simple allele specific-PCR (AS-PCR protocol for the accurate genotyping of FAD2A/FAD2B was developed to discriminate the wild and mutant allele of both genes (FAD2A 448 G > A and FAD2B 441_442insA. The results may serve to develop a feasible procedure for producing highly desired high oleate peanut cultivars through hybridization.FAD2A y FAD2B son genes homólogos de genomas A y B en cacahuete cultivado (Arachis hypogaea L. que codifican las desaturasas de ácidos grasos que convierten oleato a linoleato. Para estudiar la genética de las variedades mejoradas de cacahuete alto oleato, fue desarrollado un protocolo sencillo del alelo específico PCR-(AS-PCR para la determinación precisa del genotipo de FAD2A/FAD2B y discriminar el alelo silvestre y el mutante de ambos genes (FAD2A 448 G > A y FAD2B 441_442insA. Los resultados pueden servir para desarrollar un procedimiento viable para la producción de cultivares deseados de cacahuetes alto oleato a través de hibridación.

  2. Assay of neutralizing antibody against variola virus by the degree of focus reduction on HeLa cell cultures and its application to revaccination with smallpox vaccines of various potencies. (United States)

    Kitamura, T; Shinjo, N


    A method for assaying neutralizing antibody against variola virus was established by focus counting on HeLa cell cultures. The ND(50) titre, i.e., the serum dilution endpoint to give a 50% reduction in the number of foci, was determined with excellent reproducibility.Groups of students 19-20 years of age were revaccinated by the multiple pressure method with serial 10-fold dilutions of a smallpox vaccine and their neutralizing antibody response was assayed by the focus counting assay system and was related to the local skin reactions on the seventh day after inoculation and to the potency of the vaccine administered. There was a significant rise in the antibody level even after inoculation with a vaccine whose potency was as low as 1.3 x 10(5) pock-forming units/ml. In general, the rise in the log antibody level was proportional to the diameter of the reddening, but a significant rise was found among individuals who had no detectable skin reaction. The skin reaction was greater among individuals with a lower initial antibody level when the vaccine administered had a potency lower than 1.3 x 10(6) pock-forming units/ml.

  3. 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. (United States)

    Levanov, Lev; Vera, Cristina Pérez; Vapalahti, Olli


    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.

  4. Measurement of thyrotropin receptor antibodies (TRAK) with a second generation assay in patients with Graves' disease; Die Bestimmung von Thyreotropin-Rezeptor-Antikoerpern (TRAK) mit einem Assay der zweiten Generation bei Patienten mit Morbus Basedow

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    Zoephel, K.; Wunderlich, G.; Franke, W.G. [Klinik und Poliklinik fuer Nuklearmedizin, Technische Univ. Dresden (Germany); Koch, R. [Inst. fuer Medizinische Informatik und Biometrie, Technische Univ. Dresden (Germany)


    Aim: The detection of TSH-receptor-antibodies (TRAb) in patients (pts) with Graves' disease (GD) is routinely used in nuclear medicine laboratories. It is performed by commercial, porcine radioreceptorassays (RRA) measuring TSH binding inhibitory activity. A second generation assay using the human, recombinant TSH-receptor was developed during the last years. The manufacturer composed this new assay as a coated tube RRA (CT RRA) and claimed a higher sensitivity for GD. Methods: TRAb was measured in 207 pts with various thyroid disorders and 205 healthy controls using the new coated tube RRA (Fa. B.R.A.H.M.S. Diagnostica GmbH, Berlin, Germany) as well as a conventional RRA (Fa. Medipan Diagnostica GmbH, Selchow, Germany): 60 pts suffering from GD showing a relapse after anti-thyroid drug treatment and before radioiodine therapy, 109 pts with disseminated autonomia (DA) and 38 pts suffering from Hashimoto's thyroiditis. A ROC-analysis was performed to find the optimal decision threshold level for positivity. Results: We found 42/60 TRAb-positive pts with GD in the established RRA (threshold 6 U/L) and 52/60 in the CT RRA, respectively. The sensitivity increased from 70% (RRA) to 86,7% (CT RRA). The CT RRA found 2 false positives (one Hashimoto's and one healthy control) and the RRA detected 3 Hashimoto's and 2 healthy controls as false positive. Conclusion: The increased sensitivity of CT RRA for GD provides an advantage compared to conventional RRA, especially in GD-patients relapsing afte antithyroid drug treatment. Functional sensitivity and Interassayvariation of CT RRA are very precisely compared to conventional RRA. Handling of the new assay is also improved. (orig.) [German] Ziel: Die Bestimmung der TSH-Rezeptorantikoerper (TRAK) bei Patienten mit Morbus Basedow ist fester Bestandteil der nuklearmedizinischen In-vitro-Diagnostik. Seit kurzem ist die Bestimmung mit einem TRAK-Assay moeglich, bei dem im Gegensatz zu den herkoemmlichen

  5. [Immunofluorescence assay with Crithidia luciliae for the detection of anti-DNA antibodies. Atypical images and their relationship with Chagas' disease and leishmaniasis]. (United States)

    Griemberg, Gloria; Ferrarotti, Nidia F; Svibel, Graciela; Ravelli, Maria R; Taranto, Nestor J; Malchiodi, Emilio L; Pizzimenti, Maria C


    Anti-native DNA antibodies can be detected by indirect immunofluorescence assay with Crithidia luciliae, displaying an annular image due to a kinetoplast containing double stranded DNA. Other structures such as membrane, flagellum and basal corpuscle can be stained as well, showing what is called atypical fluorescent images. As C. luciliae belongs to the Trypanosomatidae family, which include the human pathogens Trypanosoma cruzi and Leishmania spp., it was considered that these atypical images could be caused by cross-reactions. Serological studies for Chagas' disease were performed in 105 serum samples displaying atypical images. Sixty four percent of the samples from non endemic and 78.3% from endemic areas for Chagas' disease showed fluorescence in both, membrane and flagellum (joint image). Fifty samples from normal blood donors and 57 samples from patients with conective tissue diseases were tested with C. luciliae. None of them presented the joint image except for two patients with lupus who were also chagasic. In addition, 54 samples from chagasic patients were studied and all of them presented the joint image. We also studied 46 samples from patients with leishmaniasis from whom 28 were coinfected with T. cruzi. The joint image was observed in 88.0% of the samples with leishmaniasis and in 89.3% of the co-infected samples. The results suggest that C. luciliae could be used as an economical, and of low risk, alternative substrate for the serological diagnosis of Chagas' disease, even though it does not discriminate for Leishmania spp. infection. This study also suggests that whenever atypical images are observed in C. luciliae during the search for anti-DNA antibodies, it would be convenient to submit the patient to clinical and serological tests for the diagnosis of leishmaniosis and Chagas' disease.

  6. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody. (United States)

    Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H; Hartung, John S


    '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.

  7. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Fang Ding

    Full Text Available '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.

  8. A rapid and accurate 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay for quantification of bacteriocins with nisin as an example

    Institute of Scientific and Technical Information of China (English)


    The objective of this study is to propose a more accurate and faster MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay (MCA) for quantitative measurement of polypeptide bacteriocins in solutions with nisin as an example. After an initial incubation of nisin and indicator bacterium Micrococcus luteus NCIB 8166 in tubes, MTT was added for another incubation period. After that, nisin was quantified by estimating the number of viable bacteria based on measuring the amount of purple formazan produced by cleavage of yellow tetrazolium salt MTT. Then MCA was compared to a standard agar diffusion assay (ADA). The results suggested a high correlation coefficient (r2=0.975±0.004) between optical density (OD) and the inhibitory effect of nisin on a bacterial strain Micrococcus luteus NCIB 8166 at a range of 0.125~32 IU/ml.The MCA described in this study was very quick. Quantification of nisin took only 7~8 h and the detection limit was at the level of 0.125 IU/ml when compared to 12 IU/ml and 24~28 h for ADA. The MCA provides an accurate and rapid method for quantification of nisin in solutions and is expected to be used for quantification of other antimicrobial substances.

  9. Application of the fluorescence polarization assay for detection of caprine antibodies to Brucella melitensis in areas of high prevalence and widespread vaccination. (United States)

    Ramírez-Pfeiffer, C; Nielsen, K; Smith, P; Marín-Ricalde, F; Rodríguez-Padilla, C; Gomez-Flores, R


    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.

  10. Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool

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    Zoheiry Mona K


    Full Text Available Abstract Background This research was carried out to develop a reliable monoclonal antibody (MoAb-based sandwich enzyme linked immunosorbent assay (ELISA for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes. Methods From a panel of MoAbs raised against F. gigantica excretory/secretory antigens (ES Ags, a pair (12B/11D/3F and 10A/9D/10G was chosen due to its high reactivity and strict specificity to F. gigantica antigen by indirect ELISA. Results The two MoAbs were of the IgG1 and IgG2a subclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p Conclusions These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

  11. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay. (United States)

    Adungo, Ferdinard; Yu, Fuxun; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu; Morita, Kouichi


    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.

  12. Cross-reactivity of antibodies with phenolic compounds in pistachios during quantification of ochratoxin A by commercial enzyme-linked immunosorbent assay kits. (United States)

    Lee, Hyun Jung; Meldrum, Alexander D; Rivera, Nicholas; Ryu, Dojin


    Ochratoxin A (OTA), a nephrotoxic mycotoxin, naturally occurs in wide range of agricultural commodities. Typical screening of OTA involves various enzyme-linked immunosorbent assay (ELISA) methods. Pistachio (Pistacia vera L.) is a rich source of phenolic compounds that may result in a false positive due to structural similarities to OTA. The present study investigated the cross-reactivity profiles of phenolic compounds using two commercial ELISA test kits. High-performance liquid chromatography was used to confirm the concentration of OTA in the pistachio samples and compared with the results obtained from ELISA. When the degree of interaction and 50 % inhibitory concentration of phenolic compounds were determined, the cross-reactivity showed a pattern similar to that observed with the commercial ELSIA kits, although quantitatively different. In addition, the degree of interaction increased with the increasing concentration of phenolic compounds. The ELISA value had stronger correlations with the content of total phenolic compound, gallic acid, and catechin (R(2) = 0.757, 0.732, and 0.729, respectively) compared with epicatechin (R(2) = 0.590). These results suggest that phenolic compounds in pistachio skins may cross-react with the OTA antibody and lead to a false positive or to an overestimation of OTA concentration in ELISA-based tests.

  13. Guidelines of the Office International des Epizooties for laboratory quality evaluation, for international reference standards for antibody assays and for laboratory proficiency testing. (United States)


    Three guidelines, adopted by the International Committee of the Office International des Epizooties (OIE), have been combined for publication in a single document. The Guidelines for evaluating laboratory quality (adopted in 1995) form part of the OIE Guidelines for evaluating Veterinary Services. General requirements for equipment, staffing and management of laboratories are outlined. The guidelines for international reference standards for antibody assays (adopted in 1998) provide general rules governing the preparation of immune sera by OIE Reference Laboratories. A data sheet should accompany each preparation dispatched from the laboratory, and details are given of the information to be contained in the data sheet. The guidelines are to be used in conjunction with the OIE Manual of standards for diagnostic tests and vaccines. Guidelines on the proficiency of laboratory testing (adopted in 1996) describe how the operation of a laboratory can be assessed by inter-laboratory testing, and by voluntary participation in an accreditation (quality assurance) audit, operated by an independent authority. Criteria for assessing serological testing are provided.

  14. The effect of immunoscintigraphy with monoclonal antibodies on assays of hormones and tumor markers. This is not the end of the matter!

    NARCIS (Netherlands)

    J.A.M.J.L. Janssen (Joseph); P.J. Blankestijn (Peter); R. Docter (Roel); B.G. Blijenberg (Bert); S.W.J. Lamberts (Steven); E.P. Krenning (Eric)


    textabstractThe use of monoclonal antibodies in medicine for in-vivo diagnostic methods and for therapeutic purposes will increase in the future. Although monoclonal antibodies possess a high specificity, the animal origin of these antibodies remains a problem. Repeated administrat

  15. Surface plasmon resonance-enhanced fluorescence implementation of a single-step competition assay: demonstration of fatty acid measurement using an anti-fatty acid monoclonal antibody and a Cy5-labeled fatty acid. (United States)

    Vareiro, Margarida M L M; Tranchant, Isabelle; Maplin, Sandra; Zak, Kris; Gani, M M; Slevin, Christopher J; Hailes, Helen C; Tabor, Alethea B; Cameron, Petra J; Jenkins, A Toby A; Williams, David E


    The development of a single-step, separation-free method for measurement of low concentrations of fatty acid using a surface plasmon resonance-enhanced fluorescence competition assay with a surface-bound antibody is described. The assay behavior was unexpectedly complex. A nonlinear coverage-dependent self-quenching of emission from surface-bound fluorescent label was deduced from the response kinetics and attributed to a surface plasmon-mediated energy transfer between adsorbed fluorophores, modified by the effects of plasmon interference. Principles of assay design to avoid complications from such effects are discussed. An anti-fatty acid mouse monoclonal antibody reacting to the alkyl chain was prepared and supported on a gold chip at a spacing appropriate for surface-plasmon field-enhanced fluorescence spectroscopy (SPEFS), by applying successively a self-assembled biotinylated monolayer, then streptavidin, then biotinylated protein A, and then the antibody, which was crosslinked to the protein A. Synthesis of a fluorescently (Cy5) tagged C-11 fatty acid is reported. SPEFS was used to follow the kinetics of the binding of the labeled fatty acid to the antibody, and to implement a competition assay with free fatty acid (undecanoic acid), sensitive at the 1 microM scale, a sensitivity limit caused by the low affinity of antibodies for free fatty acids, rather than the SPEFS technique itself. Free fatty acid concentration in human serum is in the range 0.1-1mM, suggesting that this measurement approach could be applied in a clinical diagnostic context. Finally, a predictive, theoretical model of fatty acid binding was developed that accounted for the observed "overshoot" kinetics.

  16. Error in anti-DNA antibody radioimmunoassay after gallium scanning

    Energy Technology Data Exchange (ETDEWEB)

    Torretti, D.; Rooney, P.; Williams, G.; Decker, J.L.


    Significant interference with the accurate measurement of anti-DNA antibodies occurs after gallium-67 scanning. The observed effect is dependent on the radioimmunoassay used. False-negative results are observed with a modified Farr assay whereas false-positive results are noted in the millipore filter assay. These spurious values are the result of persistent radioactivity in the patients' sera after administration of /sup 67/Ga citrate.

  17. 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. (United States)

    Chaka, H; Thompson, P N; Goutard, F; Grosbois, V


    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

  18. 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. (United States)

    Bhullar, S S; Chandak, N H; Baheti, N N; Purohit, H J; Taori, G M; Daginawala, H F; Kashyap, R S


    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.

  19. Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method

    Directory of Open Access Journals (Sweden)

    Wan Zhixiang


    Full Text Available Abstract Background Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR-based assays and enzyme-linked immunosorbent assays (ELISA are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS method was applied to detect HBV and HCV antibodies rapidly and simultaneously. Methods Chemically modified glass slides were used as solid supports (named chip, on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. Results We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05 existed between the results determined by our assay and ELISA respectively. Conclusion

  20. A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk

    NARCIS (Netherlands)

    Kramps, J.A.; Maanen, van C.; Wetering, van de G.; Stienstra, G.; Quak, S.; Brinkhof, J.; Ronsholt, L.; Nylin, B.


    To detect Bovine Virus Diarrhoea Virus (BVDV)-specific antibodies in cattle serum, plasma and bulk milk, a simple, reliable and rapid blocking ELISA ("Ceditest") has been developed using two monoclonal antibodies ("WB112" and "WB103") directed to different highly conserved epitopes on the non-struct

  1. Development and Validation of ELISA Assays for Determination of Serum Antibody against Pertussis Toxin, Filamentous Hemagglutinin, Pertactin of Bordetella Pertussis%百日咳毒素丝状血凝素和百日咳黏附素血清抗体检测方法的建立及验证

    Institute of Scientific and Technical Information of China (English)

    徐颖华; 骆鹏; 王丽婵; 卫辰; 侯启明; 张庶民


    . The limit of quantification of the assays had been demonstrated for anti-PT antibody with 1.31IU/ml, for anti-FHA antibody with 1.02IU/ml, for anti-Prn antibody with 0.51IU/ml. The average intra-assay coefficient of variation of three assays was 9.99%-. 11.01% and 9.00%. The average inter-assay coefficient of variation of three assays was 11.55% -. 12.76% and 11.18%, respectively. The average recovery rates of three assays were 97.19% ■. 101.20% and 107.83%, respectively. Three antibody levels in 30 serum sample were detected by ELISA methods developed in this study, which were not difference significantly in comparison with that of similar method from other country. These methods were also applied for immunogenicity analysis of three-component acellular pertussis vaccine in the clinical trial. Conclusion Three quantitative ELISA methods had been demonstrated to be simple, accurate and reproducible. They were used for evaluation of immunogenicy of acellular pertussis vaccine andinvestigation of pertussis epidemiology.

  2. Application of the Filariasis CELISA Antifilarial IgG Antibody Assay in Surveillance in Lymphatic Filariasis Elimination Programmes in the South Pacific

    Directory of Open Access Journals (Sweden)

    Hayley Joseph


    Full Text Available Elimination of lymphatic filariasis (LF in the Pacific Island Countries and Territories (PICT has been defined as <0.1% circulating filarial antigen (CFA prevalence in children born after the implementation of successful mass drug administrations (MDAs. This research assessed the feasibility of CFA and antibody testing in three countries; Tonga, Vanuatu, and Samoa. Transmission is interrupted in Vanuatu and Tonga as evidenced by no CFA positive children and a low antibody prevalence and titre. Transmission is ongoing in Samoa with microfilaraemic (Mf and CFA positive children and a high antibody prevalence and titre. Furthermore, areas of transmission were identified with Mf positive adults, but no CFA positive children. These areas had a high antibody prevalence in children. In conclusion, CFA testing in children alone was not useful for identifying areas of residual endemicity in Samoa. Thus, it would be beneficial to include antibody serology in the PICT surveillance strategy.

  3. Evaluation of the 2. generation radio-receptional assay for anti-TSH receptor antibodies (TRAb) in autoimmune thyroid diseases. Comparison with 1. generation and anti-thyroperoxidae antibodies (AbTPO)

    Energy Technology Data Exchange (ETDEWEB)

    Giovanella, L.; Ceriani, L.; Garacini, S. [University Hospital Ospedale di Circolo e Fondazione Macchi, Dept. of Nuclear Medicine, Lab. of Endocrinology and Thyroid Unit, Varese (Italy)


    The detection of autoantibodies to the TSH-receptor (TRAb) by radio-receptor assays (RRA) is widely requested in clinical practice for the diagnostic work-up of Graves' disease and its differentiation from diffuse thyroid autonomy. Additionally, TRAb measurement can be useful during antithyroid drug treatment of Graves' disease to evaluate the risk of relapse after therapy discontinuation. Nevertheless, some patients affected by Graves' disease are TRAb-negative when 1. generation assay is used. In this study the diagnostic performance of a newly developed 2. generation TRAb assay (TRAK human DYNOtest(R), BRAHMS Diagnostica GmbH, Berlin, Germany) was evaluated in 74 untreated patients affected by Graves' disease, in 53 untreated patients affected by Hashimoto's thyroiditis and in 88 patients affected by euthyroid nodular goiter. It was also compared the new TRAb assay with the 1. generation test (TRAK(R) Assay, BRAHMS Diagnostica GmbH, Berlin, Germany) and anti-thyroperoxidase assay (AbTPO DYNOtest(R), BRAHMS GmbH, Berlin). The 2. generation TRAb assay showed the better diagnostic sensitivity in Graves' disease (97%) with respect to the 1. generation assay (85%) and AbTPO assay (64%). The AbTPO assay was positive in 50 of 53 (94%) patients affected by autoimmune thyroiditis. The 1. and 2. generation TRAb assays were positive in 4 (7%) and 7 (13%) of 53 patients affected by autoimmune thyroiditis, respectively. No patients affected by nodular goiter showed positive 1. and 2. generation TRAb assay while AbTPO levels were positive in 8 of 88 patients (specificity 91%). In conclusion, the 2. generation TRAb assay is clearly more sensitive than the 1. generation test and should be used in clinical practice to minimize the incidence of TRAb-negative Graves' disease. Long term prospective studies are needed to evaluate the prognostic role of 2. generation TRAb assay in Graves' disease. The assay of AbTPO is the best marker for

  4. Development and application of a blocking enzyme-linked immunosorbent assay (ELISA) to differentiate antibodies against live and inactivated porcine reproductive and respiratory syndrome virus. (United States)

    Cong, Yanlong; Huang, Zhiqiang; Sun, Yixue; Ran, Wei; Zhu, Lisai; Yang, Guilian; Ding, Xuemei; Yang, Zhanqing; Huang, Xiao; Wang, Chunfeng; Ding, Zhuang


    The aim of this study was to establish a method that could differentiate antibodies against live and inactivated vaccines of porcine reproductive and respiratory syndrome virus (PRRSV). A blocking ELISA (b-ELISA) was established using the PRRSV non-structural protein, Nsp9, as the antigen and a monoclonal antibody, 2D6, against the Nsp9 protein as the capture antibody. The test was validated by using 415 clinical sera in the b-ELISA compared to a commercial kit based on the indirect ELISA using the nucleocapsid (N) protein as antigen. Significant differences were observed for the data obtained by the two detection methods. This may be due to the commercial kit detecting antibodies elicited by live and inactivated virus, whereas the b-ELISA only detects antibodies produced by any active viral replication, such as natural infection or live vaccination. Therefore, the b-ELISA in this study is able to distinguish between antibodies against live and inactivated viruses in pigs.

  5. [[Virus-like particle-based immunoglobulin M capture enzyme-linked immunosorbent assay for the detection of IgM antibodies against Chikungunya virus]. (United States)

    Li, Jian-dong; Zhang, Quan-fu; Zhang, Shuo; Li, Chuan; Liu, Qin-zhi; Liang, Mi-fang; Li, De-xin


    To establish a MacELISA method for the detection of IgM antibodies against Chikungunya virus (CHIKV), we prepared virus like particle (VLP) antigens of CHIKV using the whole structural protein C-E3-E2-6K-E1 encoding gene with a baculovirus expression system in Sf9 insect cells. The VLPs were purified and used to immunize Kunming mice. Then, polyclonal antibodies were purified from the samples of ascites with a protein G HiTrap SP column and labeled with horseradish peroxidase. A MacELISA method for the detection of IgM antibodies against CHIKV was assembled with goat anti-human IgM antibody, VLP antigens and an enzyme-labeled polyclonal antibody. The results were evaluated with a serum panel containing serum samples from laboratory-confirmed CHIK, HFRS patients, healthy donors, and commercially available CHIKV IgM as a quality control. It was shown that the MacELISA had a specificity of 99% (99/100), the coefficients of variation (CoV) within a plate were ELISA plates in terms of the plate variation coefficient was <15%. A comparative analysis was performed to compare the current method against a commercial CHIKV IgM antibody detection kit for IIFA-IgM. The detection limit of MacELISA was significantly lower than that of the IIFA-IgM commercial kit (P< 0.0001). Here, we demonstrate that the VLP-based MacELISA is a promising tool for the early diagnosis and epidemiological investigation of CHIKV infection, with a high level of sensitivity and specificity for the detection of IgM antibodies against CHIKV.

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

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


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

  7. Diagnostic performance of an indirect enzyme-linked immunosorbent assay (ELISA) to detect bovine leukemia virus antibodies in bulk-tank milk samples. (United States)

    Nekouei, Omid; Durocher, Jean; Keefe, Greg


    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.

  8. Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Damgaard, Dres; Palarasah, Yaseelan; Skjødt, Karsten


    The enzyme peptidylarginine deiminase 2 (PAD2) has been associated with inflammatory diseases, such as rheumatoid arthritis and neurodegenerative diseases including multiple sclerosis. To investigate the association of various diseases with extracellular PAD2, we raised monoclonal antibodies (mAb...

  9. Evaluation of a Novel Enzyme-Linked Immunosorbent Assay To Detect Immunoglobulin G Antibody to Enolase for Serodiagnosis of Invasive Candidiasis▿ (United States)

    Laín, Ana; Moragues, María D.; Ruiz, Juan Carlos García; Mendoza, Joaquín; Camacho, Ana; del Palacio, Amalia; Pontón, José


    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

  10. An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk (United States)

    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...

  11. Localization and distribution of ‘Candidatus Liberibacter asiaticus’ in citrus and periwinkle by direct tissue blot immuno assay with an anti-OmpA polyclonal antibody (United States)

    ‘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...

  12. Synthesis of an oxytetracyline-tolidin-BSA immunogen and antibodies production of anti-oxytetracyline developed for oxytetracyline residue detection with enzyme-linked immunosorbent assays technique

    Directory of Open Access Journals (Sweden)

    Widiastuti R


    Full Text Available An oxytetracycline-tolidin-bovine serum albumin (OTC-tolidin-BSA-conjugate was synthezed as immunogen for producing specific antibodies in immunized rabbits that would be used as reagent for development of OTC residue detection with enzym-linked immunoassays technique. The immunogen was prepared through diazotization tolidin and subsequently reacted with OTC. The red purple immunogen of OTC-tolidin-BSA absorbed at wave lengths of 277 nm and 488 nm under UV screening absorbances and confirmation with the high performance liquid chromatography (HPLC showed the absence of peak at retention time of 3.46 minutes. Characaterized result with SDS-PAGE showed the molecular weight of the OTC-tolidin-BSA at 69.79 kDA. Subsequently, the immunogen was immunized into New Zealand rabbits in order to produce the polyclonal antibodies. The antibodies were purified using a protein A sepharose column. The OD optimum responses of 0.92 to 1.20 were obtained from the second fractionation at dilution of 1/1000 by titrating the antibodies and OTC-tolidin-BSA coating antigen at concentration of 10 µg/mL on several bleeding times.

  13. ALS试验(TB-S A抗体)在结核性胸膜炎辅助诊断中的应用%Evaluation of ALS assay (TB-SA antibody)in diagnosis of tuberculous pleurisy

    Institute of Scientific and Technical Information of China (English)

    焦瑾; 王茂水; 吴艳华; 王新锋


    Objective To investigate the diagnostic value of the ALS assay (TB-SA antibody)in the tuberculous pleu-risy.Methods 55 patients diagnosed as tuberculous pleurisy (TPE group)and 45 healthy persons (healthy group) were enrolled.The levels of TB-SA antibody from lymphocyte secretion were detected by ELISA assay.Mann-Whitney U test was used to compare the levels of TB-SA antibody between the two groups.Receiver operating characteristic (ROC)analysis was performed to analyze the sensitivity of ALS assay (TB-SA antibody)in diagnosis of tuberculous pleurisy.Results The mean concetration of TB-SA antibody from lymphocyte secretion in TPE group was significantly higher than that in healthy group (P<0.01).The area under the curve,best cutoff point,sensitivity,specificity,posi-tive likehood ratio and negtive likehood ratio of the ALS assay (TB-SA)were 0.900 (95%CI:0.824-0.95 1 ),0.04, 77.8%,92.7%,10.69 and 0.24,respectively.Conclusion ALS (TB-SA antibody)assay performs well in the di-agnosis of tuberculous pleurisy.%目的:探讨ALS试验(TB-SA抗体)在结核性胸膜炎辅助诊断中的应用价值。方法选取初治结核性胸膜炎患者(结核组)55例和健康体检者(对照组)45例,采用ELISA法测定淋巴细胞分泌液中结核抗体的含量。采用Mann-Whitney U检验比较两组之间 TB-SA 抗体的差异,绘制受试者工作特征(ROC )曲线评价 ALS 试验(TB-SA抗体)对结核性胸膜炎诊断的敏感性。结果结核组TB-SA抗体的浓度显著高于正常组(P<0.01)。根据两组数据绘制受试者工作特征曲线(ROC曲线),曲线下面积为0.900(95%CI:0.824~0.951),截断点为0.04,敏感性和特异性分别为77.8%和92.7%,阳性似然比和阴性似然比分别为10.69和0.24。结论 ALS(TB-SA抗体)试验在结核性胸膜炎的诊断中具有一定的价值。

  14. Clinical Evaluation of BioPlex 2200 HIV Ag-Ab, an Automated Screening Method Providing Discrete Detection of HIV-1 p24 Antigen, HIV-1 Antibody, and HIV-2 Antibody


    Salmona, Maud; Delarue, Severine; Delaugerre, Constance; Simon, François; Maylin, Sarah


    Early and accurate diagnosis is essential for optimal therapeutic outcomes in patients infected with HIV. Currently, none of the commercially available fourth-generation assays differentiate HIV-1 and HIV-2 antibodies (Ab) or the HIV-1 p24 antigen (Ag). The aim of this study was to evaluate the performance of a novel assay, the BioPlex 2200 HIV Ag-Ab. This assay uses a multiplex flow immunoassay design allowing the simultaneous detection and identification of antibodies to HIV-1 (groups M and...

  15. Pseudotype-based neutralization assays for influenza: a systematic analysis

    Directory of Open Access Journals (Sweden)

    George William Carnell


    Full Text Available 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 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 towards 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 to 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 hemagglutinin inhibition (HI, single radial hemolysis (SRH and microneutralization (MN. 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.

  16. Impact of de novo donor-specific HLA antibodies detected by Luminex solid-phase assay after transplantation in a group of 88 consecutive living-donor renal transplantations. (United States)

    Dieplinger, Georg; Ditt, Vanessa; Arns, Wolfgang; Huppertz, Andrea; Kisner, Tuelay; Hellmich, Martin; Bauerfeind, Ursula; Stippel, Dirk L


    De novo donor-specific HLA antibodies (DSA) after renal transplantation are known to be correlated with poor graft outcome and the development of acute and chronic rejection. Currently, data for the influence of de novo DSA in patient cohorts including only living-donor renal transplantations (LDRT) are limited. A consecutive cohort of 88 LDRT was tested for the occurrence of de novo DSA by utilizing the highly sensitive Luminex solid-phase assay for antibody detection. Data were analyzed for risk factors for de novo DSA development and correlated with acute rejection (AR) and graft function. Patients with de novo DSA [31 (35%)] showed a trend for inferior graft function [mean creatinine change (mg/dL/year) after the first year: 0.15 DSA (+) vs. 0.02 DSA (-) (P = 0.10)] and a higher rate of AR episodes, especially in case of de novo DSA of both class I and II [6 (55%), (P = 0.05)]. Antibody-mediated rejection (AMR) appeared in five patients and was significantly correlated with de novo DSA (P = 0.05). Monitoring for de novo DSA after LDRT may help to identify patients at risk of declining renal function. Especially patients with simultaneous presence of de novo DSA class I and class II are at a high risk to suffer AR episodes.

  17. [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]. (United States)

    Ogata, Norio


    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.

  18. 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. (United States)

    Noguchi, Tomoaki; Nishida, Yuichi; Takizawa, Keiji; Cui, Yue; Tsutsumi, Koki; Hamada, Takashi; Nishi, Yoshisuke


    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

  19. 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

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou, E-mail:


    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{sup −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. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO{sub 2} particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors.

  20. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.


    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the uni

  1. Enzyme inhibition assay for pyruvate dehydrogenase complex: Clinical utility for the diagnosis of primary biliary cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Katsuhisa Omagri; Hiroaki Hazama; Shigeru Kohno


    Primary biliary cirrhosis (PBC) is usually diagnosed by the presence of characteristic histopathological features of the liver and/or antimitochondrial antibodies (AMA) in the serum traditionally detected by immunofluorescence.Recently, new and more accurate serological assays for the detection of AMA, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, and enzyme inhibition assay, have been developed. Of these,the enzyme inhibition assay for the detection of antipyruvate dehydrogenase complex (PDC) antibodies offers certain advantages such as objectivity, rapidity,simplicity, and low cost. Since this assay has almost 100% specificity, it may have particular applicability in screening the at-risk segment of the population in developing countries. Moreover, this assay could be also used for monitoring the disease course in PBC. Almost all sera of PBC-suspected patients can be confirmed for PBC or non-PBC by the combination results of immunoblotting and enzyme inhibition assay without histopathological examination. For the development of a "complete" or "gold standard" diagnostic assay for PBC, similar assays of the enzyme inhibition for anti2-oxoglutarate dehydrogenase complex (OGDC) and anti-branched chain oxo-acid dehydrogenase complex (BCOADC) antibodies will be needed in future.

  2. Enzyme-Linked Immunosorbent Assay To Differentiate the Antibody Responses of Animals Infected with Brucella Species from Those of Animals Infected with Yersinia enterocolitica O9


    Erdenebaatar, Janchivdorj; Bayarsaikhan, Balgan; Watarai, Masahisa; Makino, Sou-ichi; Shirahata, Toshikazu


    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.

  3. Enzyme-linked immunosorbent assay to differentiate the antibody responses of animals infected with Brucella species from those of animals infected with Yersinia enterocolitica O9. (United States)

    Erdenebaatar, Janchivdorj; Bayarsaikhan, Balgan; Watarai, Masahisa; Makino, Sou-ichi; Shirahata, Toshikazu


    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.

  4. Evaluation of a new antibody-based enzyme-linked immunosorbent assay for the detection of bovine leukemia virus infection in dairy cattle

    NARCIS (Netherlands)

    Monti, G.E.; Frankena, K.; Engel, B.; Buist, W.; Tarabla, H.D.; Jong, de M.C.M.


    The objective of this study was to validate a new blocking enzyme-linked immunosorbent assay (ELISA) (designated M108 for milk and S108 for serum samples) for detecting bovine leukemia virus (BLV) infection in dairy cattle. Milk, serum, and ethylenediaminetetraacetic acid-blood samples were collecte

  5. 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). (United States)

    Mentasti, M; Kese, D; Echahidi, F; Uldum, S A; Afshar, B; David, S; Mrazek, J; De Mendonça, R; Harrison, T G; Chalker, V J


    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.

  6. A survey for antibodies to equine arteritis virus in donkeys, mules and zebra using virus neutralisation (VN) and enzyme linked immunosorbent assay (ELISA). (United States)

    Paweska, J T; Binns, M M; Woods, P S; Chirnside, E D


    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.

  7. Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins. (United States)

    Rosen, Osnat; Ozeri, Eyal; Barnea, Ada; David, Alon Ben; Zichel, Ran


    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 vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p 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.

  8. Comparative analysis of antinuclear antibody spectrum by immunoblotting assays%免疫印迹法检测抗核抗体谱比对分析

    Institute of Scientific and Technical Information of China (English)

    蒋跃根; 应葳; 王峰来; 蔡秀玲; 李安生


    目的 比较抗核抗体谱-12S检测(antinuclear antibody profile,ANA-12S)试剂盒和抗核抗体谱线性免疫分析法检测(antinuclear antibody spectrum linear immunoassay,IMTEC-ANA-LIA)试剂盒对血清抗核抗体谱(antinuclear antibody spectrum, ANAs)检测结果的一致性,从而验证ANA-12S检测ANAs的有效性.方法 收集于该院就诊的间接免疫荧光法(indirect immunofluorescence,IIF)检测抗核抗体(ANA)阳性患者标本144例,其中男21例,女123例;ANA阴性患者标本10例,其中男2例,女8例.用IMTEC-ANA-LIA和ANA-12S分别进行ANAs检测.比较和分析两种方法所得的检测结果.结果 ANA阳性标本中两者检测阳性率均为100%,其中ANA-12S法检测阳性项目占28.8% (497/1 728),IMTEC-ANA-LIA法检测阳性项目占27.7%(479/1 728),差异无统计学意义(x2=0.46,P>0.05),ANA-12S法与IMTEC-ANA-LIA法检测结果总符合率为97.1%,Kappa值为0.929;ANA阴性标本中两者检测的阴性率均为70%(7/10),其中3例系统性红斑狼疮(SLE)疑似患者抗SmD1阳性,阴性项目占97.5%(117/120),总符合率100%,Kappa值为1.结论 ANA-12S与IMTEC-ANA-LIA的检测结果具有较高符合率和准确性;ANA-12S试剂盒可以作为快速、简便、经济的检测方法应用于自身抗体的检测.

  9. 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. (United States)

    Galula, Jedhan U; Chang, Gwong-Jen J; Chuang, Shih-Te; Chao, Day-Yu


    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

  10. 化学发光检测梅毒特异性抗体反应性结果分析%Reactive results of Treponema pallidum antibody in chemiluminescent assay

    Institute of Scientific and Technical Information of China (English)

    顾春瑜; 刘刚; 赵乔妹; 李少增; 于勇


    Objective To find the cause of false positive results during the detection of Treponema pallidum antibody(TP) in chemiluminescent assay. Methods We put 129 TP reactive samples to chemiluminscent assay, reexamined them by treponema pallidum hemaglutination assay (TPHA) before comparing them. Results When cut - off S/CO was 1, the difference was significant, but there was no significant difference when cut - off S/CO was 5. Conclusions When anti - TP is reactive, especially in the samples of low S/CO value(1.0 -5.0), multiple factors should be taken into consideration, such as age, history of disease, and clinical symptoms , followed by re - examination of the sample by other means. In this way, the chance of false positivity will be reduced.%目的 分析梅毒特异性抗体(treponema pallidum antibody,TP)的有反应性结果,尤其是COI值较低的标本,为减少假阳性的发生提供依据.方法 选取129例用化学发光法检测梅毒特异性抗体有反应性的术前筛查标本,用梅毒螺旋体血凝试剂盒(treponema pallidum hemaglutination assay,TPHA)进行复检,分析比对两者的结果.结果 当切点S/CO值为5时,Syphilis TP阳性为96例,阴性为33例,两种方法差异无统计学意义(P>0.05).结论 当TP有反应性时(尤其是S/CO值为1.0 ~5.0的标本),要综合考虑患者的年龄、病史,以及临床表现,可再用TPHA做复检,如两者均为阳性再报阳性,这样大大减少了假阳性的发生,降低了医疗纠纷的可能.

  11. Real time assays for quantifying cytotoxicity with single cell resolution.

    Directory of Open Access Journals (Sweden)

    Sonny C Hsiao

    Full Text Available A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC, antibody dependent cellular cytotoxicity (ADCC, and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.

  12. UltramicroELISA indirecto para la deteccion de anticuerpos totales a citomegalovirus en suero humano Indirect Ultramicroelisa assay for the detection of human antibodies to cytomegalovirus using human serum samples

    Directory of Open Access Journals (Sweden)

    Jose Laferte


    Full Text Available Se normalizó un ultramicroELISA indirecto para la detección de anticuerpos a Citomegalovirus (CMV humano (UMELISA CMV. Se determinó la concentración óptima de antígeno en 30 ug/ml, la dilución de los sueros fue de 1:40 y la dilución de trabajo del conjugado fue de 1:1500. El UMELISA CMV fue comparado con las técnicas de aglutinación de latex para anticuerpos anti-CMV (Dupont de Neumors y la inmunofluorescencia indirecta (EFT. Los resultados mostraron un alto grado de concordancia y elevada copositividad y conegatividad del UMELISA con respecto a estos dos ensayos. El método es válido para el pesquisaje de anticuerpos en banco de sangre asi como para el diagnóstico de la infección mediante sueros pareados.We have standardized an indirect ultramicro ELISA assay for detecting antibodies to human Cytomegalovirus (CMV using human serum samples (UMELISA CMV. The optimal concentration of coating antigen (30 ug/ml, serum dilution (1:40 and anti-human conjugate working dilution (1:1500, were determined by a check board titration method. The UMELISA CMV was compared with the latex agglutination test for antibodies to CMV (Dupont de Nemours and with an indiret immunofluorescent method. The results have showed the high coincidence, sensitivity and especificity of the proposed assay regarding the two methods compared with, and supporting its use either for a blood donors screening or in the serological diagnosis of this infection by paired serum samples.

  13. 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 (United States)

    Chen, Weiye; Li, Cuicui; Xie, Meimei; Bu, Zhigao


    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

  14. Development of a novel protein biochip enabling validation of immunological assays and detection of serum IgG and IgM antibodies against Treponema pallidum pathogens in the patients with syphilis. (United States)

    Huang, Na-Li; Ye, Lei; Schneider, Marion E; Du, Yi-Xin; Xu, Yuan-Hong; Fan, Li-Bin; Du, Wei-Dong


    In this study, we developed a novel protein biochip methodology that was characterized by dithiobis (succinimidyl undecanoate) (DSU) and specialized for detection of serum IgG and IgM antibodies against Treponema pallidum pathogens in the patients with syphilis, respectively. The biochips were validated by a dimension of atomic force microscope (AFM). The visualized detection limit of IgG antibody on the biochip was 0.39μg/ml. Finally, 286 serum samples from the patients with syphilis were simultaneously tested on the rTpN15-17-47 coated biochips. The results were evaluated in comparison with the assays of T. pallidum particle agglutination (TPPA) and the toluidine red unheated serum test (TRUST). The result demonstrated that the relative positive rate in the 286 patients by biochip was 99.0%, similar to that by TPPA (97.9%, P>0.05) and higher than that by TRUST, (76.2%, Ppallidum on a larger scale, but also for monitoring therapy modality of the disease.

  15. Prospective study of SEVA TB peroxidase assay for cocktail antigen and antibody in the diagnosis of Tuberculosis in suspected patients attending a tertiary care hospital located in rural area

    Institute of Scientific and Technical Information of China (English)

    Anindita Majumdar; Pranita D Kamble; CM Badole; BC Harinath


    Objective:To evaluate inhouse developed SEVA TB peroxidase enzyme immunoassay using cocktail of mycobacterial excretory-secretory antigens (ES-31, ES-43&EST-6) for antibody detection and their affinity purified antibodies for antigen detection in tuberculosis suspected patients. Methods:Inhouse developed SEVA TB peroxidase enzyme immunoassay was evaluated prospectively in 73 suspected pulmonary and 46 extra-pulmonary tuberculosis patients during November 2008~March 2009 in a tertiary hospital located in rural area. Results: Assay on prospective analysis showed 100% correlation of pulmonary tuberculosis (PTB) and extra-pulmonary tuberculosis (EPTB) acid fast bacilli positivity and antitubercular treatment in 11 cases. Thirty nine PTB and 12 EPTB cases showed negative for ELISA test and were also not given antitubercular therapy. However 30 PTB and 27 EPTB cases showing ELISA positivity were neither acid fast bacilli positive nor antitubercular therapy treated. These cases may possibly have dormant infection and need further diagnosis. In EPTB cases ELISA was observed to be more useful than AFB smear test. Conclusions:This inhouse developed user-friendly peroxidase ELISA can be used as an adjunct test of smear microscopy or culture techniques for routine screening of patients suspected of PTB or EPTB.

  16. Effect of using heat-inactivated specimens with several HIV antibody screening and confirmatory assay kits%样品热灭活对HIV抗体筛查和确证试验结果的影响研究

    Institute of Scientific and Technical Information of China (English)

    郎文文; 张桂云; 邱景富; 曹薇; 李华荣; 邱茂锋; 蒋岩


    目的 研究血浆样品经热灭活(56℃30min)后,其艾滋病病毒(HIV)抗体筛查和确证试验结果是否会受到影响.方法 取5份HIV抗体阳性血浆样品,各分为2管,其中1管进行热灭活处理,然后对这2组样品分别进行10倍系列稀释,用1种HIV抗体酶联免疫吸附试验(ELISA)试剂检测,比较S/CO比值.取300份血浆样品,分为灭活组和普通组,分别用5种HIV抗体ELISA试剂(其中第三代试剂3种、第四代试剂2种)和3种HIV抗体快速检测试剂进行筛查检测,出现筛查阳性反应的样品进一步做确证试验.结果 随着稀释度的增加,5份HIV抗体阳性样品灭活前后的ELISA检测结果(S/CO比值)都逐渐减小,直至转为阴性反应,其中4份样品灭活后比灭活前早一个稀释度转阴,1份同时转阴,提示灭活过程会略微降低HIV抗体的浓度.用5种ELISA试剂、3种快速检测试剂检测300份样品,样品灭活前后的检测结果差异均无统计学意义;对出现筛查阳性反应的所有样品,灭活前后的确证试验检测结果一致.结论 经56℃30min热灭活后,尽管HIV阳性血浆样品中的HIV抗体浓度略有降低,但对于未经稀释的常规临床血浆样品来说,热灭活处理不会明显影响目前常用筛查、确证试剂的检测结果.%Objective To study the effect of using heat-inactivated (56 ℃ , 30min) plasma specimens with several HIV antibody screening and confirmatory assay kits. Methods Five HIV antibody-positive plasma specimens were aliquoted into 2 vials, respectively. One group of them was heat-inactivated by 56 ℃, 30min. Both groups were 1 : 10 serially diluted and then detected by an ELISA kit for HIV antibody. Three hundred plasma specimens were aliquoted into 2 vials, respectively, and one group of them was heat-inactivated. Both groups were detected by 5 ELISA kits (including 3 HIV antibody and 2 HIV antigen/antibody ELISAs) and 3 HIV rapid tests, respectively. The repeat-reactive specimens

  17. Diagnosis of canine leishmaniasis in the endemic area of Belo Horizonte, Minas Gerais, Brazil by parasite, antibody and DNA detection assays. (United States)

    da Silva, E S; van der Meide, W F; Schoone, G J; Gontijo, C M F; Schallig, H D F H; Brazil, R P


    Canine leishmaniasis caused by Leishmania chagasi (L. infantum) is found throughout the South American continent, including Brazil, and dogs are considered to be the main reservoir host for this parasite. To support the implementation of a diagnostic protocol for surveillance of the disease in the region of Belo Horizonte (Minas Gerais, Brazil) we have compared the sensitivity and specificity of two serological tests, indirect immunofluorescent antibody test (IFAT) and direct agglutination test (DAT), with the combination of direct microscopy-culture-PCR as the gold standard, using samples obtained from 103 dogs in the city of Belo Horizonte, Minas Gerais. The currently used standard serodiagnostic test, IFAT, had a sensitivity of 100% and its specificity was 74% compared to the gold standard of the study. The sensitivity and specificity of the DAT were 100% and 91%, respectively. On the basis of this study it is recommended to change from the IFAT to DAT for the serodiagnosis of canine leishmaniasis because of the superior specificity of the test combined with its user-friendliness.

  18. Evaluation and Comparison of Enzyme Immunoassay (Eia and Acid Fast Staining with Confirmation by Immunofluorescent Antibody Assay for Detection of Cryptosporidium Species in Infants and Young Children.

    Directory of Open Access Journals (Sweden)

    D Dorostcar Moghaddam


    Full Text Available Introduction: Cryptosporidiosis is prevalent world wide, causing a variety of problems ranging from acute, self-limiting diarrhea to fatal cases in immunocompromised persons, particulary those with acquired immunodeficiency (AIDS. Diagnosis of Cryptosporidium is made by identification of oocysts in stool specimens. The detection is most commonly made by the acid-fast staining method followed by microscopic examination which has low specificity and sensitivity. Material and Methods: In the present study, we evaluated diagnostic utility of a commercially available enzyme immunoassay (EIA, which detects Cryptosporidium-Specific antigen (CSA in 204 unprocessed stool specimens obtained from patients less than 3 years of age. Results: When compared with the routine screening procedure applied in this field study (screening by acid-fast staining and microscopy after concentration of positive results by IFA, both sensitivity and specificity were 98%. Of the 139 specimens negative by microscopy, 13 (9.3% were positive by EIA, 11 of which were confirmed by inhibition with antibody to Cryptosporidia-specific antigen. Conclusion: The EIA is an important tool for identifying Cryptosporidium in fecal specimens in field studies since it is sensitive, specific, simple to use and unaffected by the presence of a preservative.

  19. Community Laboratory Testing for Cryptosporidium: Multicenter Study Retesting Public Health Surveillance Stool Samples Positive for Cryptosporidium by Rapid Cartridge Assay with Direct Fluorescent Antibody Testing (United States)

    Roellig, Dawn M.; Yoder, Jonathan S.; Madison-Antenucci, Susan; Robinson, Trisha J.; Van, Tam T.; Collier, Sarah A.; Boxrud, Dave; Monson, Timothy; Bates, Leigh Ann; Blackstock, Anna J.; Shea, Shari; Larson, Kirsten; Xiao, Lihua; Beach, Michael


    Cryptosporidium is a common cause of sporadic diarrheal disease and outbreaks in the United States. Increasingly, immunochromatography-based rapid cartridge assays (RCAs) are providing community laboratories with a quick cryptosporidiosis diagnostic method. In the current study, the Centers for Disease Control and Prevention (CDC), the Association of Public Health Laboratories (APHL), and four state health departments evaluated RCA-positive samples obtained during routine Cryptosporidium testing. All samples underwent “head to head” re-testing using both RCA and direct fluorescence assay (DFA). Community level results from three sites indicated that 54.4% (166/305) of Meridian ImmunoCard STAT! positives and 87.0% (67/77) of Remel Xpect positives were confirmed by DFA. When samples were retested by RCA at state laboratories and compared with DFA, 83.3% (155/186) of Meridian ImmunoCard STAT! positives and 95.2% (60/63) of Remel Xpect positives were confirmed. The percentage of confirmed community results varied by site: Minnesota, 39.0%; New York, 63.9%; and Wisconsin, 72.1%. The percentage of confirmed community results decreased with patient age; 12.5% of community positive tests could be confirmed by DFA for patients 60 years of age or older. The percentage of confirmed results did not differ significantly by sex, storage temperature, time between sample collection and testing, or season. Findings from this study demonstrate a lower confirmation rate of community RCA positives when compared to RCA positives identified at state laboratories. Elucidating the causes of decreased test performance in order to improve overall community laboratory performance of these tests is critical for understanding the epidemiology of cryptosporidiosis in the United States (US). PMID:28085927

  20. Laboratory Building for Accurate Determination of Plutonium

    Institute of Scientific and Technical Information of China (English)


    <正>The accurate determination of plutonium is one of the most important assay techniques of nuclear fuel, also the key of the chemical measurement transfer and the base of the nuclear material balance. An

  1. Circulating filarial antigen in the hydrocele fluid from individuals living in a bancroftian filariasis area - Recife, Brazil: detected by the monoclonal antibody Og4C3-assay

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    Abraham Rocha


    Full Text Available The purpose of this study was to examine the circulating filarial antigen (CFA detected by the monoclonal antibody (mAb Og4C3-ELISA in paired samples of serum and hydrocele fluid from 104 men with hydrocele, living in an endemic area of Wuchereria bancrofti. Nocturnal blood specimens were filtered and examined for microfilariae (MF and ultrasound was used in order to identify the presence of adult worms (the filaria dance sign - FDS in the lymphatic vessels of the scrotal area. Four groups were selected according to their parasitological status: group I - 71 MF- and FDS-; group II - 21 MF+ and FDS+; group III - 10 MF- and FDS+ and group IV- 2 MF+ and FDS-. CFA was identified simultaneously (fluid and serum in 11 (15.5%, 21 (100%, 3 (30%, and 1 (50% in groups I, II, III, and IV, respectively. In despite of high CFA+ level (antigen Og4C3 units/ml, the Geometrical Mean (GM = 2696 in the sera of these 36/104 paired samples, when compared to the hydrocele fluid, (GM = 1079, showed a very good correlation between the CFA level in the serum and CFA level in the fluid (r = 0.731. CFA level in the serum of the 23 microfilaremics (groups II and IV was extremely high (GM = 4189 and was correlated with MF density (r = 0.442. These findings report for the first time the potential alternative use of the hydrocele fluid to investigate CFA using the mAb Og4C3-ELISA.

  2. Development of a highly-sensitive multi-plex assay using monoclonal antibodies for the simultaneous measurement of kappa and lambda immunoglobulin free light chains in serum and urine. (United States)

    Campbell, John P; Cobbold, Mark; Wang, Yanyun; Goodall, Margaret; Bonney, Sarah L; Chamba, Anita; Birtwistle, Jane; Plant, Timothy; Afzal, Zaheer; Jefferis, Roy; Drayson, Mark T


    Monoclonal κ and λ immunoglobulin free light chain (FLC) paraproteins in serum and urine are important markers in the diagnosis and monitoring of B cell dyscrasias. Current nephelometric and turbidimetric methods that use sheep polyclonal antisera to quantify serum FLC have a number of well-observed limitations. In this report, we describe an improved method using specific mouse anti-human FLC monoclonal antibodies (mAbs). Anti-κ and anti-λ FLC mAbs were, separately, covalently coupled to polystyrene Xmap® beads and assayed, simultaneously, in a multi-plex format by Luminex® (mAb assay). The mAbs displayed no cross-reactivity to bound LC, the alternate LC type, or other human proteins and had improved sensitivity and specificity over immunofixation electrophoresis (IFE) and Freelite™. The assay gives good linearity and sensitivity (<1 mg/L), and the competitive inhibition format gave a broad calibration curve up to 437.5 mg/L and prevented anomalous results for samples in antigen excess i.e. high FLC levels. The mAbs displayed good concordance with Freelite™ for the quantitation of normal polyclonal FLC in plasma from healthy donors (n=249). The mAb assay identified all monoclonal FLC in serum from consecutive patient samples (n=1000; 50.1% with monoclonal paraprotein by serum IFE), and all FLC in a large cohort of urine samples tested for Bence Jones proteins (n=13090; 22.8% with monoclonal κ, 9.0% with monoclonal λ, and 0.8% with poly LC detected by urine IFE). Importantly this shows that the mAbs are at least close to the ideal of detecting FLC from all patients and neoplastic plasma cell clones. Given the overall effectiveness of the anti-FLC mAbs, further clinical validation is now warranted on serial samples from a range of patients with B cell disorders. Use of these mAbs on other assay platforms should also be investigated.

  3. Evaluation of three commercial enzyme-linked immunosorbent assays for the detection of antibodies against Salmonella spp. in meat juice from finishing pigs in Spain. (United States)

    Vico, J P; Engel, B; Buist, W G; Mainar-Jaime, R C


    The control of animal salmonellosis is considered as a major objective in Europe and indirect ELISAs will be important tools for the implementation of control programs for this infection in pigs. We analyse the results yielded by three commercial ELISAs (Herdcheck Swine Salmonella, SALMOTYPE Pig Screen, and PrioCHECK Salmonella) on meat juice samples from a population of slaughter pigs of Aragon, NW Spain, to assess their efficacy using traditional and latent-class approaches. Overall, the Herdcheck Swine Salmonella detected more Salmonella-infected pigs than the other two tests, but its relative sensitivity was low (65.9%). A similar result was observed when only serotypes detectable by this test were considered (69.1%). When a Bayesian approach was used the Herdcheck Swine Salmonella showed also the highest overall accuracy (sensitivity = 88% and specificity = 74%). Our results suggest that a relatively small proportion of the observed prevalence in herds would be explained by using these ELISAs. Also, this study points out that when different ELISA tests are used within the same herd, results may differ substantially. Thus, caution is advised if it is decided to use these assays for herd health classification in Spanish Salmonella control programs.

  4. Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of serum antibodies against Akabane virus in cattle. (United States)

    Kittelberger, Reinhold; McFadden, Andrew M J; Kirkland, Peter D; Hannah, Michaela J; Orr, Della; Bueno, Rudolfo; Swainsbury, Richard; Keen, Denise; Jenner, Judy; French, Jennifer; Pigott, Clive J


    In New Zealand, an arbovirus surveillance program has been operating for more than 20 years, which includes testing of cattle with the Akabane virus neutralization test. With the aim to replace this laborious test by an easier-to-perform enzyme-linked immunosorbent assay (ELISA), 2 commercial ELISA kits, ELISA-1 from France (originally from Australia) and ELISA-2 from Japan, were compared, using 334 serum samples from noninfected New Zealand cattle, and 548 serum samples from naturally infected cattle herds in Australia. Diagnostic specificities for the test methods were high, ranging from 99.4% to 100%. The diagnostic sensitivities varied considerably between the test methods and differed from the values reported by the manufacturers (94% for each ELISA). The diagnostic sensitivities relative to the virus neutralization test (n = 378) were 96.0% for ELISA-1 or 98.9% when suspect samples were included, and 78.0% for ELISA-2. Differences in the commercial ELISA kits may be explained by the presence of other Simbu serogroup viruses in Australian cattle herds, causing cross-reactions in ELISA-1. Both commercial ELISA kits would be fit for purpose and could replace the virus neutralization test for Akabane virus surveillance in New Zealand. ELISA-1 may be able to detect other Simbu serogroup viruses, should they be present. The current study shows that despite comparable ELISA test characteristics given by the manufacturers, evaluation on the target population revealed marked differences in the ELISA kits test methods' characteristics.

  5. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas


    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  6. 基因重组HCV抗原斑点酶免疫法检测血清中抗-HCV%Immunoblotting Assay Using Recombinant Antigens for Detecting Antibodies against HCV

    Institute of Scientific and Technical Information of China (English)

    王百锁; 胡兴菊; 向前; 邢爱华; 王敬军


    目的 建立检测抗-HCV的斑点酶免疫法。方法 将基因重组HCV核心、NS3/NS4、NS5抗原和马抗人IgG、人IgG以最佳浓度固定在硝酸纤维膜条带上,用辣根过氧化酶和改进的底物进行斑点酶免疫反应。结果 检测了14份经ORTHO第三代抗-HCVELISA检测的血清,符合率为100%;72份经UBI第三代抗-HCVELISA监测的血清,符合率95.83%;103份经国产厦门新创抗-HCVELISA监测的血清,符合率95.14%。54份自然人群血清,阳性率3.47%。89份阳性标本中,三种抗体同时阳性的占30.34%,两种抗体阳性占69.66%,没有检出单一抗体阳性标本。核心抗体检出率最高87.64%,NS3/NS4抗体次之76.40%,NS5抗体最低61.80%。结论 用基因重组抗原建立的斑点酶免疫法与国内外广泛应用的ELISA试剂有较高的符合率,对进一步研究HCV感染诊断的确认试剂有意义。%Objective To set up a configuration of immunoblo tting assay forcletecting serum antibodies against HCV.Methods The proteins of the said virus that Core and NS3/NS4 and NS5 expres sed in E.coli cells were immobilized separately on nitrocellulose filter as antigens.HRP and a modified preparation of chromogenic substrate were used in the following procedure.Results Among 189 samples of sera tested by this Blotting Assay,14 were tested also by using ORTHO 3rd Generation ELISA regent and the coincident rate was 100% between the two assays,72 were tested by UBI 3rd Generation ELISA and their coincident rate was 95.83%,103 were tested by Xia Meng Xin Chuang ELISA and the coincident rate was 95.14%,Among 89 posi tive sera determined by Blotting Assay,those showed positive for all three antig ens accounted for 30.34%,and those positive for two of three antigens accounted for 69.66%,positive for only one antigen was not found.Among the 3 antibodies of Core was most efficient that 78 out of 89 positive sera reacted to it,the next was NS3/NS4with that number of 68

  7. Evaluation of a new antibody-based enzyme-linked immunosorbent assay for the detection of bovine leukemia virus infection in dairy cattle. (United States)

    Monti, Gustavo E; Frankena, Klaas; Engel, Bas; Buist, Willem; Tarabla, Héctor D; de Jong, Mart C M


    The objective of this study was to validate a new blocking enzyme-linked immunosorbent assay (ELISA) (designated M108 for milk and S108 for serum samples) for detecting bovine leukemia virus (BLV) infection in dairy cattle. Milk, serum, and ethylenediaminetetraacetic acid-blood samples were collected from 524 adult Holstein cows originating from 6 dairy herds in Central Argentina. The M108 and S108 were compared with agar gel immunodiffusion (AGID), polymerase chain reaction and a commercial ELISA. Because there is currently no reference test capable of serving as a gold standard, the test sensitivity (SE) and specificity (SP) were evaluated by the use of a latent class model. Statistical inference was performed by classical maximum likelihood and by Bayesian techniques. The maximum-likelihood analysis was performed assuming conditional independence of tests, whereas the Bayesian approach allowed for conditional dependence. No clear conclusion could be drawn about conditional dependence of tests. Results with maximum likelihood (under conditional independence) and posterior Bayes (under conditional dependence) were practically the same. Conservative estimates of SE and SP (with 95% confidence intervals) for M108 were 98.6 (96.7; 99.6) and 96.7 (92.9; 98.8) and for S108 99.5 (98.2; 99.9) and 95.4 (90.9; 98.1), respectively. The ELISA 108 using either milk or serum to detect BLV-infected animals had comparable SE and SP with the official AGID and a commercial ELISA test, which are currently the most widely accepted tests for the serological diagnosis of BLV infection. Therefore, ELISA 108 can be used as an alternative test in monitoring and control programs.

  8. Antimitochondrial antibody (United States)

    ... page: // Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  9. Evaluation of the Architect Epstein-Barr Virus (EBV) viral capsid antigen (VCA) IgG, VCA IgM, and EBV nuclear antigen 1 IgG chemiluminescent immunoassays for detection of EBV antibodies and categorization of EBV infection status using immunofluorescence assays as the reference method. (United States)

    Corrales, Isabel; Giménez, Estela; Navarro, David


    Commercial immunoassays for detecting IgG and IgM antibodies against Epstein-Barr virus (EBV), viral capsid antigens (VCA), and IgGs toward EBV nuclear antigen-1 (EBNA-1) are routinely used in combination to categorize EBV infection status. In this study, we evaluated the performances of the Architect EBV VCA IgG, VCA IgM, and EBNA-1 IgG chemiluminescent microparticle assays (CMIAs) in EBV serological analyses using indirect immunofluorescence assays and anticomplement immunofluorescence assays as the reference methods for VCA IgG, VCA IgM, and EBNA-1 IgG antibody detection, respectively. A total of 365 serum samples representing different EBV serological profiles were included in this study. The κ values (concordances between the results) obtained in the Architect CMIA and those in the reference assays were 0.905 (P EBV infection, and 92.42% and 97.82% for diagnosing the absence of an EBV infection. In summary, we demonstrated that the Architect EBV antibody panel performs very well for EBV antibody detection and correctly categorizes clinically relevant EBV infection states.

  10. The diagnostic performance of an antibody enzyme-linked immunosorbent assay using serum and colostrum to determine the disease status of a Jersey dairy herd infected with Mycobacterium avium subspecies paratuberculosis. (United States)

    Jenvey, Caitlin J; Reichel, Michael P; Cockcroft, Peter D


    Colostrum may have the ability to improve the diagnostic accuracy of some tests when compared to serum for important livestock diseases because of the high concentrations of immunoglobulins present within this sample type. The ELISA for Johne's disease is one such test, as it suffers from low sensitivity when testing serum samples collected during the subclinical stage of infection. Blood and colostrum samples were collected from 34 Jersey dairy cows and tested for antibodies against Mycobacterium avium subspecies paratuberculosis (MAP) by ELISA. Fecal samples were also collected and tested by a high-throughput Johne's polymerase chain reaction (HT-J PCR) assay and fecal culture (FC), with the latter being used as the reference test. A receiver operating characteristic (ROC) analysis was performed, and the area under the curve (AUC) was calculated. The HT-J PCR and FC results were also compared. Of the 34 cows in this study, 4 had FC results consistent with MAP infection. The HT-J PCR did not identify any FC-positive cows. Using a 1:20 dilution and sample-to-positive (S/P) ratio cutoff threshold of 0.15, the relative sensitivity values of both serum (AUC 0. 56) and colostrum (AUC 0.63) were 0%. With lower sample dilutions, the relative sensitivity values of serum were 0% (1:2, AUC 0.62; 1:5, AUC 0.55); however, the relative sensitivity value of colostrum was 75% (95% confidence interval [CI]: 19-99%) at a dilution of 1:5, S/P ratio cutoff threshold of 0.15, and AUC of 0.73 (95% CI: 0.55-0.87). The testing of colostrum samples for MAP-specific antibodies by ELISA may provide improved identification of animals in the early stages of infection with MAP when compared with serum samples.

  11. Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016 (United States)

    Steinhagen, Katja; Probst, Christian; Radzimski, Christiane; Schmidt-Chanasit, Jonas; Emmerich, Petra; van Esbroeck, Marjan; Schinkel, Janke; Grobusch, Martin P; Goorhuis, Abraham; Warnecke, Jens M; Lattwein, Erik; Komorowski, Lars; Deerberg, Andrea; Saschenbrecker, Sandra; Stöcker, Winfried; Schlumberger, Wolfgang


    Serological diagnosis of Zika virus (ZIKV) infections is challenging due to high cross-reactivity between flaviviruses. We evaluated the diagnostic performance of a novel anti-ZIKV ELISA based on recombinant ZIKV non-structural protein 1 (NS1). Assay sensitivity was examined using sera from 27 patients with reverse transcription (RT)-PCR-confirmed and 85 with suspected ZIKV infection. Specificity was analysed using sera from 1,015 healthy individuals. Samples from 252 patients with dengue virus (n = 93), West Nile virus (n = 34), Japanese encephalitis virus (n = 25), chikungunya virus (n = 19) or Plasmodium spp. (n = 69) infections and from 12 yellow fever-vaccinated individuals were also examined. In confirmed ZIKV specimens collected ≥ 6 days after symptom onset, ELISA sensitivity was 58.8% (95% confidence interval (CI): 36.0–78.4) for IgM, 88.2% (95% CI: 64.4–98.0) for IgG, and 100% (95% CI: 78.4–100) for IgM/IgG, at 99.8% (95% CI: 99.2–100) specificity. Cross-reactivity with high-level dengue virus antibodies was not detected. Among patients with potentially cross-reactive antibodies anti-ZIKV positive rates were 0.8% (95% CI: 0–3.0) and 0.4% (95% CI: 0–2.4) for IgM and IgG, respectively. Providing high specificity and low cross-reactivity, the NS1-based ELISA has the potential to aid in counselling patients, pregnant women and travellers after returning from ZIKV-endemic areas. PMID:28006649

  12. High throughput functional assays of the variant antigen PfEMP1 reveal a single domain in the 3D7 Plasmodium falciparum genome that binds ICAM1 with high affinity and is targeted by naturally acquired neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Andrew V Oleinikov


    Full Text Available Plasmodium falciparum-infected erythrocytes bind endothelial receptors to sequester in vascular beds, and binding to ICAM1 has been implicated in cerebral malaria. Binding to ICAM1 may be mediated by the variant surface antigen family PfEMP1: for example, 6 of 21 DBLbetaC2 domains from the IT4 strain PfEMP1 repertoire were shown to bind ICAM1, and the PfEMP1 containing these 6 domains are all classified as Group B or C type. In this study, we surveyed binding of ICAM1 to 16 DBLbetaC2 domains of the 3D7 strain PfEMP1 repertoire, using a high throughput Bioplex assay format. Only one DBL2betaC2 domain from the Group A PfEMP1 PF11_0521 showed strong specific binding. Among these 16 domains, DBL2betaC2(PF11_0521 best preserved the residues previously identified as conserved in ICAM1-binding versus non-binding domains. Our analyses further highlighted the potential role of conserved residues within predominantly non-conserved flexible loops in adhesion, and, therefore, as targets for intervention. Our studies also suggest that the structural/functional DBLbetaC2 domain involved in ICAM1 binding includes about 80 amino acid residues upstream of the previously suggested DBLbetaC2 domain. DBL2betaC2(PF11_0521 binding to ICAM1 was inhibited by immune sera from east Africa but not by control US sera. Neutralizing antibodies were uncommon in children but common in immune adults from east Africa. Inhibition of binding was much more efficient than reversal of binding, indicating a strong interaction between DBL2betaC2(PF11_0521 and ICAM1. Our high throughput approach will significantly accelerate studies of PfEMP1 binding domains and protective antibody responses.

  13. Characterization of Antibodies for Grain-Specific Gluten Detection. (United States)

    Sharma, Girdhari M; Rallabhandi, Prasad; Williams, Kristina M; Pahlavan, Autusa


    Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation.

  14. Colorimetric protein assay techniques. (United States)

    Sapan, C V; Lundblad, R L; Price, N C


    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  15. Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin-DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody. (United States)

    Dou, Shuping; Virostko, John; Greiner, Dale L; Powers, Alvin C; Liu, Guozheng


    Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)⊃-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)⊃-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to ∼95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)⊃-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in

  16. Recombinant renewable polyclonal antibodies. (United States)

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M


    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  17. Comparison of immunofluorescence and enzyme-linked immunosorbent assay and immunoglobulin G avidity techniques for screening of anti: Toxoplasma antibodies among single serum sample pregnant women in Tabriz, Iran

    Directory of Open Access Journals (Sweden)

    Mehrangiz Rajaii


    Full Text Available Background: Congenital toxoplasmosis is that pregnant women acquire the infection during gestation; diagnosis of the acute infection during pregnancy is a complex subject of maternal toxoplasmosis. Thus, the presence of immunoglobulin G (IgG and/or IgM Toxoplasma antibodies in a single serum sample drawn during gestation cannot be used to define whether the infection was recently acquired or chronic. Materials and Methods: At this cross-sectional descriptive study, sera of 391 pregnant women examined and compared. They were in an age range of 21-35 years, referred by gynecologists and infectious disease specialists, during March 2012-April 2013. They have referred, 215 (54.98%, 102 (26%, 74 (18.92% in the first, second and third trimesters of gestation, respectively. For each of them, a questionnaire was completed and serum samples were prepared in an equal condition, examined according to the procedures of indirect immunofluorescence (IIF, enzyme-linked immunosorbent assay (ELISA and IgG Avidity techniques. Results: We have found 111 (28.38% seronegative and 280 (71.61% seropositive cases by IIF and 124 (31.70% seronegative, 267 (68.28% seropositive cases by ELISA. The IgG avidity test confirmed 45 (69.23% and 7 (10.76% doubtful cases of IgM test in IIF and ELISA techniques. Conclusions: This study highlights how to manage pregnant women with toxoplasmosis, especially in a single serum sample condition.

  18. Artificial antigen synthesis and the development of polyclonal antibody-based immunoassay for citreoviridin determination. (United States)

    Zhuang, Zhen Hong; Que, Shan Jin; Gao, Yue Ming; Yuan, Jun; Ye, Zhou; Du, Min; Lin, Guang Mei; Liu, Li Cai; Wang, Shi Hua


    Citreoviridin, a mycotoxin produced by Penicillium citreonigrum is a common contaminant of wide range of agri-products and detrimental to human and animal health. Therefore it is important to develop a rapid, sensitive, and specific immunoassay for citreoviridin detection. In this study, polyclonal antibody against citreoviridin was developed. For the preparation of citreoviridin-bovine serum albumin conjugate (CIT-BSA), hydroxyl groups on adjacent carbon atoms were oxidized by sodium periodate, so the product with reactive aldehyde residues was suitable for coupling with amine. Anti-citreoviridin polyclonal antibody was prepared by immunizing mice with CIT-BSA conjugate. The specificity and sensitivity of the polyclonal antibody was determined by indirect competitive ELISA. Results showed that the IC50 value of the polyclonal antibody was 0.56 μg/mL and no cross-reactivity was found between antiserum and other mycotoxins used in the experiment. The citreoviridin recovery rates by this polyclonal antibody were calculated through rice powder spiked by artificial citreoviridin. The recovery rates ranged were found from 70.5 ± 0.08 % to 94.7 ± 0.09% for inter-assay, and from 77.5 ± 0.04% to 95.4 ± 0.18% for intra-assay, which indicated that this polyclonal antibody could detect trace amount of CIT from the tested samples. Consequently, this study provided a specific and sensitive anti-citreoviridin polyclonal antibody, which made the determination of citreoviridin easier, quicker, and more accurate.

  19. Barcoded microchips for biomolecular assays. (United States)

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu


    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  20. C1Q Assay Results in Complement-Dependent Cytotoxicity Crossmatch Negative Renal Transplant Candidates with Donor-Specific Antibodies: High Specificity but Low Sensitivity When Predicting Flow Crossmatch

    Directory of Open Access Journals (Sweden)

    José M. Arreola-Guerra


    Full Text Available The aim of the present study was to describe the association of positive flow cross match (FXM and C1q-SAB. Methods. In this observational, cross-sectional, and comparative study, patients included had negative AHG-CDC-XM and donor specific antibodies (DSA and were tested with FXM. All pretransplant sera were tested with C1q-SAB assay. Results. A total of 50 donor/recipient evaluations were conducted; half of them had at least one C1q+ Ab (n=26, 52%. Ten patients (20.0% had DSA C1q+ Ab. Twenty-five (50% FXMs were positive. Factors associated with a positive FXM were the presence of C1q+ Ab (DSA C1q+ Ab: OR 27, 2.80–259.56, P=0.004, and no DSA C1q+ Ab: OR 5, 1.27–19.68, P=0.021 and the DSA LABScreen-SAB MFI (OR 1.26, 95% CI 1.06–1.49, P=0.007. The cutoff point of immunodominant LABScreen SAB DSA-MFI with the greatest sensitivity and specificity to predict FXM was 2,300 (sensitivity: 72% and specificity: 75%. For FXM prediction, DSA C1q+ Ab was the most specific (95.8%, 85–100 and the combination of DSA-MFI > 2,300 and C1q+ Ab was the most sensitive (92.0%, 79.3–100. Conclusions. C1q+ Ab and LABScreen SAB DSA-MFI were significantly associated with FXM. DSA C1q+ Ab was highly specific but with low sensitivity.

  1. Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross-reacting antibodies. (United States)

    Gown, A M; Vogel, A M


    Monoclonal antibodies were generated against the intermediate filament proteins of different human cells. The reactivity of these antibodies with the different classes of intermediate filament proteins was determined by indirect immunofluorescence on cultured cells, immunologic indentification on SDS polyacrylamide gels ("wester blot" experiments), and immunoperoxidase assays on intact tissues. The following four antibodies are described: (a) an antivimentin antibody generated against human fibroblast cytoskeleton; (b), (c) two antibodies that recognize a 54-kdalton protein in human hepatocellular carcinoma cells; and (d) an antikeratin antibody made to stratum corneum that recognizes proteins of molecular weight 66 kdaltons and 57 kdaltons. The antivimentin antibody reacts with vimentin (58 kdaltons), glial fibrillary acidic protein (GFAP), and keratins from stratum corneum, but does not recognize hepatoma intermediate filaments. In immunofluorescence assays, the antibody reacts with mesenchymal cells and cultured epithelial cells that express vimentin. This antibody decorates the media of blood vessels in tissue sections. One antihepatoma filament antibody reacts only with the 54 kdalton protein of these cells and, in immunofluorescence and immunoperoxidase assays, only recognizes epithelial cells. It reacts with almost all nonsquamous epithelium. The other antihepatoma filament antibody is much less selective, reacting with vimentin, GFAP, and keratin from stratum corneum. This antibody decorates intermediate filaments of both mesenchymal and epithelial cells. The antikeratin antibody recognizes 66-kdalton and 57-kdalton proteins in extracts of stratum corneum and also identifies proteins of similar molecular weights in all cells tested. However, by immunofluorescence, this antibody decorates only the intermediate filaments of epidermoid carcinoma cells. When assayed on tissue sections, the antibody reacts with squamous epithelium and some, but not all

  2. Magnetic bead-based phage anti-immunocomplex assay (PHAIA) for the detection of the urinary biomarker 3-phenoxybenzoic acid to assess human exposure to pyrethroid insecticides. (United States)

    Kim, Hee-Joo; Ahn, Ki Chang; González-Techera, Andrés; González-Sapienza, Gualberto G; Gee, Shirley J; Hammock, Bruce D


    Noncompetitive immunoassays are advantageous over competitive assays for the detection of small molecular weight compounds. We recently demonstrated that phage peptide libraries can be an excellent source of immunoreagents that facilitate the development of sandwich-type noncompetitive immunoassays for the detection of small analytes, avoiding the technical challenges of producing anti-immunocomplex antibody. In this work we explore a new format that may help to optimize the performance of the phage anti-immunocomplex assay (PHAIA) technology. As a model system we used a polyclonal antibody to 3-phenoxybenzoic acid (3-PBA) and an anti-immunocomplex phage clone bearing the cyclic peptide CFNGKDWLYC. The assay setup with the biotinylated antibody immobilized onto streptavidin-coated magnetic beads significantly reduced the amount of coating antibody giving identical sensitivity (50% saturation of the signal (SC(50))=0.2-0.4ng/ml) to the best result obtained with direct coating of the antibody on ELISA plates. The bead-based assay tolerated up to 10 and 5% of methanol and urine matrix, respectively. This assay system accurately determined the level of spiked 3-PBA in different urine samples prepared by direct dilution or clean-up with solid-phase extraction after acidic hydrolysis with overall recovery of 80-120%.

  3. Cell viability assays: introduction. (United States)

    Stoddart, Martin J


    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  4. A novel enzyme-linked immunosorbent assay for quantitative detection of anti-thyroid peroxidase antibodies in serum%血清抗甲状腺过氧化物酶抗体ELISA定量方法的建立

    Institute of Scientific and Technical Information of China (English)

    孙颖; 李会强; 陈寅; 仁杰; 李婵


    Objective To establish a novel enzyme-linked immunosorbent assay (ELISA) for quantitative detection of the concentra tion of anti-thyroid peroxidase antibody (TPOAb) in serum. Methods The microtiter plate was coated with biotinylated bovine serum albumin (BSA) and streptavidin. The biotinylated TPO antigen and standardized anti-TPOAb or test sera were successively added into the wells of plate. The HRP-anti-IgG was then added into the plate for colorization. The optimal concentrations of biotinylated TPO an tigen and HRP-anti-IgG were screened by chessboard titration, and the reaction conditions were optimized for the method evaluation. Results In the indirect-coating mode, the amount of coated antigen was 0. 083 μg/mL. The sensitivity of the assay was 0. 165 IU/mL. The coefficient of variations (CV) of inter-assay in high and low concentration of serum mixture were 9.2% and 9.0% , and the CV of intra-assay were 4.6% and 5.6% respectively. The recovery rate was between 96% and 104%. The coated ELISA plate re mained stable for 5 days at 37 ℃. The rate of cross-reaction with anti-thyroid globulin (TGAb) was 0. 22%. The reference range in serum was less than 65. 7 IU/mL. The correlation coefficient of the experimental results with those of Abbott kit was 0. 985 (P < 0.01). Conclusion In the developed ELISA of indirect-coated mode, the amount of needed purified antigen significantly reduced. The sensitivity and specificity of the assay were satisfied. The method is simple and cost-saving, so it should be very suitable for anti TPOAb detection in primary hospitals.%目的 建立一种ELISA方法用于定量分析血清抗甲状腺过氧化物酶(TPO)抗体(TPOAb).方法 用生物素化牛血清清蛋白(BSA)和链霉亲合素包被微孔板,同时加入生物素化TPO抗原和待检血清,再加入酶标记抗人IgG,建立间接包被模式酶联免疫法测定抗TPO抗体.经方阵滴定确定生物素化抗原和酶标抗体的最适浓度,优化反应条件,

  5. High-throughput epitope binning assays on label-free array-based biosensors can yield exquisite epitope discrimination that facilitates the selection of monoclonal antibodies with functional activity.

    Directory of Open Access Journals (Sweden)

    Yasmina Noubia Abdiche

    Full Text Available Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs. In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic

  6. Preexisting Antibodies to an F(ab′2 Antibody Therapeutic and Novel Method for Immunogenicity Assessment

    Directory of Open Access Journals (Sweden)

    Jane Ruppel


    Full Text Available Anti-therapeutic antibodies (ATAs may impact drug exposure and activity and induce immune complex mediated toxicity; therefore the accurate measurement of ATA is important for the analysis of drug safety and efficacy. Preexisting ATAs to the hinge region of anti-Delta like ligand 4 (anti-DLL4 F(ab′2, a potential antitumor therapeutic, were detected in cynomolgus monkey serum, which presented a challenge in developing assays for detecting treatment induced ATA. A total ATA assay was developed using a bridging ELISA that detected both anti-CDR and anti-framework ATA including anti-hinge reactivity. A competition assay that could detect 500 ng/mL of anti-CDR ATA in the presence of preexisting ATA was also developed to determine ATA specific to the anti-DLL4 F(ab′2 CDR using anti-DLL4 F(ab′2 and a control F(ab′2. We used these assay methods in a cynomolgus monkey in vivo study to successfully evaluate total and anti-CDR ATA. The preexisting anti-hinge reactivity was also observed to a lesser extent in human serum, and a similar approach could be applied for specific immunogenicity assessment in clinical trials.

  7. Antibody microarrays for native toxin detection. (United States)

    Rucker, Victor C; Havenstrite, Karen L; Herr, Amy E


    We have developed antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aureus enterotoxin B, and tetanus toxin C fragment in spiked samples. Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) a competition assay that employed unlabeled toxins as reporters for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending from the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Sigmoidal calibration curves and detection limits were established for both assay formats. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples.

  8. Methods and devices for protein assays (United States)

    Chhabra, Swapnil; Cintron, Jose M.; Shediac, Renee


    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  9. Enzyme assays. (United States)

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie


    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  10. Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA (United States)

    Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi


    Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

  11. Varicella-zoster virus-specific, cell-mediated immunity with interferon-gamma release assay after vaccination of college students with no or intermediate IgG antibody response. (United States)

    Terada, Kihei; Itoh, Yuri; Fujii, Akihide; Kitagawa, Seiko; Ogita, Satoko; Ouchi, Kazunobu


    This study measured Varicella-zoster virus (VZV) specific cell-mediated immunity (CMI) and antibodies to clarify immune response after vaccination in 68 college students with negative or intermediate IgG antibody status. The enrolled numbers of negative, intermediate, and positive VZV-IgG antibody were 27, 41, and 28 students, respectively. The positive rates of CMI were 3.7% (1/27), 41.5% (17/41), and 96.4% (27/28) before vaccination, respectively. After vaccination, the IgG antibody titers became significantly higher in the intermediate IgG group compared to those in the negative IgG group (P vaccination (P vaccination, the IgG antibody and interferon-gamma values increased significantly in the positive CMI group compared to the negative CMI group after vaccination (P vaccination (P vaccination (P history of vaccination became IgG seropositive after a second dose of vaccination, but 22% (5/23) of them remained negative for CMI. CMI is valuable information to identify potential non-responders to vaccination and to predict risk of clinical VZV infection.

  12. Radioimmunoprecipitation polyethylene glycol assay for circulating Entamoeba histolytica antigens

    Energy Technology Data Exchange (ETDEWEB)

    Pillai, S.; Mohimen, A.; Mehra, S. (Calcutta Medical Research Inst., Calcutta (India). Kothari Centre of Gastroenterology)


    An assay capable of detecting circulating Entamoeba histolytica antigens in amoebiasis is described. This assay utilised a radiolabelled affinity purified rabbit anti-E. histolytica antibody that had been depleted of antibodies that cross-react with human serum proteins, and a polyethylene glycol precipitation step.

  13. Speaking Fluently And Accurately

    Institute of Scientific and Technical Information of China (English)



    Even after many years of study,students make frequent mistakes in English. In addition, many students still need a long time to think of what they want to say. For some reason, in spite of all the studying, students are still not quite fluent.When I teach, I use one technique that helps students not only speak more accurately, but also more fluently. That technique is dictations.

  14. Evaluation of the automated ADVIA centaur® XP syphilis assay for serological testing. (United States)

    Saw, Sharon; Zhao, Huiqin; Tan, Phyllis; Saw, Betty; Sethi, Sunil


    We evaluated the performance of the ADVIA Centaur XP Syphilis assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) using samples previously tested on the ARCHITECT i4000SR system (Abbott Diagnostics, Lake Forest, IL, USA) and confirmed by the Treponema pallidum particle agglutination assay (TPPA) (SERODIA-TPPA, Fujirebio Diagnostics Inc., Malvern, PA, USA). Clinical patient information was included to aid resolution of discordant samples where available. Precision, interference, and cross-reactivity were also assessed. Relative to patient clinical status, the sensitivity of both the ADVIA Centaur XP and the ARCHITECT assays was 100% (95% CI, 93.9-100), and the specificity of the ADVIA Centaur XP assay was 95.5% (95% CI, 90.4-98.3), which was slightly higher than that of the ARCHITECT assay at 93.9% (95% CI, 88.4-97.3). Overall agreement relative to patient clinical status was 96.9% (95% CI, 93.3-98.8) for the ADVIA Centaur XP assay and 95.8% (95% CI, 91.9-98.2) for the ARCHITECT assay. Overall agreement between the two automated assays was 96.9% (95% CI, 93.3-98.8). ADVIA Centaur XP assay precision was <5% at all index values tested. No significant interference was observed for lipemia or hemolysis; a small effect was seen with some samples for bilirubin. The assay exhibited no significant cross-reactivity with a number of potential interfering factors. The ADVIA Centaur XP Syphilis assay can be considered a sensitive and accurate assay for identification of treponemal antibodies in screening populations as well as patients presenting with suspicion of syphilitic infection.

  15. Thyroid Antibodies (United States)

    ... e.g., at regular intervals after thyroid cancer treatment) Thyroid stimulating hormone receptor antibody, Thyroid Stimulating Immunoglobulin TRAb, TSHR Ab, TSI Graves disease When a person has symptoms of hyperthyroidism If a pregnant woman has a known autoimmune ...

  16. Anti-DNA antibodies--quintessential biomarkers of SLE. (United States)

    Pisetsky, David S


    Antibodies that recognize and bind to DNA (anti-DNA antibodies) are serological hallmarks of systemic lupus erythematosus (SLE) and key markers for diagnosis and disease activity. In addition to common use in the clinic, anti-DNA antibody testing now also determines eligibility for clinical trials, raising important questions about the nature of the antibody-antigen interaction. At present, no 'gold standard' for serological assessment exists, and anti-DNA antibody binding can be measured with a variety of assay formats, which differ in the nature of the DNA substrates and in the conditions for binding and detection of antibodies. A mechanism called monogamous bivalency--in which high avidity results from simultaneous interaction of IgG Fab sites with a single polynucleotide chain--determines anti-DNA antibody binding; this mechanism might affect antibody detection in different assay formats. Although anti-DNA antibodies can promote pathogenesis by depositing in the kidney or driving cytokine production, they are not all alike, pathologically, and anti-DNA antibody expression does not necessarily correlate with active disease. Levels of anti-DNA antibodies in patients with SLE can vary over time, distinguishing anti-DNA antibodies from other pathogenic antinuclear antibodies. Elucidation of the binding specificities and the pathogenic roles of anti-DNA antibodies in SLE should enable improvements in the design of informative assays for both clinical and research purposes.

  17. Developement of an Immunoradiometric Assay for Tumour Marker CA19-9

    Institute of Scientific and Technical Information of China (English)


    A two-site sandwich immunoradiometric assay for quantifying CA19-9 in serum is developed byusing two monoclonal antibodies. One of CA19-9 antibodies is labeled with 125I as a tracer and the othermonoclonal antibody is immobilized on plastic tubes as the solid phase. The sensitivity of the assay is 2

  18. Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies

    NARCIS (Netherlands)

    Burgess, J K; Lopez, J A; Gaudry, L E; Chong, B H


    The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because (1

  19. "Unconventional" Neutralizing Activity of Antibodies Against HIV

    Institute of Scientific and Technical Information of China (English)


    Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.

  20. The radial immunodiffusion assay for plasma Histidine-rich Glycoprotein (HRG) based on a polyclonal antibody, shows a different specificity towards the two variants of a common amino acid polymorphism

    NARCIS (Netherlands)

    Hennis, B.C.; Hoffmann, J.J.M.L.; Kluft, C.


    Recently, two molecular weight forms of Histidine-rich Glycoprotein have been described which explain 59% of the variation in plasma HRG levels. Here, we demonstrate that this can partly be ascribed to a difference in specificity of the immuno assay for HRG towards the two alleles of the polymorphis

  1. A rapid radioimmunoassay using /sup 125/I-labeled staphylococcal protein A for antibody to varicella-zoster virus

    Energy Technology Data Exchange (ETDEWEB)

    Richman, D.D.; Cleveland, P.H.; Oxman, M.N.; Zaia, J.A.


    A sensitive radioimmunoassay for serum antibody to varicella-zoster virus is described; it uses 125I-labeled staphylococcal protein A and a specially designed immunofiltration apparatus. The assay accurately distinguishes between individuals who are susceptible and those who are immune to infection with varicella-zoster virus. In addition, it can detect passive antibody in recipients of varicella-zoster immune globulin. This radioimmunoassay also detects the heterologous antibody responses that occasionally occur in patients infected with herpes simplex virus, which also have been detected by other antibody assays. The particular advantages of this assay are the use of noninfectious reagents, the speed of execution (less than 3 hr), the requirement for only small quantities of serum (30 microliters), the objectivity of end-point determination, and the capability of screening large numbers of sera. Consequently, this radioimmunoassay is especially useful for the rapid identification of susceptible individuals, which is essential for the appropriate management of patients and hospital personnel after exposure to varicella.

  2. Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins* (United States)

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias


    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope–labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A–coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope–labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed. PMID:24722731

  3. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins. (United States)

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias


    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

  4. Antiparietal cell antibody test (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

  5. Evaluation of the Sensibility and Specificity of the Complement Fixation Enzyme Linked Immunosorbent Assay Kit for Detecting Brucellosis Antibodies%布鲁氏菌病补体结合酶联免疫吸附试验抗体检测试剂盒敏感性和特异性评价

    Institute of Scientific and Technical Information of China (English)

    王楠; 姚学军; 马立峰; 王秀丽; 程君生; 蒋玉文; 赵心力; 毛开荣


    To analysis the sensibility, specificity and coincidence rate of the complement fixation enzyme linked immunosorbent assay kit ( CF-ELISA kit ) for detecting brucellosis antibodies, 400 bovine and sheep serum specimens were collected from brucellosis-affected areas selecting CF-ELISA kit to detect brucellosis antibodies, where cases had already been identified. Another total of 200 bovine and sheep serum specimens for detecting antibodies were collected from no brucellosis-affected areas, where no cases had been identified. Results showed that there was no significant difference (P>0.05) among the CF-ELISA kit, iELISA kit and CGS for detecting antibodies of brucellosis-affected areas serum. Kappa statistics result showed that the method of CF-ELISA and iELISA had highly coincidence rate. The specificity and coincidence rate were 100% between the CF-ELISA kit and iELISA kit for detecting antibodies of no brucellosis-affected areas serum.%为评价布鲁氏菌病补体结合酶联免疫吸附试验抗体检测试剂盒( CF-ELISA试剂盒)的敏感性、特异性及与其他试剂盒的符合率,用CF-ELISA试剂盒对布病感染地区牛、羊血清各200份,布病净化地区牛、羊群血清各100份进行抗体检测,同时与其他商品化检测试剂进行了比较。结果表明,感染地区牛、羊血清抗体的McNemar检验结果表明CF-ELISA试剂盒与间接酶联免疫吸附试验( iELISA)试剂盒和CGS的敏感性和特异性差异均不显著( P>0.05),Kappa一致性检验分析结果表明,CF-ELISA试剂盒与iELISA试剂盒检测结果有高度的符合性。检测净化地区牛、羊群血清抗体产品间的特异性和符合率均为100%。

  6. Enzyme-linked immunosorbent assays using recombinant TgSAG2 and NcSAG1 to detect Toxoplasma gondii and Neospora caninum-specific antibodies in domestic animals in Turkey (United States)

    ZHOU, Mo; CAO, Shinuo; SEVINC, Ferda; SEVINC, Mutlu; CEYLAN, Onur; LIU, Mingming; WANG, Guanbo; MOUMOUNI, Paul Franck Adjou; JIRAPATTHARASATE, Charoonluk; SUZUKI, Hiroshi; NISHIKAWA, Yoshifumi; XUAN, Xuenan


    Considering the scarce information on occurrences of Toxoplasma gondii and Neospora caninum in domestic animals from Turkey, the aim of this study was to investigate the seroprevalence of these parasite infections in cattle, horses, sheep, goats and dogs in Turkey. The specific antibodies against T. gondii and N. caninum were detected by iELISAs based on the recombinant TgSAG2 or NcSAG1 in a total of 2,039 serum samples from eleven provinces. The seroprevalence of T. gondii infections was 46.3%, 4.0%, 20.0%, 12.9% and 19.8%, that of N. caninum infections was 0.3%, 7.4%, 2.1%, 3.2% and 16.6% in the horses, cattle, sheep, goats and dogs, respectively. These results indicated that T. gondii and N. caninum infections are prevalent in Turkish domestic animals. PMID:27523504

  7. Rapid, targeted and culture-free viral infectivity assay in drop-based microfluidics. (United States)

    Tao, Ye; Rotem, Assaf; Zhang, Huidan; Chang, Connie B; Basu, Anindita; Kolawole, Abimbola O; Koehler, Stephan A; Ren, Yukun; Lin, Jeffrey S; Pipas, James M; Feldman, Andrew B; Wobus, Christiane E; Weitz, David A


    A key viral property is infectivity, and its accurate measurement is crucial for the understanding of viral evolution, disease and treatment. Currently viral infectivity is measured using plaque assays, which involve prolonged culturing of host cells, and whose measurement is unable to differentiate between specific strains and is prone to low number fluctuation. We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification to detect infection events. Using murine noroviruses (MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests. However, the fast, low-cost, highly accurate genomic-based assay promises to be a superior method for drug screening and isolation of resistant viral strains. Moreover our technique can be adapted to measuring the infectivity of other pathogens, such as bacteria and fungi.

  8. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  9. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A; Thompson, Vicki S


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  10. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  11. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  12. Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays. (United States)

    Karakosta, Theano D; Soosaipillai, Antoninus; Diamandis, Eleftherios P; Batruch, Ihor; Drabovich, Andrei P


    Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples

  13. Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute Scrub Typhus infection.

    Directory of Open Access Journals (Sweden)

    Daniel H Paris


    Full Text Available BACKGROUND: There is an urgent need to develop rapid and accurate point-of-care (POC technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy. METHODOLOGY/PRINCIPAL FINDINGS: In this study we present the clinical evaluation of loop-mediated isothermal PCR assay (LAMP in the context of a prospective fever study, including 161 patients from scrub typhus-endemic Chiang Rai, northern Thailand. A robust reference comparator set comprising following 'scrub typhus infection criteria' (STIC was used: a positive cell culture isolate and/or b an admission IgM titer ≥1∶12,800 using the 'gold standard' indirect immunofluorescence assay (IFA and/or c a 4-fold rising IFA IgM titer and/or d a positive result in at least two out of three PCR assays. Compared to the STIC criteria, all PCR assays (including LAMP demonstrated high specificity ranging from 96-99%, with sensitivities varying from 40% to 56%, similar to the antibody based rapid test, which had a sensitivity of 47% and a specificity of 95%. CONCLUSIONS/SIGNIFICANCE: The diagnostic accuracy of the LAMP assay was similar to realtime and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course. The combination of DNA- and antibody-based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub typhus.

  14. Advances in monoclonal antibody application in myocarditis

    Institute of Scientific and Technical Information of China (English)

    Li-na HAN; Shuang HE; Yu-tang WANG; Li-ming YANG; Si-yu LIU; Ting ZHANG


    Monoclonal antibodies have become a part of daily preparation technologies in many laboratories.Attempts have been made to apply monoclonal antibodies to open a new train of thought for clinical treatments of autoimmune diseases,inflammatory diseases,cancer,and other immune-associated diseases.This paper is a prospective review to anticipate that monoclonal antibody application in the treatment of myocarditis,an inflammatory disease of the heart,could be a novel approach in the future.In order to better understand the current state of the art in monoclonal antibody techniques and advance applications in myocarditis,we,through a significant amount of literature research both domestic and abroad,developed a systematic elaboration of monoclonal antibodies,pathogenesis of myocarditis,and application of monoclonal antibodies in myocarditis.This paper presents review of the literature of some therapeutic aspects of monoclonal antibodies in myocarditis and dilated cardiomyopathy to demonstrate the advance of monoclonal antibody application in myocarditis and a strong anticipation that monoclonal antibody application may supply an effective therapeutic approach to relieve the severity of myocarditis in the future.Under conventional therapy,myocarditis is typically associated with congestive heart failure as a progressive outcome,indicating the need for alternative therapeutic strategies to improve long-term results.Reviewing some therapeutic aspects of monoclonal antibodies in myocarditis,we recently found that monoclonal antibodies with high purity and strong specificity can accurately act on target and achieve definite progress in the treatment of viral myocarditis in rat model and may meet the need above.However,several issues remain.The technology on howto make a higher homologous and weak immunogenic humanized or human source antibody and the treatment mechanism of monoclonal antibodies may provide solutions for these open issues.If we are to further stimulate

  15. An ELISA test for the detection of antibodies to Legionella pneumophila.


    Wreghitt, T. G.; Nagington, J.; Gray, J


    An enzyme-linked immunosorbent assay (ELISA) test has been developed to detect antibodies to Legionella pneumophila serogroup 1. There is good correlation between indirect fluorescent antibody (IFA) and ELISA titres but ELISA is more sensitive.

  16. Neutralizing antibodies to Haemophilus ducreyi cytotoxin.


    Lagergård, T; Purvén, M


    Neutralizing antibodies against cytotoxin produced by Haemophilus ducreyi bacteria were studied in rabbits by an assay employing HEp-2 cells and diluted crude cytotoxin preparations from the organism. Antisera to 12 different H. ducreyi strains were prepared by immunization of rabbits with bacterial sonicates combined with Freund's adjuvant. The antibody response during infection with H. ducreyi was studied in two groups of rabbits which were infected with five live strains by either single o...

  17. 粗提抗原检测炭疽血清抗体ELISA方法的初步评价%Preliminary evaluation on the detection of serum level of antibody to Bacillus anthracis by enzyme-linked immunosorbent assay using crude antigen

    Institute of Scientific and Technical Information of China (English)

    魏建春; 张慧娟; 马凤琴; 张恩民; 俞东征


    Objective To evaluate the method of detecting antibodies to Bacillus anthracis by enzymelinked immunosorbent assay(ELISA)using crude antigen.Methods The anti-Bacillus anthracis antibody levels in sera of 42 healthy people and 42 patients were detected by indirect ELISA.Standard curve was plotted using the data from positive controls,based on which the relative content of each serum was calculated and compared with the result of rLF.Results The median of antibody's relative content in patient group and healthy people group are 1.19 and 0.24,the differences being statistically significant(uc=7.643,P<0.05).The result of crude antigen is in concordance with rLF(but not parallel absolutely).Conclusions Crude antigen can distinguish most of patients with healthy population effectively.The results suggested that crude antigen is applicable in anti-Bacillus anthracis antibody surveillance.%目的 对使用粗提抗原检测炭疽血清抗体的酶联免疫吸附试验(ELISA)方法进行初步评价.方法 用间接ELISA方法检测人群血清(健康人血清42份、炭疽病人血清42份)特异性抗体,用阳性血清对照绘制标准曲线,按照标准曲线计算出每份血清标本的抗体相对含量,所得结果与重组致死因子(rLF)方法的检测结果进行比较.结果 病人组血清抗体相对含量中位数为1.19,健康人组血清抗体相对含量中位数为0.24,两组比较差异有统计学意义(uc=7.643,P<0.05).粗提抗原检测与rLF检测结果并不完全对应,但两种方法显示出较高的一致性.结论 粗提抗原检测炭疽血清抗体的方法能区分大部分的病人和健康人,有一定的应用潜力,可用在炭疽疾病监测工作中.

  18. The elephant interferon gamma assay: a contribution to diagnosis of tuberculosis in elephants. (United States)

    Angkawanish, T; Morar, D; van Kooten, P; Bontekoning, I; Schreuder, J; Maas, M; Wajjwalku, W; Sirimalaisuwan, A; Michel, A; Tijhaar, E; Rutten, V


    Mycobacterium tuberculosis (M. tb) has been shown to be the main causative agent of tuberculosis in elephants worldwide. M. tb may be transmitted from infected humans to other species including elephants and vice versa, in case of prolonged intensive contact. An accurate diagnostic approach covering all phases of the infection in elephants is required. As M. tb is an intracellular pathogen and cell-mediated immune (CMI) responses are elicited early after infection, the skin test is the CMI assay of choice in humans and cattle. However, this test is not applicable in elephants. The interferon gamma (IFN-γ) assay is considered a good alternative for the skin test in general, validated for use in cattle and humans. This study was aimed at development of an IFN-γ assay applicable for diagnosis of tuberculosis in elephants. Recombinant elephant IFN-γ (rEpIFN-γ) produced in eukaryotic cells was used to immunize mice and generate the monoclonal antibodies. Hybridomas were screened for IFN-γ-specific monoclonal antibody production and subcloned, and antibodies were isotyped and affinity purified. Western blot confirmed recognition of the rEpIFN-γ. The optimal combination of capture and detection antibodies selected was able to detect rEpIFN-γ in concentrations as low as 1 pg/ml. The assay was shown to be able to detect the native elephant IFN-γ, elicited in positive-control cultures (pokeweed mitogen (PWM), phorbol myristate acetate plus ionomycin (PMA/I)) of both Asian and African elephant whole-blood cultures (WBC). Preliminary data were generated using WBC from non-infected elephants, a M. tb infection-suspected elephant and a culture-confirmed M. tb-infected elephant. The latter showed measurable production of IFN-γ after stimulation with ESAT6/CFP10 PPDB and PPDA in concentration ranges as elicited in WBC by Mycobacterium tuberculosis complex (MTBC)-specific antigens in other species. Hence, the IFN-γ assay presented potential as a diagnostic tool for the

  19. Radioimmunoassay for detection of VP1 specific neutralizing antibodies of foot and mouse disease virus

    Energy Technology Data Exchange (ETDEWEB)

    Patzer, E.J.; Jackson, M.L. (Genentech, Inc., South San Francisco CA (USA)); Moore, D.M. (U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, NY (USA))


    A solid-phase radioimmunoassay was developed for the detection of antibodies against a specific region of the VP1 protein of the A24 and O1 serotypes of foot and mouth disease virus. The antibody titers from the radioimmunoassay showed a positive correlation with neutralizing antibody titers determined by a mouse protection assay. The specificity of the assay resides in the peptide used as antigen. The assay is rapid, reproducible and does not require the use of whole virions.

  20. Laboratory evaluation of the Chembio Dual Path Platform HIV-Syphilis Assay

    Directory of Open Access Journals (Sweden)

    Mireille B. Kalou


    Full Text Available Background: Use of rapid diagnostic tests for HIV and syphilis has increased remarkably in the last decade. As new rapid diagnostic tests become available, there is a continuous need to assess their performance and operational characteristics prior to use in clinical settings.Objectives: In this study, we evaluated the performance of the Chembio Dual Path Platform (DPP® HIV–Syphilis Assay to accurately diagnose HIV, syphilis, and HIV/syphilis co-infection.Method: In 2013, 990 serum samples from the Georgia Public Health Laboratory in Atlanta, Georgia, United States were characterised for HIV and syphilis and used to evaluate the platform. HIV reference testing combined third-generation Enzyme Immunoassay and Western Blot, whereas reference testing for syphilis was conducted by the Treponema pallidum passive particle agglutination method and the TrepSure assay. We assessed the sensitivity and specificity of the DPP assay on this panel by comparing results with the HIV and syphilis reference testing algorithms.Results: For HIV, sensitivity was 99.8% and specificity was 98.4%; for syphilis, sensitivity was 98.8% and specificity was 99.4%. Of the 348 co-infected sera, 344 (98.9% were detected accurately by the DPP assay, but 11 specimens had false-positive results (9 HIV and 2 syphilis due to weak reactivity.Conclusion: In this evaluation, the Chembio DPP HIV–Syphilis Assay had high sensitivity and specificity for detecting both HIV and treponemal antibodies. Our results indicate that this assay could have a significant impact on the simultaneous screening of HIV and syphilis using a single test device for high-risk populations or pregnant women needing timely care and treatment.

  1. Antiphospholipid antibody: laboratory, pathogenesis and clinical manifestations

    Directory of Open Access Journals (Sweden)

    T. Ziglioli


    Full Text Available Antiphospholipid antibodies (aPL represent a heterogeneous group of antibodies that recognize various antigenic targets including beta2 glycoprotein I (β2GPI, prothrombin (PT, activated protein C, tissue plasminogen activator, plasmin and annexin A2. The most commonly used tests to detect aPL are: lupus anticoagulant (LAC, a functional coagulation assay, anticardiolipin antibody (aCL and anti-β2GPI antibody (anti-β2GPI, which are enzyme-linked immunoassay (ELISA. Clinically aPL are associated with thrombosis and/or with pregnancy morbidity. Apparently aPL alone are unable to induce thrombotic manifestations, but they increase the risk of vascular events that can occur in the presence of another thrombophilic condition; on the other hand obstetrical manifestations were shown to be associated not only to thrombosis but mainly to a direct antibody effect on the trophoblast.

  2. Accurate, noninvasive detection of Helicobacter pylori DNA from stool samples: potential usefulness for monitoring treatment. (United States)

    Shuber, Anthony P; Ascaño, Jennifer J; Boynton, Kevin A; Mitchell, Anastasia; Frierson, Henry F; El-Rifai, Wa'el; Powell, Steven M


    A novel DNA assay demonstrating sensitive and accurate detection of Helicobacter pylori from stool samples is reported. Moreover, in three individuals tested for therapeutic response, the assay showed the disappearance of H. pylori DNA during treatment. Thus, this noninvasive molecular biology-based assay has the potential to be a powerful diagnostic tool given its ability to specifically identify H. pylori DNA.

  3. Accurate, Noninvasive Detection of Helicobacter pylori DNA from Stool Samples: Potential Usefulness for Monitoring Treatment


    Shuber, Anthony P; Ascaño, Jennifer J.; Boynton, Kevin A.; Mitchell, Anastasia; Frierson, Henry F.; El-Rifai, Wa’el; Powell, Steven M


    A novel DNA assay demonstrating sensitive and accurate detection of Helicobacter pylori from stool samples is reported. Moreover, in three individuals tested for therapeutic response, the assay showed the disappearance of H. pylori DNA during treatment. Thus, this noninvasive molecular biology-based assay has the potential to be a powerful diagnostic tool given its ability to specifically identify H. pylori DNA.

  4. Quantitative immunobinding assay for vitamin D-dependent calcium-binding protein (calbindin-D28k) using nitrocellulose filters

    Energy Technology Data Exchange (ETDEWEB)

    Varghese, S.; Christakos, S.


    A sensitive dot immunobinding assay has been developed for the quantitative determination of vitamin D-dependent calcium-binding protein (calbindin-D28k; CaBP) in rat and human kidney and brain. Protein samples are spotted onto nitrocellulose sheets, fixed, and then rinsed with Tris-buffered saline. The remaining protein binding sites are blocked with bovine serum albumin, gelatin, or nonfat dry milk protein and the filters are then incubated sequentially with antiserum to calbindin-D28k (1:500 dilution) and /sup 125/I-protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by counting in a gamma counter. The sensitivity of the assay is such that 10 ng calbindin-D28k can be accurately quantitated. The highest levels of CaBP were detected in kidney (7.8 +/- 0.5 micrograms/mg protein) and cerebellum (22.1 +/- 1.4 micrograms/mg protein). Ten micrograms calmodulin, lactalbumin, or parvalbumin and 100 micrograms liver extract showed no reactivity in the assay. The assay is precise (intraassay variability, 4.0%) and reproducible (interassay variability, 8.8%). There was good agreement between the data in this assay and the data we obtained using radioimmunoassay (RIA). The assay has several advantages over the RIA. Iodination of pure antigen is not required and it is possible to detect membrane-bound and insoluble antigens using this assay. Also, the antiserum and /sup 125/I-protein A solutions can be saved and reused. This assay represents a major modification of the original immunobinding assays which used the less sensitive peroxidase stain. It is also an improvement over previous /sup 125/I immunobinding assays which were not quantitative but were used as antigen spot tests or which required iodination of the antibody.



    Talat Mokhtari Azad; Anahid Ehteda; Parvin Yavari; R Hamkar; Zahra Safar Pour; M. Essalat Rakhsheh Nategh


    Laboratory diagnosis of acute measles is usually achieved by serology assays for measle-specific IgM antibody. For comparison of measle-specific IgM antibody in saliva and serum, 95 paired blood and saliva samples were collected 1-14 days after the onset of rash. The specimens were tested for specific IgM antibody by an IgM antibody-capture Enzyme Immunoassay (EIA). Measles IgM antibody was detected in 89 (93.7%) of serum samples and in 85(89.5%) of saliva specimens. Of the 6(6.3%) serum samp...

  6. A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens.

    Directory of Open Access Journals (Sweden)

    Emmanuel Y Dotsey

    Full Text Available In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001 with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.

  7. Trans-splicing as a novel method to rapidly produce antibody fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Iwasaki, Ryohei; Kiuchi, Hiroki [Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Ihara, Masaki [Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Mori, Toshihiro; Kawakami, Masayuki [Lifescience Lab. R and D, Fujifilm Co., 577 Ushijima, Kaisei-machi, Ashigarakami-gun, Kanagawa 258-8577 (Japan); Ueda, Hiroshi, E-mail: [Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)


    To cultivate the use of trans-splicing as a novel means to rapidly express various antibody fusion proteins, we tried to express antibody-reporter enzyme fusions in a COS-1 co-transfection model. When a vector designed to induce trans-splicing with IgH pre-mRNA was co-transfected with a vector encoding the mouse IgM locus, the expression of V{sub H}-secreted human placental alkaline phosphatase (SEAP) as well as Fab-SEAP were successfully expressed both in mRNA and protein levels. Especially, the vectors encoding complementary sequence to S{mu} as a binding domain was accurate and efficient, producing trans-spliced mRNA of up to 2% of cis-spliced one. Since S{mu} sequence should exist in every IgH pre-mRNA, our finding will lead to the rapid production and analysis of various antibody-enzyme fusions suitable for enzyme-linked immunosorbent assay (ELISA) or antibody-dependent enzyme prodrug therapy (ADEPT).

  8. [Antinuclear antibodies]. (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A


    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  9. Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire. (United States)

    Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei


    High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies.

  10. Immunity to rhabdoviruses in rainbow trout: the antibody response

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lapatra, S.E.


    to their occasional detrimental effect on rainbow trout farming. Research efforts have been focused on understanding the mechanisms involved in protective immunity. Several specific and nonspecific cellular and humoral parameters are believed to be involved, but only the antibody response has been characterised...... in detail so far. Analysis of the specificity of anti-virus trout antibodies has been complicated by a generally insufficient ability of the antibodies to bind the viral proteins in assays such as immunoblotting. However, other assays, specifically designed for detection of fish anti IHNV/VHSV antibodies......, have demonstrated that rainbow trout can produce specific and highly functional antibodies that are able to neutralise virus pathogenicity in vitro as well as in vivo. The apparently more restricted antibody response to IHNV and VHSV antigens in fish compared to mammals could possibly be explained...

  11. Accurate strand-specific quantification of viral RNA.

    Directory of Open Access Journals (Sweden)

    Nicole E Plaskon

    Full Text Available The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (- strand RNA with this assay when higher levels of cDNA generated from the (+ strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (- strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR(R Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV, the recent cause of large outbreaks of disease in the Indian Ocean

  12. Selection of antibodies from synthetic antibody libraries. (United States)

    Harel Inbar, Noa; Benhar, Itai


    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  13. Comparison of automated chemiluminescence immunoassays with capture enzyme immunoassays for the detection of measles and mumps IgM antibodies in serum. (United States)

    Haywood, Becky; Patel, Mauli; Hurday, Samantha; Copping, Ruth; Webster, Daniel; Irish, Dianne; Haque, Tanzina


    Outbreaks of measles and mumps occur regularly in the UK. Rapid diagnosis of acute infection is important for both infection control and epidemiological purposes. The objective of this study was to compare the performance of an automated platform (DiaSorin Liaison(®), Saluggia, Italy) with a manual capture enzyme immunoassay (EIA; Microimmune, Hounslow, UK) for the detection of measles and mumps IgM antibodies in serum from symptomatic individuals. Ninety sera tested previously for measles (n=50) and mumps (n=40) IgM using the manual EIA were tested retrospectively using the DiaSorin Liaison(®) and the results compared. Sensitivity, specificity, inter-assay variability and intra-assay variability of the Liaison(®) assays were calculated. Sensitivity and specificity of the Liaison(®) assay for measles IgM were 92% and 100% respectively, with inter-assay variation of 14.1% and intra-assay variation of 12.5%. The sensitivity and specificity of the mumps IgM Liaison(®) assay were 88% and 95% respectively, with an inter-assay and intra-assay variation of 13.9% and 5.3% respectively. Both the measles and mumps IgM Liaison(®) assays gave fewer equivocal results than the EIA. Neither Liaison(®) IgM assay showed any cross-reactivity with sera positive against other viruses, however the measles IgM EIA cross-reacted with parvovirus IgM. The automated Liaison(®) assays are more specific, cheaper and less labour-intensive compared to the manual EIA. The Liaison(®) assays benefit from reduced number of equivocal results compared to the EIA for both measles and mumps IgM. This allows clinical decisions to be made accurately and in a timely manner.

  14. Avaliação de ensaio molecular para determinação de carga viral em indivíduos sorologicamente negativos para o HIV-1 Evaluation of a molecular assay for determining viral load on HIV-1 antibody negative patients

    Directory of Open Access Journals (Sweden)

    José Moreira Pereira


    Full Text Available O teste de carga viral foi concebido para acompanhar a evolução e o tratamento do paciente com diagnóstico confirmado de HIV-1. Contudo, sua especificidade diagnóstica não foi ainda avaliada em pessoas que apresentam um teste sorológico negativo. Mesmo assim, ele tem sido erroneamente utilizado para o diagnóstico da infecção primária pelo HIV-1. Este trabalho relata quatro pacientes em que a carga viral plasmática NucliSens (Organon Teknika foi repetidamente positiva na ausência de anticorpos para HIV e chama atenção para o fato de que a carga viral abaixo de 10 mil cópias/ml é de difícil interpretação, como tem sido assinalado em numerosos artigos, em que foram utilizadas outras metodologias.The plasma viral load test for HIV-1,a exquisitely high sensitive assay, were neither developed nor evaluated for the diagnosis of primary HIV infection; therefore, their diagnostic specificity is not well delineated when applied to persons who are negative for HIV antibody. This article reported four cases of false positive results obtained by using NucliSens viral load assay (Organon Teknika and emphasize the importance that low positive plasma viral load (< 10 000 copies/ml may be difficult to interpret how has been assinalated in numerous articles in the medical literature, using other methodologies.

  15. Development of monoclonal antibodies that recognize Treponema pallidum.


    Saunders, J M; Folds, J D


    We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay.


    Serum samples from 251 wild carnivores from different regions of Spain were tested for antibodies to Neospora caninum by the commercial competitive screening enzyme linked immunosorbent assay (c-ELISA) and confirmed by Neospora agglutination test (NAT) and/or by indirect fluorescent antibody test (I...

  17. Studies on Purification of Methamidophos Monoclonal Antibodies and Comoarative Immunoactivity of Purified Antibodies

    Institute of Scientific and Technical Information of China (English)



    Objective To purify Methamidophos (Met) monoclonal antibodies with two methods andcompare immune activity of purified antibodies. Method Caprylic acid ammonium sulphateprecipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method wereused to purify Met monoclonal antibodies, UV spectrum scanning was used to determine proteincontent and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gelelectrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linkedimmunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mLand 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASPmethod. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was181.26 μg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01μg/mL and 1.03 μg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82μg/mL, and the linear working range and LOD were 10.91-11412.29 ug/mL and 3.42 μg/mL,respectively. Conclusion Antibodies purified by SPA method are better than those by CAASPmethod, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelledtesting paper for analyzing Met residue in vegetable and drink water.

  18. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond


    Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

  19. Antibody-based biological toxin detection

    Energy Technology Data Exchange (ETDEWEB)

    Menking, D.E.; Goode, M.T. [Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD (United States)


    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a direct competition assay, antibodies against Cholera toxin, Staphylococcus Enterotoxin B or ricin were noncovalently immobilized on quartz fibers and probed with fluorescein isothiocyanate (FITC) - labeled toxins. In the indirect competition assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified anti-toxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or anti-toxin IgG in a dose dependent manner and the detection of the toxins was in the nanomolar range.

  20. Establishment of an Enzyme Linked Immunosorbent Assay for Neonatal Thyrotropin

    Institute of Scientific and Technical Information of China (English)


    A sensitive and specific ELISA for neonatal thyrotropin(Neonatal TSH) is established with using twoanti-TSH monoclonal antibody. One of them is coated on the microtiter plate, the other is conjugate ofbiotin. The label is horseradish peroxidase(HRP) conjugate of streptavidin. TMB-H2O2 solution is used asthe substrate of HRP.The sensitivity of the assay is 0.5 mIU/L, the intra-assay CVs and the intre-assay

  1. 抗血小板膜糖蛋白Ib单克隆抗体的制备、鉴定及其功能的初步研究%Development,identification and function assay of monoclonal antibody against platelet membrane glycoprotein Ib

    Institute of Scientific and Technical Information of China (English)

    谷颖; 季顺东; 赵益明; 沈飞; 阮长耿


    AIM: To prepare and identify a monoclonal antibody (mAb) against human platelet glycoprotein Ib and make its application. METHODS: BALB/c mice were immunized with human platelets washed, and the spleen cells of them were fused with myloma cells. A hybridoma cell was screened by indirect ELISA and cloned, and the mAb were purified from the ascites of mice. Ig subclass was analysed by double immunodiffusion. The antigen recognized by monoclonal antibody was identified by flow cytometry and radioimmunoassy, respectively. The inhibition of mAb on plasma von Willebrand factor ristocetin cofactor activity (vWF: Rcof) was investigated by enzyme linked immunosorbent assay (ELISA). RESULTS: A mudne mAb against human platelet membrane glycoprotein (GP) lb was developed and denominated as SZ-151. SZ-151 belonged to IgG1 subclass and its titer in ascites was 1:20 000. Flow cytometry and radioimmunoassy showed that the antigen recognized by monoclonal antibody SZ-151 was platelet membrane GPIb. ELISA showed that SZ-151 did not inhibit plasma von Willebrand factor ristocetin cofactor activity. CONCLUSION: A mAb, SZ-151 against platelet glycoprotein Ib was developed, which could be useful in assays of plasma von Willebrand factor ristocetin cofactor activity(vWF: Rcof)and can be used for diagnose patients with vWD.%目的:制备抗人血小板膜糖蛋白Ib(GPIb)单克隆抗体(mAb),并进行生化鉴定与初步探讨其临床应用.方法:采用血小板免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞融合.经间接ELISA法筛选和克隆化培养,获得1株抗人GPIb mAb的杂交瘤细胞,纯化其腹水获得mAb;免疫双扩散鉴定其抗体亚类;流式细胞术和免疫放射法鉴定其识别的抗原;ELISA法检测该抗体对血浆血管性血友病因子(vWF)的瑞斯托霉素辅因子活性的影响.结果:成功制备了1株抗人GPIb mAb,命名为SZ-151.ELISA法测定其腹水效价为1:20 000,免疫双扩散鉴定其为IgG1亚类.免疫放射法和流式

  2. First steps in the standardization of immunoglobulin IgG myeloperoxidase-anti-neutrophil cytoplasmic antibody measurements. (United States)

    Hutu, D P; Tuddenham, E; Monogioudi, E; Meroni, P; Schimmel, H; Sheldon, J; Zegers, I


    The standardization of immunoassays for immunoglobulin (Ig)G myeloperoxidase-anti-neutrophil cytoplasmic antibodies (MPO-ANCA) could contribute to a more accurate diagnosis and follow-up of small vessels-associated vasculitis, a systemic autoimmune disorder that leads to necrosis of blood vessel walls. Despite significant efforts by different groups, the level of comparability of results from commercially available immunoassays used for IgG MPO-ANCA detection is still poor. Therefore, the potential for improvement using reference materials was assessed. The evaluation of a set of 30 patient samples with 11 assays showed that differences between assays result in different interpretations for individual patients. Only 10 of 30 patient samples had the same clinical interpretation among 11 assays applying the cut-off values provided by each respective manufacturer. The correlation between results from 13 different assays was assessed in a pairwise manner. The correlation between results from patient samples was systematically very good for combinations of seven of those assays. The correlation of results ranged from reasonable to good for combinations with four other assays, therefore it should be possible to improve the comparability of results using a commutable reference material for calibration. Feasibility studies were conducted in order to find a reference material format most suitable for a calibrator. Two sets of candidate reference materials were produced from different raw materials, and assessed according to their suitability. A final format was selected, and a candidate reference material was produced.

  3. Generation, use, and validation of receptor-selective antibodies. (United States)

    Mackrill, John J


    Antibodies have proved invaluable in the study of G-protein-coupled receptors (GPCRs). The utility of these immunoglobulin probes for investigation of protein structures and functions arises from their selectivity as well as their versatility. Antibodies can be used to analyze GPCR size, abundance, distribution, turnover, modification, interaction with other proteins, and functional properties. In this chapter, techniques for the generation and characterization of receptor-selective antibodies are described. Two protocols are given for the generation of antibodies: (1) development of polyclonal antibodies (PAbs) against synthetic peptides corresponding to a specific site within a GPCR and (2) selection of synthetic single-chain fragment variable (scFv) monoclonal antibodies (MAbs) from libraries expressed on the surface of bacteriophage. Immunoblot and enzyme-linked immunosorbent assays for characterization of the selectivity and affinity of such antibodies are described. Finally, methods are given for improvement of the titer and specificity of PAbs.

  4. Multiplexed measurement of serum antibodies using an array biosensor. (United States)

    Moreno-Bondi, Maria C; Taitt, Chris Rowe; Shriver-Lake, Lisa C; Ligler, Frances S


    The array biosensor provides the capability for simultaneously measuring titers of antibody against multiple antigens. Human antibodies against four different targets, tetanus toxin, diphtheria toxin, staphylococcal enterotoxin B (SEB) and hepatitis B, were measured simultaneously in sera from eight different donors in a single assay and titers were determined. The assays could measure amounts of bound antibody as low as approximately 100 fg. Each individual serum exhibited a different pattern of reactivity against the four target antigens. Applications of this biosensor capability include monitoring for exposure to pathogens and for efficacy of vaccination.

  5. Comparison of Techniques for Monitoring Infliximab and Antibodies Against Infliximab in Crohn's Disease

    DEFF Research Database (Denmark)

    Steenholdt, Casper; Ainsworth, Mark A; Tovey, Michael


    Several techniques are used to measure infliximab (IFX) and anti-IFX antibodies (Abs) in Crohn's disease. The aim of this study was to compare different assays for this purpose.......Several techniques are used to measure infliximab (IFX) and anti-IFX antibodies (Abs) in Crohn's disease. The aim of this study was to compare different assays for this purpose....


    Directory of Open Access Journals (Sweden)

    S. N. Depamede


    Full Text Available Clostridium botulinum neurotoxin (BoNTs is one of the causes of economic loss in the livestock industry. This economic loss would be as a direct result when animals poisoned by BoNTs or indirectly when the livestock products are contaminated by BoNTs, which end up with the products are banned by authority. Therefore a routine surveillance of BoNTs in the farm and in livestock product processing industry is urgently needed. One of the most relatively quick and accurate methods to perform a routine detection of the presence of BoNTs is enzyme-linkage immunosorbant assay (ELISA. In this article we describe the results of the development of ELISA, using polyclonal antibodies against BoNTs-B produced locally. Antibodies were generated from six Balb/c mice with standard immunological methods. Mice were immunized three times for a period of 8 weeks with a commercial type B Clostridium botulinum toxoid at a dose of 100 ng per mouse per injection. The resulting antibody was purified by a combination of ammonium sulfate precipitation 50% (w/v technique and a protein A column method. The results of this preliminary study indicated that the developed ELISA method capable of detecting type B Clostridium botulinum toxin up to 1.0 ng/ml.

  7. A Strategy for Screening Monoclonal Antibodies for Arabidopsis Flowers (United States)

    Shi, Qian; Zhou, Lian; Wang, Yingxiang; Ma, Hong


    The flower is one of the most complex structures of angiosperms and is essential for sexual reproduction. Current studies using molecular genetic tools have made great advances in understanding flower development. Due to the lack of available antibodies, studies investigating the localization of proteins required for flower development have been restricted to use commercial antibodies against known antigens such as GFP, YFP, and FLAG. Thus, knowledge about cellular structures in the floral organs is limited due to the scarcity of antibodies that can label cellular components. To generate monoclonal antibodies that can facilitate molecular studies of the flower, we constructed a library of monoclonal antibodies against antigenic proteins from Arabidopsis inflorescences and identified 61 monoclonal antibodies. Twenty-four of these monoclonal antibodies displayed a unique band in a western blot assay in at least one of the examined tissues. Distinct cellular distribution patterns of epitopes were detected by these 24 antibodies by immunofluorescence microscopy in a flower section. Subsequently, a combination of immunoprecipitation and mass spectrometry analysis identified potential targets for three of these antibodies. These results provide evidence for the generation of an antibody library using the total plant proteins as antigens. Using this method, the present study identified 61 monoclonal antibodies and 24 of them were efficiently detecting epitopes in both western blot experiments and immunofluorescence microscopy. These antibodies can be applied as informative cellular markers to study the biological mechanisms underlying floral development in plants. PMID:28293248

  8. Application of colloidal gold based immunochromatography assay to test hepatitis B surface antigen,hepatitis C virus antibody and human immunodeficiency virus antibody%胶体金免疫层析试验在检测急诊患者血液传播疾病中的应用

    Institute of Scientific and Technical Information of China (English)



    目的 探讨胶体金免疫层析试验检测法(gold immunochromatography assay,GICA)在检测急诊病人乙型肝炎病毒表面抗原、丙型肝炎病毒抗体、人类免疫缺陷病毒抗体中的临床应用价值.方法 随机选择2010年2月至2013年2月在四川省双流县第一人民医院就诊患者1080人作为研究对象,采集患者血清标本进行GICA与酶联免疫吸附测定(enzyme-linked immuno sorbent assay,ELISA).以ELISA法检测结果为金标准,计算GICA检测乙型肝炎病毒表面抗原、丙型肝炎病毒抗体、人类免疫缺陷病毒抗体的灵敏度、特异度、漏诊率和准确性.结果 用GICA检测乙型肝炎病毒表面抗原的灵敏度为91.3% (21/23),特异度为99.6%(1053/1057),漏诊率为8.7% (2/23),准确性为99.4%(1074/1080);应用GICA检测抗丙型肝炎病毒抗体的灵敏度为57.1%(4/7),特异度为99.8%(1071/1073),漏诊率为42.9%(3/7),准确性为99.5%(1075/1080).应用GICA检测人类免疫缺陷病毒抗体的灵敏度、特异度和准确性为100.0%.结论 GICA可用于乙型肝炎病毒表面抗原和人类免疫缺陷病毒抗体的筛检,但对丙型肝炎病毒抗体的检测漏诊率高,不适宜用于丙型肝炎病毒抗体的筛检.%Objective To access the value of GICA in testing HBsAg,HCV-Ab and HIV-Ab in the emergency cases.Methods A total of 1080 serum specimens were collected from February 2010 to February 2013 in Shuangliu First People's Hospital.ELISA and GICA were used to test.Taken ELISA as the gold standard,sensitivity,specificity,rate of fail to diagnosis and accuracy were calculated by using GICA.Resuits The sensitivity,specificity,rate of fail to diagnosis and accuracy were 91.3%,99.6%,8.7% and 99.4% respectively in testing HBsAg using GICA.In terms of HCV-Ab testing,the sensitivity,specificity,rate of fail to diagnosis and accuracy were 57.1%,99.8%,42.9% and 99.5% respectively.The indexs of sensitivity,specificity and accuracy were

  9. Baculovirus display of functional antibody Fab fragments. (United States)

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


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

  10. Antibodies to West Nile Virus in Wild and Farmed Crocodiles in Southeastern Mexico


    Machain-Williams, Carlos; Padilla-Paz, Sergio E.; Weber, Manuel; Cetina-Trejo, Rosa; Juarez-Ordaz, José Alfredo; Loroño-Pino, María Alba; ULLOA, ARMANDO; Wang,Chong; Garcia-Rejon, Julián; Blitvich, Bradley J.


    Surveillance for evidence of West Nile virus (WNV) infection in Morelet’s crocodiles (Crocodylus moreletii) was conducted in Campeche State, Mexico, in 2007. Sera from 62 crocodiles (32 free-ranging and 30 captive) were assayed for antibodies to WNV by epitope-blocking enzyme-linked immunosorbent assay. Antibodies to WNV were detected in 13 (41%) wild and nine (30%) captive crocodiles, and the overall antibody prevalence was 35%. Although evidence of WNV infection in captive crocodiles has be...

  11. Purification of Murine Monoclonal IgM Antibody

    Institute of Scientific and Technical Information of China (English)


    This paper presents the purification of a monoclonal IgM antibody against human tumor associated antigen Lewis-Y by ion exchange chromatography and gel filtration.Enzyme-linked immunosorbent assay (ELISA) and SDS-polyacrylamide gel electrophoresis (PAGE) were used to identify purified IgM antibody.In flow cytometry analysis, the purified IgM antibody recognizes human breast tumor cell line MCF-7 which expresses Lewis-Y antigen.This work presents a new way for the purification of murine monoclonal IgM antibody.

  12. Immunoreagents and competitive assays to fludioxonil


    Abad Fuentes, Antonio; Agulló, Consuelo; Esteve Turrillas, Francesc Albert; Abad Somovilla, Antonio; Mercader Badia, Josep Vicent


    Fludioxonil is a new-generation fungicide widely used for postharvest fruit protection. The aim of this study was to produce hitherto unreported immunoreagents for Fludioxonil analysis by immunoassay. Derivatives of this agrochemical were synthesized with different linker tethering sites. Those functionalized haptens were activated, and the purified active esters were efficiently conjugated to different carrier proteins for immunogen and assay antigen preparation. Antibodies to Fludioxonil we...

  13. Assays for Determination of Protein Concentration. (United States)

    Olson, Bradley J S C


    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc.

  14. Acetylcholine receptor antibody (United States)

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