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Sample records for antigen hbsag assays

  1. Evaluation of two new automated assays for hepatitis B virus surface antigen (HBsAg) detection: IMMULITE HBsAg and IMMULITE 2000 HBsAg.

    PubMed

    Weber, Bernard; Dengler, Thomas; Berger, Annemarie; Doerr, Hans Wilhelm; Rabenau, Holger

    2003-01-01

    In recent years the diagnostic industry has developed new automated immunoassays for the qualitative detection of hepatitis B virus (HBV) surface antigen (HBsAg) in serum and plasma samples that are performed on analyzers that permit a high-speed throughput, random access, and primary tube sampling. The aim of the present study was the evaluation of two new automated HBsAg screening assays, IMMULITE HBsAg and IMMULITE 2000 HBsAg, from Diagnostic Products Corporation. The new HBsAg assays were compared to well-established tests (Auszyme Monoclonal [overnight incubation, version B], IMx HBsAg, AxSYM HBsAg, and Prism HBsAg [all from Abbott] and Elecsys HBsAg [Roche Diagnostics]). In the evaluation were included seroconversion panels, sera from the acute and chronic phases of infection, dilution series of various HBsAg standards, HBV subtypes and S gene mutants. To challenge the specificity of the new assays, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. IMMULITE HBsAg and IMMULITE 2000 HBsAg, although not as sensitive as the Elecsys HBsAg assay, were equivalent to the AxSYM HBsAg assay and showed a higher sensitivity than the Auszyme Monoclonal B and IMx HBsAg systems for detection of acute infection in seroconversion panels. The specificities (100%) of both IMMULITE assays on unselected blood donors and potentially interfering samples were comparable to those of the alternative assays after repeated testing. In conclusion, the new IMMULITE HBsAg and IMMULITE 2000 HBsAg assays show a good sensitivity for HBsAg detection compared to other well-established tests. The specificity on repeatedly tested samples was equivalent to that of the alternative assays. The rapid turnaround time, primary tube sampling, and on-board dilution make it an interesting assay system for clinical laboratory diagnosis.

  2. [Clinical evaluation of a novel HBsAg quantitative assay].

    PubMed

    Takagi, Kazumi; Tanaka, Yasuhito; Naganuma, Hatsue; Hiramatsu, Kumiko; Iida, Takayasu; Takasaka, Yoshimitsu; Mizokami, Masashi

    2007-07-01

    The clinical implication of the hepatitis B surface antigen (HBsAg) concentrations in HBV-infected individuals remains unclear. The aim of this study was to evaluate a novel fully automated Chemiluminescence Enzyme Immunoassay (Sysmex HBsAg quantitative assay) by comparative measurements of the reference serum samples versus two independent commercial assays (Lumipulse f or Architect HBsAg QT). Furthermore, clinical usefulness was assessed for monitoring of the serum HBsAg levels during antiviral therapy. A dilution test using 5 reference-serum samples showed linear correlation curve in range from 0.03 to 2,360 IU/ml. The HBsAg was measured in total of 400 serum samples and 99.8% had consistent results between Sysmex and Lumipulse f. Additionally, a positive linear correlation was observed between Sysmex and Architect. To compare the Architect and Sysmex, both methods were applied to quantify the HBsAg in serum samples with different HBV genotypes/subgenotypes, as well as in serum contained HBV vaccine escape mutants (126S, 145R). Correlation between the methods was observed in results for escape mutants and common genotypes (A, B, C) in Japan. Observed during lamivudine therapy, an increase in HBsAg and HBV DNA concentrations preceded the aminotransferase (ALT) elevation associated with drug-resistant HBV variant emergence (breakthrough hepatitis). In conclusion, reliability of the Sysmex HBsAg quantitative assay was confirmed for all HBV genetic variants common in Japan. Monitoring of serum HBsAg concentrations in addition to HBV DNA quantification, is helpful in evaluation of the response to lamivudine treatment and diagnosis of the breakthrough hepatitis.

  3. Prevalence of hepatitis B surface antigen (HBsAg) in blood donors from Bombay.

    PubMed

    Satoskar, A; Ray, V

    1992-01-01

    Analysis of serum samples from 3104 blood donors from Bombay screened for hepatitis B surface antigen (HBsAg) by ELISA. HBsAg was detected in 4.7% of the subjects. Relatives showed a significantly higher prevalence of HBsAg than volunteer donors. There was no significant association between HBsAg positivity and a particular blood group.

  4. [Comparison of the clinical performance of the ECLusys HBsAg II assay with the Lumipulse f and HISCL 2000-i HBsAg screening assays].

    PubMed

    Sugiura, Aya; Iwahara, Kunihiro; Suga, Yasuyuki; Uchiyama, Sachinori; Maekawa, Masato

    2012-02-01

    We compared the ECLusys HBsAgII (ECL HBsAg) assay to the Lumipulse Forte (LPf HBsAg) and HISCL (HIS HBsAg) assays. Measurement of dilution panels for which the WHO HBsAg international reference panel was the parent specimen revealed that the ECL and HIS assays enabled detection to a theoretical level of 0.04 IU/mL, whereas the LPf assay enabled detection to a level of 0.08 IU/mL. In a specificity test using high RF positive specimens (n = 33), pregnancy specimens (n = 35), cytomegalovirus antibody positive specimens (n = 36), and high M protein positive specimens (n = 21) that were confirmed negative for HBsAg by the LPf assay, negative results were obtained for all specimens on the HIS assay, but the ECL assay yielded a positive result for one of the high RF positive specimens. This individual was suggested on further testing to be an HBV carrier who was strongly positive for HBc antibody. In HBsAg mutants detection test, the detection rate was 92.3% with the ECL assay and 69.2% with the HIS assay. In a correlation test using routinely collected clinical specimens (n = 155), including positive stock specimens, aside from the one case where the LPf assay gave a negative result but both the ECL and HIS assays gave positive results, all of the results were consistent for all specimens. The above results confirmed that the ECL assay is both highly sensitive and specific, and also enables a high rate of HBsAg mutant detection.

  5. Improved Detection of Hepatitis B Virus Surface Antigen by a New Rapid Automated Assay

    PubMed Central

    Weber, Bernard; Bayer, Anja; Kirch, Peter; Schlüter, Volker; Schlieper, Dietmar; Melchior, Walter

    1999-01-01

    The performance of hepatitis B virus (HBV) surface antigen (HBsAg) screening assays is continuously improved in order to reduce the residual risk of transfusion-associated hepatitis B. In a multicenter study, a new automated rapid screening assay, Elecsys HBsAg (Roche Diagnostics), was compared to well-established tests (Auszyme Monoclonal [overnight incubation] version B and IMx HBsAg [Abbott]). Included in the evaluation were 23 seroconversion panels; sera from the acute and chronic phases of infection; dilution series of various HBsAg standards, HBV subtypes, and S gene mutants; and isolated anti-HBV core antigen-positive samples. To challenge the specificity of the new assay, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. Elecsys HBsAg showed a higher sensitivity for HBsAg subtypes ad, ay, adw2, adw4, ayw1, ayw2, ayw4, and adr detection in dilution series of different standards or sera than Auszyme Monoclonal version B and/or IMx HBsAg. Acute hepatitis B was detected in 11 to 16 of 23 seroconversion panels between 2 and 16 days earlier with Elecsys HBsAg than with the alternative assays. Elecsys HBsAg and Auszyme Monoclonal version B detected HBsAg surface mutants with equal sensitivity. The sensitivity and specificity of Elecsys HBsAg were 100%. Auszyme Monoclonal version B had a 99.9% specificity, and its sensitivity was 96.6%. IMx HBsAg showed a poorer sensitivity and specificity than the other assays. In conclusion, Elecsys HBsAg permits earlier detection of acute hepatitis B and different HBV subtypes than the alternative assays. By using highly sensitive HBsAg screening assays, low-level HBsAg carriers among isolated anti-HBV core antigen-positive individuals can be detected. PMID:10405414

  6. A vaccine prepared from the 22 nm particles of surface hepatitis B antigen (HBsAg)

    SciTech Connect

    Karelin, V.P.; Babaeva, E.E.; Gubenko, E.F.; Kaulen, D.K.; Zhdanov, V.M.

    1980-01-01

    A method for obtaining a subunit inactivated vaccine preparation from the 22-nm particles of HBsAg is proposed. For inactivation of the residual infectious hepatitis B virus (HBV) the preparations were successively treated with 1% sodium dodecyl sulfate (SDS) and nucleases. In addition, thermal denaturation and ultraviolet irradiation of HBV DNA were used. As a control the biologic activity of a reference virus (SV40) was tested after the same treatment. The effectiveness of DNA inactivation was monitored by adding 3H-thymidine labeled reference virus to the vaccine preparations. The purified and inactivated HBsAg was adsorbed on Al2O3. Antigenicity was calculated on the basis of the determination of antibody in guinea pigs immunized with various doses of the vaccine, and the release of /sup 125/I- HBsAg from blood and kidneys in immunized and control mice was analyzed. Possible methods of inactivation and control of HBV vaccine is discussed.

  7. The underlying mechanisms for the "isolated positivity for the hepatitis B surface antigen (HBsAg)" serological profile.

    PubMed

    Pondé, Robério Amorim de Almeida

    2011-02-01

    During HBV infection, four structural antigen/antibody systems are observed: hepatitis B surface antigen (HBsAg) and its antibody (anti-HBs); the pre-S antigens associated with HBsAg particles and their antibodies; the particulate nucleocapsid antigen (HBcAg) and anti-HBc; and an antigen structurally related to HBcAg, namely HBeAg and its antibody (anti-HBe). Through the examination of this antigen-antibodies system, hepatitis B infection is diagnosed and the course of the disorder may be observed. Isolated HBsAg seropositivity is a peculiar serological pattern in HBV infection observed some times in routine laboratory. In most cases is not clear how this profile should be interpreted neither its significance. This pattern, however, may be associated with some clinical and laboratorial situations of great relevance, some of which will be addressed in this article.

  8. Multicenter Evaluation of the Elecsys Hepatitis B Surface Antigen Quantitative Assay

    PubMed Central

    Zacher, B. J.; Moriconi, F.; Bowden, S.; Hammond, R.; Louisirirotchanakul, S.; Phisalprapa, P.; Tanwandee, T.; Wursthorn, K.; Brunetto, M. R.; Wedemeyer, H.; Bonino, F.

    2011-01-01

    The Elecsys hepatitis B surface antigen (HBsAg) II quantitative assay is a new quantitative electrochemiluminescence immunoassay which uses onboard dilution and a simple algorithm to determine HBsAg levels expressed in international units (IU)/ml (standardized against the World Health Organization [WHO] Second International Standard). This study evaluated its performance using routine serum samples from a wide range of HBsAg carriers and patients with chronic hepatitis B (CHB). HBsAg levels were measured in serum samples collected independently by five centers in Europe, Australia, and Asia. Serial dilution analyses were performed to assess the recommended dilution algorithm and determine the assay range free of hook effect. Assay precision was also established. Following assessment of serial dilutions (1:100 to 1:1,000,000) of the 611 samples analyzed, 70.0% and 85.6% of samples tested with analyzers incorporating 1:100 (Elecsys 2010 and cobas e 411) and 1:400 (Modular Analytics E170) onboard dilution, respectively, fell within the linear range of the assay, providing a final result on the first test. No high-dose hook effect was seen up to the maximum HBsAg serum level tested (870,000 IU/ml) using the dilution algorithm. HBsAg levels were reliably determined across all hepatitis B virus (HBV) genotypes, phases of HBV infection, and stages of disease tested. Precision was high across all analyzers (% coefficient of variation [CV], 1.4 to 9.6; HBsAg concentrations, 0.1 to 37,300 IU/ml). The Elecsys HBsAg II quantitative assay accurately and reliably quantifies HBsAg in routine clinical samples. Onboard dilution minimizes retesting and reduces the potential for error. PMID:21880853

  9. Multicenter evaluation of the Elecsys hepatitis B surface antigen quantitative assay.

    PubMed

    Zacher, B J; Moriconi, F; Bowden, S; Hammond, R; Louisirirotchanakul, S; Phisalprapa, P; Tanwandee, T; Wursthorn, K; Brunetto, M R; Wedemeyer, H; Bonino, F

    2011-11-01

    The Elecsys hepatitis B surface antigen (HBsAg) II quantitative assay is a new quantitative electrochemiluminescence immunoassay which uses onboard dilution and a simple algorithm to determine HBsAg levels expressed in international units (IU)/ml (standardized against the World Health Organization [WHO] Second International Standard). This study evaluated its performance using routine serum samples from a wide range of HBsAg carriers and patients with chronic hepatitis B (CHB). HBsAg levels were measured in serum samples collected independently by five centers in Europe, Australia, and Asia. Serial dilution analyses were performed to assess the recommended dilution algorithm and determine the assay range free of hook effect. Assay precision was also established. Following assessment of serial dilutions (1:100 to 1:1,000,000) of the 611 samples analyzed, 70.0% and 85.6% of samples tested with analyzers incorporating 1:100 (Elecsys 2010 and cobas e 411) and 1:400 (Modular Analytics E170) onboard dilution, respectively, fell within the linear range of the assay, providing a final result on the first test. No high-dose hook effect was seen up to the maximum HBsAg serum level tested (870,000 IU/ml) using the dilution algorithm. HBsAg levels were reliably determined across all hepatitis B virus (HBV) genotypes, phases of HBV infection, and stages of disease tested. Precision was high across all analyzers (% coefficient of variation [CV], 1.4 to 9.6; HBsAg concentrations, 0.1 to 37,300 IU/ml). The Elecsys HBsAg II quantitative assay accurately and reliably quantifies HBsAg in routine clinical samples. Onboard dilution minimizes retesting and reduces the potential for error.

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

    PubMed Central

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

    2014-01-01

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

  11. Delayed type hypersensitivity (DTH) skin reaction to hepatitis B surface antigen (HBsAg) in patients with type B acute and chronic hepatitis.

    PubMed Central

    Nagafuchi, S; Kashiwagi, S; Hayashi, S; Inoue, T; Imayama, S; Takeshita, M; Kikuchi, M

    1985-01-01

    Skin reactivity to HBsAg was studied in patients with type B acute and chronic hepatitis and in healthy controls. HBsAg preparations containing 50 micrograms/ml of antigen both with and without alum elicited positive skin reactions in all seven anti-HBs+ persons. Histological examination of skin tissue from the reactive area using monoclonal antibodies to the cell surface of lymphocytes revealed accumulation of Leu-3a positive lymphocytes, showing that the inflammation was a typical delayed type hypersensitivity (DTH) reaction. Irrespective of the presence of HBeAg and anti-HBe, patients with chronic type B hepatitis responded clearly to PPD and SK/SD antigens, whereas they did not exhibit DTH skin reactivity to HBsAg. In contrast, although patients with acute type B hepatitis did not exhibit specific DTH a reaction to HBsAg in the acute period, they began to develop DTH skin reactivity to HBsAg in the convalescent phase of the disease preceding the appearance of anti-HBs antibody. It is suggested that DTH reaction to HBsAg might play an important role in the pathogenesis of type B viral hepatitis. Images Fig. 2 PMID:4075587

  12. Immunofluorescence detection of new antigen-antibody system (delta/anti-delta) associated to hepatitis B virus in liver and in serum of HBsAg carriers.

    PubMed Central

    Rizzetto, M; Canese, M G; Aricò, S; Crivelli, O; Trepo, C; Bonino, F; Verme, G

    1977-01-01

    A new antigen-antibody system associated with the hepatitis B virus and immunologically distinct from the HB surface, core, and e systems is reported. The new antigen, termed delta, was detected by direct immunofluorescence only in the liver cell nuclei of patients with HBsAg positive chronic liver disease. At present, the intrahepatic expression of HBcAg and delta antigen appears to be mutually exclusive. No ultrastructural aspect corresponding to the delta antigen could be identified under the electron microscope. delta antibody was found in the serum of chronic HBsAg carriers, with a higher prevalence in patients with liver damage. The nuclear fluorescence patterns of HBcAg and delta antigen were similar; it is only possible to discriminate between the two antigens by using the respective specific antisera. Images Figure PMID:75123

  13. Performance evaluation of new automated hepatitis B viral markers in the clinical laboratory: two quantitative hepatitis B surface antigen assays and an HBV core-related antigen assay.

    PubMed

    Park, Yongjung; Hong, Duck Jin; Shin, Saeam; Cho, Yonggeun; Kim, Hyon-Suk

    2012-05-01

    We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P < .0001). This observation could provide impetus for further research to elucidate the clinical usefulness of the qHBsAg and HBcrAg assays.

  14. Application of a Newly Developed High-Sensitivity HBsAg Chemiluminescent Enzyme Immunoassay for Hepatitis B Patients with HBsAg Seroclearance

    PubMed Central

    Shinkai, Noboru; Matsuura, Kentaro; Sugauchi, Fuminaka; Watanabe, Tsunamasa; Murakami, Shuko; Iio, Etsuko; Ogawa, Shintaro; Nojiri, Shunsuke; Joh, Takashi

    2013-01-01

    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring. PMID:23946517

  15. Application of a newly developed high-sensitivity HBsAg chemiluminescent enzyme immunoassay for hepatitis B patients with HBsAg seroclearance.

    PubMed

    Shinkai, Noboru; Matsuura, Kentaro; Sugauchi, Fuminaka; Watanabe, Tsunamasa; Murakami, Shuko; Iio, Etsuko; Ogawa, Shintaro; Nojiri, Shunsuke; Joh, Takashi; Tanaka, Yasuhito

    2013-11-01

    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring.

  16. Quantification of HBsAg: basic virology for clinical practice.

    PubMed

    Lee, Jung Min; Ahn, Sang Hoon

    2011-01-21

    Hepatitis B surface antigen (HBsAg) is produced and secreted through a complex mechanism that is still not fully understood. In clinical fields, HBsAg has long served as a qualitative diagnostic marker for hepatitis B virus infection. Notably, advances have been made in the development of quantitative HBsAg assays, which have allowed viral replication monitoring, and there is an opportunity to make maximal use of quantitative HBsAg to elucidate its role in clinical fields. Yet, it needs to be underscored that a further understanding of HBsAg, not only from clinical point of view but also from a virologic point of view, would enable us to deepen our insights, so that we could more widely expand and apply its utility. It is also important to be familiar with HBsAg variants and their clinical consequences in terms of immune escape mutants, issues resulting from overlap with corresponding mutation in the P gene, and detection problems for the HBsAg variants. In this article, we review current concepts and issues on the quantification of HBsAg titers with respect to their biologic nature, method principles, and clinically relevant topics.

  17. Defective antigen presentation by monocytes in ESRD patients not responding to hepatitis B vaccination: impaired HBsAg internalization and expression of ICAM-1 and HLA-DR/Ia molecules

    PubMed Central

    Barth, C.; Pollok, M.; Michałkiewicz, J.; Madaliński, K.; Maciejewski, J.; Baldamis, C. A.

    1995-01-01

    This study was undertaken to evaluate the monocyte function of uraemic non-responders to hepatitis B vaccination. Therefore, some parameters concerning antigen processing by monocytes (Mo) as antigen presenting cells (APC) were analysed. It was found that in uraemic non-responders, (1) the internalization of HBsAg by monocytes was significantly decreasjed—HBsAg complexed with specific IgG or as immune complex isolated from patients is better internalized compared with free HBsAg; (2) during antigen presentation the expression of adhesion (ICAM-1) and accessory (HLA-DR/Ia) molecules was significantly decreased in uraemic patients, especially in non-responders; and (3) impaired internalization of HBsAg as well as a decrease in ICAM-1 and HLA-DR/Ia expression, correlated well with the blunted proliferation of CD4+ T cells stimulated by autologous monocytes induced by HBsAg. PMID:18475616

  18. Comparison of Serum HBsAg Quantitation by Four Immunoassays, and Relationships of HBsAg Level with HBV Replication and HBV Genotypes

    PubMed Central

    Tuaillon, Edouard; Mondain, Anne-Marie; Nagot, Nicolas; Ottomani, Laure; Kania, Dramane; Nogue, Erika; Rubbo, Pierre-Alain; Pageaux, Georges-Philippe; Van de Perre, Philippe; Ducos, Jacques

    2012-01-01

    Background The decline in hepatitis B virus surface antigen (HBsAg) may be an early predictor of the viral efficacy of Hepatitis B virus (HBV) therapy. The HBsAg levels obtained by different immunoassays now need comparing and the relationships between levels of HBsAg and HBV DNA alongside HBsAg and genotype must be evaluated. Methodology/Principal Findings HBsAg levels were compared among 80 patients using the Abbott Architect assay, a commercial immunoassay approved for HBsAg detection and quantitation, and three other assays derived from immunoassays approved for HBsAg detection (manufactured by Diasorin, Bio-Rad and Roche). Good correlation was found between the Abbot vs. Diasorin, Bio-Rad and Roche assays with narrow 95% limits of agreement and small mean differences: −0.06 to 0.11, −0.09 log10 IU/mL; −0.57 to 0.64, −0.04 log10 IU/mL; −0.09 to 0.45, −0.27 log10 IU/mL, respectively. These agreements were not affected by genotypes A or D. HBsAg was weakly correlated with HBV DNA, whatever the HBsAg assay used: Abbott, ρ = 0.36 p = 0.001, Diasorin ρ = 0.34, p = 0.002; Bio-Rad ρ = 0.37, p<0.001; or Roche ρ = 0.41, p<0.001. This relationship between levels of HBsAg and HBV DNA seemed to depend on genotypes. Whereas HBsAg (Abbott assay) tended to correlate with HBV DNA for genotype A (ρ = 0.44, p = 0.02), no such correlation was significant for genotypes D (ρ = 0.29, p = 0.15). Conclusion/Significance The quantitation of HBsAg in routine clinical samples is comparable between the reference assay and the adapted assays with acceptable accuracy limits, low levels of variability and minimum discrepancy. While HBsAg quantitation is not affected by HBV genotype, the observed association between levels of HBsAg and HBV DNA seems genotype dependent. PMID:22403628

  19. Evaluation of turkey erythrocyte hemagglutination assay for the detection of hepatitis B antigen.

    PubMed Central

    Ramirez, R I; Tiku, M L; Ogra, P L

    1975-01-01

    A recently described hemagglutination test for hepatitis B antigen (HBsAg) using turkey erythrocytes coated with horse antibody to HBsAg purified by affinity column chromatography was evaluated on a comparative basis with 100 HBsAg-positive and -negative serum samples. The turkey erythrocyte hemagglutination test (TEHA) was found to be less sensitive than radioimmunoassay (RIA) but gave far better results than counterimmunoelectrophoresis. Quantitative titration of HBsAg in serial dilutions of the samples appeared to be more reliably performed by TEHA than by RIA. TEHA is a simple and sensitive technique for the detection of HBsAg and may offer several practical advantages over RIA. PMID:1236871

  20. Introduction to Antigen and Antibody Assays.

    PubMed

    Day, Michael J

    2015-12-01

    Serological tests are used widely in veterinary practice; most often in the diagnosis of infectious disease. Such tests may be used to detect antigen from an infectious agent within a biological sample or to detect the presence of serum antibody specific for the pathogen as evidence of immunological exposure. These tests are all based on the fundamental principles of interaction between antigenic epitopes and antibodies of either the immunoglobulin (Ig) G, IgM, IgA, or IgE classes. The relative concentration of specific antibody within a sample is traditionally determined by calculation of the titer of antibody. With few exceptions, the primary interaction between an antigen and antibody in vitro cannot be visualized and so serological tests generally employ a secondary indicator system based on the use of a polyclonal antiserum or monoclonal antibody. A range of such tests has been developed, but many in veterinary medicine are based on the principle of the enzyme-linked immunosorbent assay, which is described in detail in this article. The interpretation of serological tests must be made carefully, taking into consideration the sensitivity and specificity of the test and the possible reasons for false-positive and false-negative outcomes. PMID:27154595

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

    PubMed

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

    2016-02-01

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

  2. Immunochromatographic assay for quantitative and sensitive detection of hepatitis B virus surface antigen using highly luminescent quantum dot-beads.

    PubMed

    Shen, Jun; Zhou, Yaofeng; Fu, Fen; Xu, Hengyi; Lv, Jiaofeng; Xiong, Yonghua; Wang, Andrew

    2015-09-01

    Hepatitis B virus infection is one of the major causes of hepatitis, liver cirrhosis and liver cancer. In this study, we used highly luminescent quantum dot-beads (QBs) as signal amplification probes in the sandwich immunochromatographic assay (ICA) for ultrasensitive and quantitative detection of hepatitis B virus surface antigen (HBsAg) in human serum. Various parameters that influenced the sensitivity and stability of the QB-based ICA (QB-ICA) sensor were investigated. Two linear independent regression equations for detection of serum HBsAg were expressed with Y=0.3361X-0.0059 (R(2)=0.9983) for low HBsAg concentrations between 75 pg mL(-1) and 4.8 ng mL(-1), and Y=0.8404 X-2.9364 (R(2)=0.9939) for high HBsAg concentrations in the range from 4.8 ng mL(-1) to 75 ng mL(-1). The detection limit of the proposed ICA sensor achieved was 75 pg mL(-1), which is much higher than that of the routinely-used gold nanoparticle based ICA. The intra- and inter-assays recovery rates for spiked serum samples at HBsAg concentrations of 75 pg mL(-1), 3.75 ng mL(-1) and 18.75 ng mL(-1) ranged from 90.14% to 97.6%, and coefficients of variation were all below 7%, indicating that the QB-ICA sensor has an acceptable accuracy for HBsAg detection. Additionally, the quantitative method developed showed no false positive results in an analysis of 49 real HBsAg-negative serum samples, and exhibited excellent agreement (R(2)=0.9209) with a commercial chemiluminescence immunoassay kit in identifying 47 HBsAg-positive serum samples. In summary, due to its high fluorescence intensity, the sandwich QB-ICA sensor is a very promising point-of-care test for rapid, simple and ultrasensitive detection of HBsAg, as well as other disease-related protein biomarkers.

  3. Distribution of hepatitis B e antigen (HBeAg) and anti-HBe in carriers with different levels of HBsAg.

    PubMed

    Nath, N; Fang, C T; Fields, H A; Doto, I L; Maynard, J E

    1980-01-01

    Blood samples from 154 asymptomatic carriers of HBsAg were studied for the presence of HbeAg and anti-HBe using techniques of rheophoresis and a micro solid phase radioimmunoassay (micro-SPRIA). The level of HBsAg in each sample was determined by titration using reverse passive hemagglutination (RPHA) test. The significance of relationship between the titer of HBsAg, HBeAg, anti-HBe, and anti-HBc were statistically analyzed. Micro-SPRIA detected almost twice as many reactives for HBeAg and anti-HBe as were found by rheophoresis; the difference in sensitivity was significant (P less than 0.001). The mean HBsAg titer of 41 samples reactive for HBeAg was 11,181, while it was 3,032 for 92 samples reactive for anti-HBe. The remaining 23 samples with no detectable HBeAg or anti-HBe had a mean HBsAg titer of 1,018. The differences in the distribution of HBsAg among the three categories is statistically significant (P less than 0.005). HBeAg was most likely to be found in samples with higher concentrations of HBsAg.

  4. Hepatitis B surface antigen-specific cell-mediated immune responses in human chronic hepatitis B surface antigen carriers.

    PubMed Central

    Beutner, K R; Tiku, M L; Ogra, P L

    1978-01-01

    The presence of hepatitis B surface antigen (HBsAg) and antibody (anti-HBs), hepatitis B e antigen (HBeAg) and antibody (anti-HBe), the nature of T-cell function, and specific cell-mediated immunity to HBsAg were determined and evaluated serially in groups of subjects with chronic HBsAg carrier states and in seronegative controls. The techniques of in vitro lymphocyte transformation, spontaneous rosette formation, radioimmunoassay, reverse passive hemagglutination, passive hemagglutination, rheophoresis, and liver function tests were employed for these studies. For the lymphocyte transformation assay, multiple concentrations of phytohemagglutinin and purified HBsAg were used as stimulants. Cell-mediated immunity to HBsAg was detectable in 50% of the chronic HBsAg carriers (responders) at one or more concentrations of HBsAg. The remaining carriers (nonresponders) and controls failed to manifest HBsAg-specific lymphocyte transformation activity. The profile of the responders was characterized by elevated serum glutamic pyruvic transaminase levels, the presence of anti-HBe, high HBsAg titers, and the conspicuous absence of HBeAg in the serum. The nonresponders were characterized by normal serum glutamic pyruvic transaminase levels, the presence of HBeAg and anti-HBe, and lower HBsAg titers. These observations demonstrate the presence of specific cell-mediated immunity to HBsAg in chronic HBsAg carriers who manifest biochemical evidence of liver disease. PMID:80380

  5. The Lumipulse G HBsAg-Quant assay for screening and quantification of the hepatitis B surface antigen.

    PubMed

    Yang, Ruifeng; Song, Guangjun; Guan, Wenli; Wang, Qian; Liu, Yan; Wei, Lai

    2016-02-01

    Qualitative HBsAg assay is used to screen HBV infection for decades. The utility of quantitative assay is also rejuvenated recently. We aimed to evaluate and compare the performance of a novel ultra-sensitive and quantitative assay, the Lumipulse assay, with the Architect and Elecsys assays. As screening methods, specificity was compared using 2043 consecutive clinical routine samples. As quantitative assays, precision and accuracy were assessed. Sera from 112 treatment-naïve chronic hepatitis B patients, four patients undergoing antiviral therapy and one patient with acute infection were tested to compare the correlations. Samples with concurrent HBsAg/anti-HBs were also quantified. The Lumipulse assay precisely quantified ultra-low level of HBsAg (0.004 IU/mL). It identified additional 0.98% (20/2043) clinical samples with trance amount of HBsAg. Three assays displayed excellent linear correlations irrespective of genotypes and S-gene mutations (R(2)>0.95, P<0.0001), while minor quantitative biases existed. The Lumipulse assay did not yield higher HBsAg concentrations in samples with concomitant anti-HBs. Compared with other assays, the Lumipulse assay is sensitive and specific for detecting HBsAg. The interpretation of the extremely low-level results, however, is challenging. Quantitative HBsAg results by different assays are highly correlated, but they should be interpreted interchangeably only after conversion to eliminate the biases.

  6. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Plasmodium species antigen detection assays. 866.3402 Section 866.3402 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  7. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Plasmodium species antigen detection assays. 866.3402 Section 866.3402 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  8. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Plasmodium species antigen detection assays. 866.3402 Section 866.3402 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  9. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Plasmodium species antigen detection assays. 866.3402 Section 866.3402 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  10. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Plasmodium species antigen detection assays. 866.3402 Section 866.3402 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  11. Laboratory Validation of the Sand Fly Fever Virus Antigen Assay.

    PubMed

    Reeves, Will K; Szymczak, Mitchell Scott; Burkhalter, Kristen L; Miller, Myrna M

    2015-12-01

    Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses. PMID:26675463

  12. Laboratory Validation of the Sand Fly Fever Virus Antigen Assay.

    PubMed

    Reeves, Will K; Szymczak, Mitchell Scott; Burkhalter, Kristen L; Miller, Myrna M

    2015-12-01

    Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses.

  13. Monoclonal platelet antigen capture assays (MAIPA) and reagents: a statement.

    PubMed

    Kaplan, C; Freedman, J; Foxcroft, Z; Husebekk, A; Metcalfe, P; Muniz-Diaz, E; Ouwehand, W; Panzer, S; Rozman, P; Skogen, B

    2007-11-01

    This statement concerning the monoclonal-specific immobilization of platelet antigens (MAIPA) has been written on behalf of the International Society of Blood Transfusion--Working Party on Platelet Immunology. The MAIPA technique is considered as the gold standard reference technique in platelet immunology. The assay performed with reagents labelled for 'research only' is acceptable as long as it is regularly evaluated by participation of laboratories in national or international workshops held with reference laboratories. PMID:18070272

  14. Prevalence of hepatitis B surface antigen, hepatitis B e antigen and antibody, and antigen subtypes in atomic bomb survivors

    SciTech Connect

    Neriishi, K.; Kodama, K.; Akiba, S. |

    1995-11-01

    On the basis of previous studies showing an association between hepatitis B surface antigen (HBsAg) positivity and radiation exposure in atomic bomb (A-bomb) survivors, we investigated further the active state of hepatitis B virus (HBV) infection by incorporating tests of hepatitis B e antigen (HBeAg) and hepatitis B e antibody (anti-HBe) and HBsAg subtypes into our biennial health examinations. Among 6548 A-bomb survivors for whom HBsAg was assayed between July 1979 and July 1981, 129 persons were HBsAg positive. HBeAg and anti-HBe were measured in 104 of these persons and subtypes of HBsAg in 98 persons. Among those exposed to radiation (average liver dose 0.58 Sv), the odds ratio of HBsAg positivity tended to increase with radiation dose (P for trend = 0.024). The P values for association between the prevalence of HB e antigen and radiation dose were 0.094 and 0.17, respectively. The HB antigen subtype adr was predominant over other subtypes in both Hiroshima and Nagasaki, but the distribution of subtypes did not seem to differ in relation to radiation dose. These results suggested that A-bomb survivors remain in active state of HBV infection and that the mechanism(s) of seroconversion may be impaired. 29 refs., 6 tabs.

  15. Heterosandwich immunoswab assay for dengue virus Ns1 antigen detection.

    PubMed

    Ganguly, Advaita; Malabadi, Ravindra B; Loebenberg, Raimer; Suresh, Mavanur R; Sunwoo, Hoon H

    2014-01-01

    Dengue and the more severe dengue hemorrhagic fever have been a very critical public health problem globally. Millions of people especially in the tropical areas get infected with dengue. An efficient diagnostic is very important for early screening of dengue infection. In dengue-infected patients, the nonstructural protein NS1 is present on the surface of infected cells and secreted in plasma. The NS1 antigen is an important target for developing a quick diagnostic largely due to its long presence in the blood. We have developed a simple-to-use immunoswab-based diagnostic procedure employing monoclonal antibodies and the second-generation quadromas. The detection limit for NS1 has been established to be in the subnanogram range. The assay is very sensitive, has a visual end point, and also being extremely inexpensive. With this assay, screening time for a dengue-infected person would be very rapid. PMID:24211216

  16. Clinical utility of quantitative HBsAg in natural history and nucleos(t)ide analogue treatment of chronic hepatitis B: new trick of old dog.

    PubMed

    Tseng, Tai-Chung; Kao, Jia-Horng

    2013-01-01

    Using commercial quantitative assays, quantitative hepatitis B surface antigen (qHBsAg) has improved our understanding and management of chronic hepatitis B (CHB). The HBsAg level is highest in the immune tolerance phase, starts to decline during the immune clearance phase, and decreases slowly but progressively after hepatitis B e antigen (HBeAg) seroconversion. The HBsAg level is lowest in individuals with an inactive carrier state but higher in those who develop HBeAg-negative hepatitis. It has been shown that a reduction of HBsAg by 1 log IU/mL or more reflects improved host immune control of HBV infection. A combination of HBsAg <1000 IU/mL and HBV-DNA <2000 IU/mL can identify a 3-year inactive state in a genotype D HBeAg-negative carrier population. In the Asian-Pacific region, where HBV genotypes B and C are dominant, HBsAg levels of ≤10-100 IU/mL predict HBsAg loss over time. As to the prediction of disease progression, low-viremic carriers with HBsAg >1000 IU/mL have been shown to be at higher risks of HBeAg-negative hepatitis, cirrhosis, and hepatocellular carcinoma than those with HBsAg <1000 IU/mL. Although qHBsAg has been widely used in CHB patients receiving pegylated interferon therapy, the HBsAg decline is slow and does not correlate with HBV-DNA levels during nucleos(t)ide analogue (NUC) therapy. However, a rapid HBsAg decline during NUC therapy may identify patients who will finally clear HBsAg. A 6- to 12-monthly assessment of HBsAg level could be considered during NUC therapy. Taking these lines of evidence together, qHBsAg can complement HBV-DNA levels to optimize the management of CHB patients in our daily clinical practice.

  17. Antigen capture assay for detection of a 43-kilodalton Mycobacterium tuberculosis antigen.

    PubMed Central

    Wadee, A A; Boting, L; Reddy, S G

    1990-01-01

    This study describes the development of an enzyme-linked immunosorbent assay (ELISA) to detect Mycobacterium tuberculosis antigens in body fluids. A double-antibody sandwich procedure that used human and rabbit anti-M. tuberculosis immunoglobulin G antibodies was followed. The ELISA was able to detect as little as 0.8 micrograms of protein of M. tuberculosis sonic extract. Of 253 cerebrospinal fluid specimens submitted for analysis, 11 (4.3%) false-positive results were recorded. Analysis of 317 pleural and ascitic fluid specimens resulted in 6 (1.9%) false-positive recordings. No false-negative results were recorded for any of the body fluids tested. This technique is rapid (5.5 h) and sensitive, may be developed and used in many laboratories with limited resources, and may prove useful in the diagnosis of extrapulmonary and pulmonary tuberculoses. Analysis of these body fluids by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting indicated that the antibody used in the ELISA detects a mycobacterial antigen of 43 kDa. Such antigens were not detected in body fluids of nontuberculous patients. Images PMID:2126267

  18. Sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis.

    PubMed Central

    Espino, A M; Finlay, C M

    1994-01-01

    A sandwich enzyme-linked immunosorbent assay has been developed for the detection of Fasciola hepatica excretory secretory (ES) antigens in stool specimens of infected humans. The assay uses antibodies against F. hepatica ES antigens. A monoclonal antibody (ES78, mouse immunoglobulin G2a) was used to capture ES antigens, and a rabbit polyclonal antibody, peroxidase conjugate, was used to identify ES antigens. Thirteen of 14 patients with parasitological evidence of fascioliasis had a detectable concentration of ES antigens (more than 15 ng/ml). None of the stool specimens from controls and from patients with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay. When the 14 patients were retested 2 months after treatment, all of the specimens from the 11 parasitologically cured patients were negative by the antigen detection assay while the specimens from the 3 patients with persisting F. hepatica eggs in their stools remained positive. PMID:8126178

  19. Geographical variation in prevalence of hepatitis B virus DNA in HBsAg negative patients.

    PubMed

    Lo, Y M; Lo, E S; Mehal, W Z; Sampietro, M; Fiorelli, G; Ronchi, G; Tse, C H; Fleming, K A

    1993-04-01

    AIMS--To study the geographical variation of the prevalence of hepatitis B virus (HBV) DNA in hepatitis B surface antigen (HBsAg) negative subjects. METHODS--A nested polymerase chain reaction (PCR) assay was used to amplify the core region of HBV. The assay was able to detect 10 molecules of a full length HBV plasmid. RESULTS--When applied to HBsAg negative paraffin wax embedded liver samples from Italy, Hong Kong, and the United Kingdom, a geographical variation in the prevalence of HBV-DNA positivity was noted. Two of 18 (11%) of Italian samples and 2/29 (6.9%) of Hong Kong samples were positive for HBV-DNA while none of the 70 cases from the United Kingdom was positive by nested PCR. Contamination by plasmid DNA was excluded using a novel method based on heteroduplex formation. One HBV-DNA positive case had idiopathic chronic active hepatitis, but the diagnoses in the other three HBV-DNA positive cases did not suggest any aetiological connection between HBV-DNA positivity and liver pathology. CONCLUSIONS--HBV-DNA could be detected in the liver tissues of a proportion of HBsAg negative subjects. The prevalence of such cases is related to the endemic rate of a geographical region. The use of HBV PCR on paraffin wax embedded tissues will be valuable for future studies on the molecular epidemiology of HBV.

  20. Geographical variation in prevalence of hepatitis B virus DNA in HBsAg negative patients.

    PubMed

    Lo, Y M; Lo, E S; Mehal, W Z; Sampietro, M; Fiorelli, G; Ronchi, G; Tse, C H; Fleming, K A

    1993-04-01

    AIMS--To study the geographical variation of the prevalence of hepatitis B virus (HBV) DNA in hepatitis B surface antigen (HBsAg) negative subjects. METHODS--A nested polymerase chain reaction (PCR) assay was used to amplify the core region of HBV. The assay was able to detect 10 molecules of a full length HBV plasmid. RESULTS--When applied to HBsAg negative paraffin wax embedded liver samples from Italy, Hong Kong, and the United Kingdom, a geographical variation in the prevalence of HBV-DNA positivity was noted. Two of 18 (11%) of Italian samples and 2/29 (6.9%) of Hong Kong samples were positive for HBV-DNA while none of the 70 cases from the United Kingdom was positive by nested PCR. Contamination by plasmid DNA was excluded using a novel method based on heteroduplex formation. One HBV-DNA positive case had idiopathic chronic active hepatitis, but the diagnoses in the other three HBV-DNA positive cases did not suggest any aetiological connection between HBV-DNA positivity and liver pathology. CONCLUSIONS--HBV-DNA could be detected in the liver tissues of a proportion of HBsAg negative subjects. The prevalence of such cases is related to the endemic rate of a geographical region. The use of HBV PCR on paraffin wax embedded tissues will be valuable for future studies on the molecular epidemiology of HBV. PMID:8496385

  1. Geographical variation in prevalence of hepatitis B virus DNA in HBsAg negative patients.

    PubMed Central

    Lo, Y M; Lo, E S; Mehal, W Z; Sampietro, M; Fiorelli, G; Ronchi, G; Tse, C H; Fleming, K A

    1993-01-01

    AIMS--To study the geographical variation of the prevalence of hepatitis B virus (HBV) DNA in hepatitis B surface antigen (HBsAg) negative subjects. METHODS--A nested polymerase chain reaction (PCR) assay was used to amplify the core region of HBV. The assay was able to detect 10 molecules of a full length HBV plasmid. RESULTS--When applied to HBsAg negative paraffin wax embedded liver samples from Italy, Hong Kong, and the United Kingdom, a geographical variation in the prevalence of HBV-DNA positivity was noted. Two of 18 (11%) of Italian samples and 2/29 (6.9%) of Hong Kong samples were positive for HBV-DNA while none of the 70 cases from the United Kingdom was positive by nested PCR. Contamination by plasmid DNA was excluded using a novel method based on heteroduplex formation. One HBV-DNA positive case had idiopathic chronic active hepatitis, but the diagnoses in the other three HBV-DNA positive cases did not suggest any aetiological connection between HBV-DNA positivity and liver pathology. CONCLUSIONS--HBV-DNA could be detected in the liver tissues of a proportion of HBsAg negative subjects. The prevalence of such cases is related to the endemic rate of a geographical region. The use of HBV PCR on paraffin wax embedded tissues will be valuable for future studies on the molecular epidemiology of HBV. Images PMID:8496385

  2. Bovine viral diarrhea virus antigen detection across whole cattle hides using two antigen-capture enzyme-linked immunosorbent assays.

    PubMed

    Vander Ley, Brian L; Ridpath, Julia F; Sweiger, Shaun H

    2012-05-01

    Bovine viral diarrhea virus is a costly disease of cattle that can be controlled by vaccination, biosecurity, and removal of persistently infected cattle. Development and proficiency testing of assays to identify persistently infected cattle requires substantial quantities of known positive- and negative-sample material. The objective of this study was to determine what sections of bovine skin contained Bovine viral diarrhea virus antigen. Two commercially available antigen-capture enzyme-linked immunoassays were used to test subsamples representing the entire skin of 3 persistently infected calves. Both assays detected Bovine viral diarrhea virus antigen in the samples indicated for use by assay protocol. However, one assay identified all subsamples as positive, while the second assay identified 64.4% of subsamples as positive. These results show that use of samples other than those specified by the assay protocol must be validated for each individual assay. In this study, alternative sample sites and use of the entire hide for proficiency testing would be acceptable for only one of the assays tested.

  3. Immunologic studies of factor IX (Christmas factor). II. Immunoradiometric assay of factor IX antigen.

    PubMed

    Yang, H C

    1978-06-01

    A solid-phase two-site immunoradiometric assay has been developed which measures factor IX antigen levels as low as 0.0004 u per ml of plasma. In normal individuals, the factor IX antigen level correlated with the factor IX procoagulant level. In haemophilia B, 14 patients had markedly reduced antigen levels (less than 0.06 u/ml) and five had normal levels (greater than 0.60 u/ml).

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

    PubMed

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

    2006-02-01

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

  5. Evaluation of a novel chemiluminescent microplate enzyme immunoassay for hepatitis B surface antigen detection.

    PubMed

    Yang, Lin; Song, Liu-Wei; Fang, Lin-Lin; Wu, Yong; Ge, Sheng-Xiang; Li, Hui; Yuan, Quan; Zhang, Jun; Xia, Ning-Shao

    2016-02-01

    Hepatitis B virus surface antigen (HBsAg) is an important biomarker used in the diagnosis of hepatitis B virus (HBV) infection, but false-negative results are still reported in the detection of HBsAg using commercial assays. In this study, we evaluated the qualitative properties of a novel HBsAg chemiluminescence enzyme immunoassay (CLEIA) assay--WTultra. WHO standard sample dilution series and samples from low-level HBsAg carriers (<1 ng/mL) were used to evaluate the sensitivity of the WTultra assay. Boston Biomedica, Inc. (BBI) hepatitis B seroconversion panels were used to assess the ability of the WTultra assay to detect the window period. In addition, dilution series of 22 serum samples with different genotypes, serotypes and HBsAg mutations were used to assess the WTultra assay, and these were compared with other commercial assays. The lower detection limit of the WTultra assay was 0.012 IU/mL, and it showed a high sensitivity (97.52%, 95% CI, 94.95-99.00) in the detection of 282 low-level HBsAg carriers (<1 ng/mL). In samples with various HBV genotypes, serotypes and HBsAg mutations, the WTultra assay yielded 117 positive results in 132 samples, which was significantly higher than the results with the other four commercial assays (89, 83, 65 and 45, respectively, p<0.01). In the assays of mutant strains, the WTultra assay detected 82 positive results in 90 samples, which was significantly better than the results for the Hepanostika HBsAg Ultra (58 positive) and Architect (55 positive) (p<0.01) assays, which in turn were significantly better than the Murex V.3 (41 positive, p=0.026) and AxSYM V2 (29 positive, p<0.01) assays. However, in the detection of 42 samples of wild-type strains with various genotypes and serotypes, no significant differences were observed among the WTultra (35 positive), Architect (28 positive) and Hepanostika HBsAg Ultra (31 positive) assays. However, the WTultra assay detected significantly more samples than the Murex V.3 (24

  6. Diagnostic Accuracy of Seven Commercial Assays for Rapid Detection of Group A Rotavirus Antigens

    PubMed Central

    Fremy, Céline; Pillet, Sylvie; Mendes Martins, Lucile; Ambert-Balay, Katia; Aho, Serge L.; Pothier, Pierre

    2015-01-01

    Seven commercial immunochromatographic assays intended for the detection of group A rotavirus antigens in human stool samples were evaluated. These assays showed similar levels of diagnostic accuracy and were suitable for the detection of rotavirus in patients with acute gastroenteritis but missed some asymptomatic rotavirus shedding identified by real-time reverse transcription-PCR. PMID:26378280

  7. Optimisation of a micro-neutralisation assay and its application in antigenic characterisation of influenza viruses

    PubMed Central

    Lin, Yipu; Gu, Yan; Wharton, Stephen A; Whittaker, Lynne; Gregory, Victoria; Li, Xiaoyan; Metin, Simon; Cattle, Nicholas; Daniels, Rodney S; Hay, Alan J; McCauley, John W

    2015-01-01

    Objectives The identification of antigenic variants and the selection of influenza viruses for vaccine production are based largely on antigenic characterisation of the haemagglutinin (HA) of circulating viruses using the haemagglutination inhibition (HI) assay. However, in addition to evolution related to escape from host immunity, variants emerging as a result of propagation in different cell substrates can complicate the interpretation of HI results. The objective was to develop further a micro-neutralisation (MN) assay to complement the HI assay in antigenic characterisation of influenza viruses to assess the emergence of new antigenic variants and reinforce the selection of vaccine viruses. Design and setting A 96-well-plate plaque reduction MN assay based on the measurement of infected cell population using a simple imaging technique. Sample Representative influenza A (H1N1) pdm09, A(H3N2) and B viruses isolated between 2004 and 2013 Main outcome measures and results Improvements to the plaque reduction MN assay included selection of the most suitable cell line according to virus type or subtype, and optimisation of experimental design and data quantitation. Comparisons of the results of MN and HI assays showed the importance of complementary data in determining the true antigenic relationships among recent human influenza A(H1N1)pdm09, A(H3N2) and type B viruses. Conclusions Our study demonstrates that the improved MN assay has certain advantages over the HI assay: it is not significantly influenced by the cell-selected amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses, and it is particularly useful for antigenic characterisation of viruses which either grow to low HA titre and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells. PMID:26073976

  8. Clinical and Virological Characteristics of Chronic Hepatitis B Patients with Coexistence of HBsAg and Anti-HBs.

    PubMed

    Liu, Yong; Zhang, Le; Zhou, Jin-Yong; Pan, Jinshun; Hu, Wei; Zhou, Yi-Hua

    2016-01-01

    Coexistence of hepatitis B surface antigen (HBsAg) and antibody against HBsAg (anti-HBs) comprises an atypical serological profile in patients with chronic hepatitis B virus (HBV) infection. In this study, in total 94 patients with coexisting HBsAg and anti-HBs and 94 age- and sex-matched patients with positive HBsAg were characterized by quantitatively measuring HBsAg and HBV DNA, sequencing large S genes, and observing clinical features. Compared with common hepatitis B patients, the patients with coexisting HBsAg and anti-HBs had lower HBsAg and HBV DNA levels. These two groups had similar rate of pre-S deletion mutations. However, in patients with coexisting HBsAg and anti-HBs, more amino acid substitutions in the a determinant of S gene were observed in HBV genotype C, but not in genotype B. Fourteen patients with coexisting HBsAg and anti-HBs were followed up for an average of 15.5 months. There were no significant changes in the levels of HBsAg, anti-HBs, HBV DNA and ALT over the follow-up period. Compared with the baseline sequences, amino acid substitutions in the MHR of HBsAg occurred in 14.3% (2/14) patients. In conclusion, coexistence of HBsAg and anti-HBs may be associated with higher frequency of mutations in the a determinant of HBV genotype C.

  9. Evaluation of nonstructural 1 antigen assays for the diagnosis and surveillance of dengue in Singapore.

    PubMed

    Pok, Kwoon-Yong; Lai, Yee-Ling; Sng, Joshua; Ng, Lee-Ching

    2010-12-01

    Early and accurate diagnosis of dengue is imperative for disease surveillance, which helps in the control of dengue in endemic countries. In this study, we evaluated the performance of three commercially available dengue nonstructural 1 (NS1) antigen assays (Bio-Rad Platelia™ Dengue NS1 Antigen ELISA, PanBio Dengue Early ELISA, and Bio-Rad Dengue NS1 Antigen Strip test) and compared them with reverse-transcription polymerase chain reaction (RT-PCR) and other commercially available serological assays for the diagnosis of dengue. The analysis showed RT-PCR to be the most sensitive and specific (100%) diagnostic method during the first 3 days of fever. The overall sensitivity of dengue NS1 antigen assays within the same period was 81.7%, indicating their potential role as a cost-effective and convenient alternative method to RT-PCR for the diagnosis of dengue fever in a primary healthcare setting. However, reduced sensitivity in detecting secondary dengue infections was one of the drawbacks of dengue NS1 antigen assays. Nonetheless, it remains a useful assay for the early detection of dengue and hence could play an important role in routine surveillance efforts to control dengue outbreaks in Singapore.

  10. Immune Responses to HBsAg Conjugated to Protein D of Non-Typeable Haemophilus influenzae in Mice

    PubMed Central

    Su, Qiudong; Yi, Yao; Qiu, Feng; Lu, Xuexin; Ding, Junying; Jia, Zhiyuan; Tian, Ruiguang; Gao, Yan; Bi, Shengli

    2015-01-01

    Background Hepatitis B vaccine that contains an aluminum hydroxide adjuvant induces apoptotic death of Hepa 1–6 cells. Difficult-to-degrade chemical additives in vaccines effectively enhance vaccine immunogenicity, but also affect the host tissue. Identification of bio-molecules that are readily degraded and compatible in vivo as an adjuvant is important for vaccine research. The hapten–carrier effect suggests that stimulation of helper T (Th) cells by carrier adjuvants is feasible. Protein D (PD) of non-typeable Haemophilus influenzae covalently conjugated to some polysaccharide vaccines has been confirmed to convert T-cell independent (TI) antigens into T-cell dependent (TD) antigens, and elicit strong T-cell responses ultimately. Herein, we would substitube PD for aluminum hydroxide adjuvant in Hepatitis B vaccine. Methods and results Truncated PD (amino acids 20–364) was expressed in Escherichia coli and purified by (NH4)2SO4 precipitation and DEAE chromatography. After evaluation of antigenicity by western blotting, PD was covalently conjugated to yeast-derived recombinant HBsAg by cross-linking with glutaraldehyde. Intramuscular immunization with the conjugate induced higher level of HBsAg-specific antibody than did HBsAg alone (p < 0.05), and was comparable to commercial Hepatitis B vaccine. During the surveillance period (days 35–105), anti-HBs titers were hold high. Moreover, the conjugated vaccine enhanced Th1 immune responses, while Th2 responses were also activated and induced an antibody response, as determined by IFN-γ ELISPOT and IgG1/IgG2a ratio assays. Conclusions Recombinant truncated PD covalently conjugated to HBsAg antigen enhanced the immunogenicity of the antigen in mice simultaneously by humoral and cellular immune response, which would facilitate therapeutic hepatitis B vaccines. PMID:25689855

  11. Tuberculosis Diagnosis: Assay Optimization, Validation, and Antigens for Specific Diagnosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interferon (IFN)-gamma release assays (i.e. Bovigam®, Prionics AG) are components of tuberculosis (TB) eradication programs in many countries. Because this test relies on functional leukocytes, environmental conditions before and during the in vitro culture period have the potential to influence the...

  12. Immunoradiometric assay of factor VIII related antigen, with observations in 32 patients with von Willebrand's disease.

    PubMed

    Ruggeri, Z M; Mannucci, P M; Jeffcoate, S L; Ingram, G I

    1976-06-01

    A solid phase non-competitive immunoradiometric assay (IRMA) has been developed which allows measurement of factor VIII related antigen (VIIIR:AG) levels in normal plasma as low as 2.5 x 10(-4) U/ml. The assay is based on the extraction of VIIIR:AG from test plasma by means of polystrene tubes coated with a specific unlabelled anti-VIIIR:AG rabbit antiserum and subsequent labelling of the extracted antigen with 125I-labelled anti-VIIIR:AG rabbit IgG. PMID:1083743

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  14. Antigen specific killing assay using CFSE labeled target cells.

    PubMed

    Durward, Marina; Harms, Jerome; Splitter, Gary

    2010-11-09

    Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

  15. Stabilization and immune response of HBsAg encapsulated within poly(lactic-co-glycolic acid) microspheres using HSA as a stabilizer.

    PubMed

    Xu, Wenjuan; He, Jintian; Wu, Guanghao; Xiong, Fangfang; Du, Huijuan; Wang, Gaizhen

    2015-12-30

    The aim of this study was to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres containing hepatitis B virus surface antigen (HBsAg) using human serum albumin (HSA) as a stabilizer. Lyophilization and emulsification of HBsAg solution with dichloromethane caused a considerable loss of HBsAg antigenicity. Thus, the effects of HSA and trehalose on HBsAg recovery during lyophilization and emulsification were investigated. Adding HSA to HBsAg solutions significantly improved antigen recovery to >90% during lyophilization and emulsification. The effects of co-encapsulated HSA on the characteristics of the PLGA microspheres and stability of HBsAg released from the microspheres were also investigated. The in vitro release test showed that HBsAg was released from the PLGA microspheres continuously over seventy days. A large amount of released HBsAg was inactive without co-encapsulation of HSA. On the contrary, with HSA co-encapsulation, the released HBsAg retained approximately 90% of its antigenicity. The single injection of the HBsAg-HSA-loaded PLGA microspheres in rats resulted in higher anti-HBsAg IgG and Th1 cytokine levels than the single injection of the HBsAg-loaded microspheres or two injections of the conventional aluminum-adjuvanted HBsAg vaccine. Based on these findings, the HBsAg-HSA-loaded PLGA microspheres could be an effective carrier for HBsAg and form a promising depot system.

  16. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    PubMed

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C

    2010-01-01

    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.

  17. A simple and safe method for single HLA-antigen-typing by a solid phase assay.

    PubMed

    Häcker-Shahin, B; Giannitsis, D J

    1991-01-01

    A rapid solid phase assay for detection of single HLA-antigens on platelets was developed. The 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 incubation with commercially available anti-HLA-sera the bound anti-HLA-specific antibodies directed against HLA-antigens present on the platelets were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an HLA-antigen, was indicated by a slight red cell adherence over the reaction surface. In the absence of the HLA-antigen no binding occurred and the indicator red cells formed a small red disc-like pellet.

  18. Sensitive immunoradiometric assay for the detection of Paracoccidioides brasiliensis antigens in human sera.

    PubMed Central

    Ferreira-da-Cruz, M F; Galvão-Castro, B; Daniel-Ribeiro, C T

    1991-01-01

    In the present study we report the standardization of an immunoradiometric assay (IRMA) for detection of Paracoccidioides brasiliensis circulating antigens that could be useful in the diagnosis and prognosis of paracoccidioidomycosis. For this purpose we studied the reactivities of P. brasiliensis and other mycotic antigens with rabbit polyclonal anti-P. brasiliensis antibodies (immunoglobulin G) in order to evaluate the sensitivity and specificity of an IRMA for detecting P. brasiliensis antigens. The results were compared with those obtained by the double immunodiffusion test, the standard technique for the serodiagnosis of paracoccidioidomycosis. By using the immunoglobulin G fraction of rabbit antisera (900 ng per well), it was possible to detect up to 3.6 ng (0.12 micrograms/ml) of cellular antigen and 360 ng (12 micrograms/ml) of metabolic antigen in contrast to the double immunodiffusion test that could detect only 12 micrograms (1.2 mg/ml) of both antigens. IRMA was shown to be feasible and very sensitive and may therefore help, together with clinical data, in establishing early diagnosis and assessing disease activity. It could also allow the study of relationships between P. brasiliensis circulating antigens and host defense mechanisms during the disease. PMID:1907608

  19. [Diagnostic value of the quantitative HBsAg determination in hepatitis B infections].

    PubMed

    van Helden, J; Weiskirchen, R

    2016-04-01

    Introduction of systematic hepatitis B vaccination has lead to a strong decrease of new infections, but there are still a high numbers of chronically infected persons suffering on long-term complications. Using quantitative assays for the determination of HbsAg (qHBsAg) has improved our understanding of chronic hepatitis B (CHB). The concentrations of HBsAg are strongly varying through the different stages of infection. The quantitative determination of HBsAg does not only yield in additional information to the infection activity, but also provides data for an improved follow up independent from the virus load. As to the prediction of disease progression, low-viremic carriers with high HbsAg levels have been shown to be at higher risk of HBeAg negative hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Although, quantitative HBsAg determination has been widely used in CHB patients receiving pegylated interferon therapy, the HbsAg decline is slow compared to HBV-DNA levels during nucleos(t)ide analogue (NUC) therapy. However a rapid HbsAg decline during NUC therapy may identify patients who will finally clear HbsAg. A 6- to 12-monthly assessment of HbsAg level could be considered during NUC therapy. Taking these lines of evidence together, qHBsAg can complement HBV-DNA levels to optimize the management of CHB patients.

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

    PubMed Central

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

    2016-01-01

    Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized “gold standard” panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the “gold standard” diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population. PMID:27070145

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

    PubMed

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

    2016-01-01

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

  2. Exploring the Dynamic Range of the Kinetic Exclusion Assay in Characterizing Antigen-Antibody Interactions

    PubMed Central

    Bee, Christine; Abdiche, Yasmina N.; Stone, Donna M.; Collier, Sierra; Lindquist, Kevin C.; Pinkerton, Alanna C.; Pons, Jaume; Rajpal, Arvind

    2012-01-01

    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 (KD 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 KD 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. PMID:22558410

  3. Comparison of the Abbott Architect i2000 assay, the Roche Modular Analytics E170 assay, and an immunoradiometric assay for serum hepatitis B virus markers.

    PubMed

    Kim, Hyunjung; Oh, Eun-Jee; Kang, Mi-Sook; Kim, Sung Hoon; Park, Yeon-Joon

    2007-01-01

    Serum hepatitis B virus (HBV) markers are the most important data for epidemiological screening and clinical diagnosis of HBV infection, especially in endemic areas. We compared the results of the Roche Modular Analytics E170 assay, the Abbott Architect i2000 assay, and an immunoradiometric assay (IRMA) for HBV surface antigen (HBsAg), anti-HBV surface antigen (anti-HBs), HBV e antigen (HBeAg), and anti-HBV e antigen (anti-HBe). A number of serum samples (264, 263, 224, and 202 for HBsAg, anti-HBs, HBeAg, and anti-HBe, respectively) were studied. For samples giving discrepant results for HBeAg between methods, real-time PCR assays were performed. The concordance rates among the three methods were high for HBsAg (100%) and HBeAg (94.6), but low for anti-HBs (91.6%) and anti-HBe (82.2%). For anti-HBs, which could be measured quantitatively by the Modular E170 and Architect i2000 procedures, discrepant results were observed at low levels of anti-HBs. For anti-HBe, the positive rate was highest with Modular E170 (60.9%) followed by the IRMA kit (54.1%) and Architect i2000 (51.0%). This study shows substantial differences between the assay results by the three methods, which should be taken into account in determinations of serum HBV markers.

  4. Europium nanoparticle-based simple to perform dry-reagent immunoassay for the detection of hepatitis B surface antigen.

    PubMed

    Talha, Sheikh M; Salminen, Teppo; Juntunen, Etvi; Spangar, Anni; Gurramkonda, Chandrasekhar; Vuorinen, Tytti; Khanna, Navin; Pettersson, Kim

    2016-03-01

    Hepatitis B infection, caused by hepatitis B virus (HBV), presents a huge global health burden. Serological diagnosis of HBV mainly relies on the detection of hepatitis B surface antigen (HBsAg). Although there are high sensitivity commercial HBsAg enzyme immunoassays (EIAs) available, many low-resource laboratories lacking trained technicians continue to use rapid point-of-care assays with low sensitivities for HBsAg detection, due to their simplicity to operate. We developed a time-resolved fluorometric dry-reagent HBsAg immunoassay which meets the detection limit of high sensitivity EIAs but is simple to operate. To develop the assay, anti-HBsAg monoclonal antibody coated on europium nanoparticles was dried atop of biotinylated anti-HBsAg polyclonal antibody immobilized on streptavidin-coated microtiter wells. To test a sample in dry-reagent assay, serum sample and assay buffer were added to the wells, incubated, washed and europium signals were measured. The assay showed a detection limit of 0.25 ng/ml using HBsAg spiked in serum sample. When evaluated with 24 HBV positive and 37 negative serum samples, assay showed 100% sensitivity and specificity. Assay wells are stable for at least 26 weeks when stored at 4°C, and can tolerate elevated temperatures of up to 35°C for two weeks. The developed assay has high potential to be used in low-resource laboratories.

  5. Occult infection related hepatitis B surface antigen variants showing lowered secretion capacity

    PubMed Central

    Kim, Hong; Lee, Seoung-Ae; Won, You-Sub; Lee, HyunJoo; Kim, Bum-Joon

    2015-01-01

    AIM: To elucidate the molecular mechanisms underlying hepatitis B virus (HBV) occult infection of genotype C. METHODS: A total of 10 types of hepatitis B surface antigen (HBsAg) variants from a Korean occult cohort were used. After a complete HBV genome plasmid mutated such that it does not express HBsAg and plasmid encoding, each HBsAg variant was transiently co-transfected into HuH-7 cells. The secretion capacity and intracellular expression of the HBV virions and HBsAgs in their respective variants were analyzed using real-time quantitative polymerase chain reaction assays and commercial HBsAg enzyme-linked immunosorbent assays, respectively. RESULTS: All variants exhibited lower levels of HBsAg secretion into the medium compared with the wild type. In particular, in eight of the ten variants, very low levels of HBsAg secretion that were similar to the negative control were detected. In contrast, most variants (9/10) exhibited normal virion secretion capacities comparable with, or even higher than, the wild type. This provided new insight into the intrinsic nature of occult HBV infection, which leads to HBsAg sero-negativeness but has horizontal infectivity. Furthermore, most variants generated higher reactive oxidative species production than the wild type. This finding provides potential links between occult HBV infection and liver disease progression. CONCLUSION: The presently obtained data indicate that deficiency in the secretion capacity of HBsAg variants may have a pivotal function in the occult infections of HBV genotype C. PMID:25684944

  6. Pi overlapping ring systems contained in a homogeneous assay: a novel homogeneous assay for antigens

    NASA Astrophysics Data System (ADS)

    Kidwell, David A.

    1993-05-01

    A novel immunoassay, Pi overlapping ring systems contained in a homogeneous assay (PORSCHA), is described. This assay relies upon the change in fluorescent spectral properties that pyrene and its derivatives show with varying concentration. Because antibodies and other biomolecules can bind two molecules simultaneously, they can change the local concentration of the molecules that they bind. This concentration change may be detected spectrally as a change in the fluorescence emission wavelength of an appropriately labeled biomolecule. Several tests of PORSCHA have been performed which demonstrate this principle. For example: with streptavidin as the binding biomolecule and a biotin labeled pyrene derivative, the production of the excimer emitting at 470 nm is observed. Without the streptavidin present, only the monomer emitting at 378 and 390 nm is observed. The ratio of monomer to excimer provides the concentration of unlabeled biotin in the sample. Approximately 1 ng/mL of biotin may be detected with this system using a 50 (mu) l sample (2 X 10-16 moles biotin). The principles behind PORSCHA, the results with the streptavidin/biotin system are discussed and extensions of the PORSCHA concept to antibodies as the binding partner and DNA in homogeneous assays are suggested.

  7. An enzyme-linked immunosorbent assay for the detection of Entamoeba histolytica antigens in faecal material.

    PubMed

    Grundy, M S; Voller, A; Warhurst, D

    1987-01-01

    This paper describes a method for the detection of Entamoeba histolytica antigens in stool samples using a multi-layer ELISA. The method is sensitive and specific, showing no interference with other intestinal parasites, e.g. E. coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii, Hymenolepis nana, Giardia lamblia, Trichomonas and Ascaris. The method provides a rapid and simple screening assay for E. histolytica infections and should assist in diagnosis and epidemiological studies of the disease. PMID:2895514

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

    PubMed Central

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

    2004-01-01

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

  9. The ADVIA Centaur infectious disease assays: a technical review.

    PubMed

    Dwyer, Robert

    2004-04-01

    Bayer Healthcare, Diagnostics Division, has developed several new several new assays for hepatitis B, hepatitis C and HIV 1/O/2 on the ADVIA Centaur Immunoassay system. The panel for detection of markers for hepatitis B infection includes assays for hepatitis B surface antigen (HBsAg) and HBsAg. Confirmatory, antibodies to hepatitis B surface antigen (anti-HBs), IgM and IgG antibodies to hepatitis B core antigen (anti-HBc Total) and IgM antibodies to hepatitis B core antigen (anti-HBc IgM). The hepatitis B panel is complemented by third generation assays for anti-HCV and anti-HIV that concomitantly detects HIV-1 (including group O) and HIV-2. The features of the ADVIA Centaur and the design of the assays combine to deliver state-of-the-art performance. Features, such as disposable sample pipette tips to prevent sample carryover, clot detection and management to verify sample addition, reagent integrity checks to assure accurate reagent addition, auto repeat capabilities and the capacity to maintain up to 30 assays onboard at 2-8 degrees C all contribute to the systems's utility for infectious disease testing. Furthermore, the flexibility of the ADVIA Centaur has allowed for selection of formats to provide optimal assay performance. The anti-HBs, anti-HBc Total and anti-HIV 1/O/2 assays are antigen-bridging assays. The anti-HBc IgM assay is a class-capture two step assay configured for detection of IgM anti-HBc antibodies during acute HBV infection. The anti-HCV assay is an indirect assay utilizing streptavidin-coated microparticles preformed with a combination of recombinant and synthetic peptide antigens from the NS2, NS4, NS5 and core regions of the HCV genome. The HBsAg assay is a sandwich assay with an incubation time of 28 minutes and the HBsAg Confirmatory assay is a fully automated neutralization assay. The availability of these assays on the ADVIA Centaur Immunoassay system enables laboratories to consolidate other immunoassays along with the infectious

  10. An analytical solution to the characterization of antigen-antibody interactions by kinetic exclusion assay.

    PubMed

    Winzor, Donald J

    2013-07-01

    A simpler derivation of the basic expression for the dependence of fluorimetric response ratio (R(Ag)/Ro) on free antigen concentration has demonstrated the universal invalidity of the analysis that is incorporated into the manufacturer's software for determining immunoaffinities by kinetic exclusion assay, and traced the error to inadequate allowance for antibody bivalence in the solution phase of the assay. An analytical solution to the quantitative characterization of antigen-antibody interactions from the dependence of R(Ag)/Ro on total antigen concentration is also described, thereby eliminating the necessity for the extensive simulative procedures employed in current determinations of dissociation constants by kinetic exclusion assay. In the illustrative application of this analytical approach to published results on the interaction between a metal chelate (cadmium-ethylenediaminetetraacetic acid, Cd-EDTA) and an elicited monoclonal antibody, the analytical processing of the data has been performed on a calculator. However, there is no need to replace the more sophisticated procedure that is incorporated into the Sapidyne software provided that programming changes are made to rectify the erroneous equation on which the simulative analysis is based. PMID:23535275

  11. Evaluation of the performance of four methods for detection of hepatitis B surface antigen and their application for testing 116,455 specimens.

    PubMed

    Liu, Can; Chen, Tianbin; Lin, Jinpiao; Chen, Huijuan; Chen, Jing; Lin, Sheng; Yang, Bin; Shang, Hongyan; Ou, Qishui

    2014-02-01

    Hepatitis B surface antigen (HBsAg) is a crucial serum marker for the diagnosis of hepatitis B virus (HBV) infection. It is imperative to compare test results from different detection methods based on different principles. Four methods, chemiluminescent microparticle immunoassay (CMIA), electrochemiluminescent immunoassay (ECLIA), enzyme-linked immunosorbent assay (ELISA) and golden immunochromato-graphic assay (GICA) were applied to test the HBsAg level in 250 specimens. According to the EP12-A2 and EP15-A2 documents from Clinical and Laboratory Standards Institute (CLSI), the concentration at which repeated results are 50% positive (C50) of HBsAg detected by CMIA, ECLIA, ELISA and GICA was 0.05, 0.08, 0.15 and 15.0IU/ml, respectively. When the detection concentration of HBsAg was 0.5IU/ml, the imprecision degree of CMIA, ECLIA and ELISA was 8.1%, 5.9% and 14.9% respectively. When detecting high HBsAg level (≥20.0IU/ml) and HBsAg negative specimens, the consistency of the four methods was high, while for the low level (0.05-20.0IU/ml), the consistency was poor (except for the CMIA and ECLIA, P<0.05). When evaluation of the four methods in qualitative diagnosis of HBsAg level in the 116,455 specimens, there was no significant discrepancy among CMIA, CMIA and ECLIA, however, GICA was significantly different from the other 3 methods. Compared with CMIA, the false negative rate of ECLIA, ELISA and GICA was 0.2%, 1.3% and 12.3% respectively. In conclusion, GICA was only suitable for the preliminary screening of HBsAg positive individuals and ELISA can be applied to the qualitative diagnosis of HBsAg. Both CMIA and ECLIA were suitable for the quantitative determination of HBsAg.

  12. Immunoradiometric assay for quantitation of Dirofilaria immitis antigen in dogs with heartworm infections

    SciTech Connect

    Hamilton, R.G.; Scott, A.L.

    1984-10-01

    An immunoradiometric assay (IRMA) was developed, optimized, and validated for detection of parasite-specific antigen in sera from hosts with filarial infections, using Dirofilaria immitis in dogs as a model. The precision, reproducibility, and parallelism of the IRMA were examined, using precision profile analysis. The IRMA had acceptable precision and reproducibility (less than 15% intra-assay coefficient of variation (CV)) over a working range of 10 to 2000 ng of D immitis-antigen (AG)/ml. The IRMA parallelism (agreement between dilutions) was acceptable (less than 10% interdilutional CV) with laboratory-spiked D immitis AG sera containing no D immitis-antibody (AB). However, it was not acceptable (greater than 20% interdilutional CV) for analysis of sera from naturally infected dogs containing D immitis AB, probably due to dissociation of immune-complexed AG with increasing serum dilution. Nonparallelism limited the accuracy of binding data interpolation from the standard curve. Specificity of the IRMA was enhanced by preabsorption of the radiolabeled detection antibody with Toxocara canis AG before use. Varying amounts of D immitis AG (22 to 1000 ng/ml) were detected in 42% (20/48) of microfilaremic dogs. The presence of AG-specific AB at concentrations as low as 1 microgram/ml reduced the ability of the IRMA to detect D immitis AG. Factors that influence the accuracy and sensitivity of immunoassays for circulating filarial antigens are discussed.

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

    ERIC Educational Resources Information Center

    Russo, A. J.; And Others

    1984-01-01

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

  14. Development of simian immunodeficiency virus isolation, titration, and neutralization assays which use whole blood from rhesus monkeys and an antigen capture enzyme-linked immunosorbent assay.

    PubMed

    Lohman, B L; Higgins, J; Marthas, M L; Marx, P A; Pedersen, N C

    1991-10-01

    Assays that use rhesus macaque whole blood and an antigen capture enzyme-linked immunosorbent assay for the simian immunodeficiency virus (SIV) p27 core protein were developed for the isolation of SIV from the blood of infected animals, the titration of infectivity of SIV inocula, and the quantitation of virus neutralizing antibodies in serum. These assays required small amounts of whole blood, were adaptable to a microtiter format, and used substrates mainly of rhesus macaque origin.

  15. [Basic and clinical evaluation of an immunoradiometric competitive inhibition assay for 2----6 sialyl Lewis a antigen--1. Evaluation of assay conditions and normal values].

    PubMed

    Imura, H; Takahashi, T; Matsuda, T; Yoshida, O; Ohkura, H; Seitetsu, Y; Seino, Y; Ishii, M; Kuwabara, M; Ariyoshi, Y

    1989-06-01

    We describe an immunoradiometric competitive inhibition assay of the serum levels of the 2----6 sialyl Lewisa antigen, using "SLA 2-6 Otsuka" kits. The assay required only duplicate 50-microliters samples, and the concentration of 2----6 sialyl Lewisa antigen in serum was determined by reference to a standard curve ranging from 0 to 160 arbitrary U/ml. The intra- and inter-assays reproducibilities were good and analytical recovery of antigen were excellent. The serum levels of the antigen were highly dependent on the Lewis blood types of the tested individuals; i.e., the levels of the antigen in the sera of the Lewisa-b- individuals were significantly lower than those of the antigen obtained with the Lewisa+b- and Lewisa-b+ individuals. The cut-off value (42 U/ml) was obtained as mean + 2SD, which was carefully calculated from the antigen levels in sera of the non-Lewisa-b- individuals.

  16. Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

    PubMed

    Yeow, Natasha; McLiesh, Heather; Guan, Liyun; Shen, Wei; Garnier, Gil

    2016-07-01

    A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test. PMID:27185543

  17. Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

    PubMed

    Yeow, Natasha; McLiesh, Heather; Guan, Liyun; Shen, Wei; Garnier, Gil

    2016-07-01

    A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

  18. Evaluation of a two-site immunoradiometric assay for measuring noncomplexed (free) prostate-specific antigen.

    PubMed

    Cuny, C; Pham, L; Kramp, W; Sharp, T; Soriano, T F

    1996-08-01

    Serum prostate-specific antigen (PSA) in men is present as two different molecular forms separable by gel-filtration chromatography (GFC). We have evaluated a two-site IRMA that measures only the noncomplexed (free) form of PSA (F-PSA). Verification that the F-PSA assay measures solely F-PSA was obtained by assaying GFC-fractionated serum samples with both the F-PSA IRMA and a commercial PSA assay that measures total PSA (T-PSA: F-PSA plus alpha 1-antichymotrypsin-complexed PSA). The F-PSA assay detected only the 30-kDa peak corresponding to the free form of PSA, whereas the T-PSA assay detected two peaks: complexed PSA at approximately 90 kDa and F-PSA at approximately 30 kDa. The F-PSA assay had an analytical detection limit of 0.03 microgram/L and a measuring range up to 50 micrograms/L. The intraassay CV was 1.7-10% in the concentration range of 0.2-30 micrograms/L. The interassay CV was 3.4-12.5% in the same concentration range. Dilution and recovery studies showed no significant deviation from linearity across the assay range. The assay was insensitive to interference from hemoglobin, bilirubin, and total lipids up to concentrations of 5, 0.2, and 10 g/L, respectively. No significant loss of immunological activity (analyte stability) was seen day-to-day ( < or = 5) or after repeated freeze/thaw ( < or = 5) cycles. We conclude that the F-PSA IRMA is an accurate, precise, and reliable tool for measuring F-PSA in human serum. PMID:8697584

  19. Detection of antibodies to Anisakis simplex larvae by enzyme-linked immunosorbent assay and immunoelectrophoresis using crude or purified antigens.

    PubMed

    Gutierrez Ramos, R; Tsuji, M

    1994-12-01

    The enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis (IEP) were used for the serodiagnosis of larval Anisakis simplex infections in man and immunized rabbits. Sephacryl S-300 gel filtration was used for separating crude antigen. Four fractions were obtained. Sera from patients with other helminth infections sometimes cross reacted with Anisakis larval antigens. With IEP, crude antigen is more sensitive than purified antigens. With ELISA, the third fraction is the most sensitive for detecting antibodies to Anisakis larvae in the sera of humans and immunized rabbits.

  20. Antibodies to Hepatitis B Surface Antigen Potentiate the Response of Human T Lymphocyte Clones to the Same Antigen

    NASA Astrophysics Data System (ADS)

    Celis, Esteban; Chang, Tse Wen

    1984-04-01

    Human T-helper lymphocyte clones specific for hepatitis B virus surface antigen (HBsAg) proliferate on stimulation with HBsAg in vitro. Antibodies specific for HBsAg, but no other antibodies, augment this proliferative response. In the presence of antibodies to HBsAg, the maximum response could be achieved at HBsAg concentrations that were 1 percent of those required in the absence of the antibodies. These findings suggest that antigen-specific antibodies exert regulatory controls on T cells that recognize the same antigens.

  1. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus

    PubMed Central

    Huang, Yu-Chuen; Jiang, Chii-Ming; Chen, Yu-Jen; Chen, Yu-Yawn

    2013-01-01

    Pectinesterase inhibitor (PEI) isolated from jelly fig (Ficus awkeotsang Makino) is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV) infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg). Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B) and integrated (Huh7) HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes. PMID:24302965

  2. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus.

    PubMed

    Huang, Yu-Chuen; Jiang, Chii-Ming; Chen, Yu-Jen; Chen, Yu-Yawn

    2013-01-01

    Pectinesterase inhibitor (PEI) isolated from jelly fig (Ficus awkeotsang Makino) is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV) infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg). Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B) and integrated (Huh7) HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes. PMID:24302965

  3. Frequency of Detectable HBsAg in Fluid Adherent to the Endoscope, Gastric Juice, and Saliva Collected during Endoscopy in Patients Positive for HBsAg

    PubMed Central

    Yang, Ung-Suk; Liu, Bang-Hyun

    1986-01-01

    Gastric juice, saliva, and fluid adherent to the endoscope were collected from 50 patients who were seropositive for hepatitis B surface antigen (HBsAg) during the endoscopic examination of the upper gastrointestinal tract, and examined for HBsAg, using the radioimmunoassay. A positive test was obtained from 42.0% of the saliva samples, in 32.0% of the gastric juice specimens, and in 31.3% of the fluid adherent to the scope. These results should be taken as a warning, that calls for a more careful screening of the patients and disinfection of the endoscope. PMID:3154614

  4. Popliteal lymph node assay as a method of investigating H-Y antigen in the rat.

    PubMed

    Markwick, J R; Pegrum, G D

    1981-03-01

    The ability of the popliteal lymph node assay (PLNA) To detect host-versus-graft activity in response to H-Y antigens was investigated in the rat by using presensitized recipients. It was shown that significant popliteal lymph node (PLN) enlargement in response to H-Y-incompatible lymphocytes from syngeneic donors could be obtained in female recipients that had been preimmunized by the weekly i.p. injection of 1 x 10(7) lymphocytes from males of the same strain. In one rat strain, it was also shown that when male recipients had been similarly preimmunized with lymphocytes from females of the same strain, significant PLN enlargement could be obtained in response to lymphocytes from syngeneic females. The results that we obtained using the PLNA are discussed in comparison with those that have been reported when H-Y-incompatible skin grafts have been used to investigate H-Y antigen in the rat. These results indicate that, in the rat, the PLNA provides a useful alternative method for the investigation of H-Y antigen and may also provide a useful means to investigate the response of male rats to lymphocytes from syngeneic females.

  5. Antigenic characterisation of influenza B virus with a new microneutralisation assay: comparison to haemagglutination and sequence analysis.

    PubMed

    Ansaldi, Filippo; Bacilieri, Sabrina; Amicizia, Daniela; Valle, Laura; Banfi, Federica; Durando, Paolo; Sticchi, Laura; Gasparini, Roberto; Icardi, Giancarlo; Crovari, Pietro

    2004-09-01

    Although the haemagglutination inhibition assay is considered the "gold standard" for antigenic characterisation of influenza viruses, some limitations of this technique are well known. A new microneutralisation assay, as a tool for antigenic characterisation of influenza B viruses, has been standardised and its performance evaluated in comparison with the haemagglutination inhibition test in the light of molecular characterisation of the haemagglutinin. Twelve B viruses belonging to the two lineages and the four sub-lineages discriminated by phylogenetic analysis of HA were tested. The microneutralisation assay clearly distinguishes viruses belonging to different lineages and, in addition, discriminates strains belonging to different sub-lineages that are poorly or not discriminated using the haemagglutination inhibition test. This new microneutralisation assay could provide a useful tool for antigenic characterisation of circulating influenza viruses and contribute, together with the haemagglutination inhibition test and sequence analysis of the haemagglutinin and neuraminidase, in the choice of the strain for use in vaccine composition.

  6. [Fundamental and clinical evaluation of hepatitis B virus core-related antigen assay by LUMIPULSE f].

    PubMed

    Tanaka, Yasuhito; Takagi, Kazumi; Hiramatsu, Kumiko; Naganuma, Hatsue; Iida, Takayasu; Takasaka, Yoshimitsu; Mizokami, Masashi

    2006-07-01

    A sensitive chemiluminescence enzyme immunoassay (CLEIA) has been developed for hepatitis B virus (HBV) core-related antigens (HBcrAg) detection. The HBcrAg is designated as the precore/core gene products including HBeAg. The aim of this study is to evaluate reproducibility of HBcrAg and correlation with HBV-DNA in serum using the automatic LUMIPULSE f to estimate an assay suitable for general laboratory use. In this study, we demonstrated that HBcrAg assay had highly intra-assay reproducible [coefficients of variation(CVs); 2.8-5.2%] and inter-assay reproducible [CVs; 3.9-9.1%]. When the cutoff value was tentatively set at 1 kU/ml, all healthy controls (HBsAg/HBV-DNA negative; n=100) and anti-HCV antibody-positive (n=50) sera were identified as negative. The assay showed a detection limit of 0.5 kU/ml using four serially diluted HBV high-titer sera, indicating higher sensitivity than HBV-DNA (transcription-mediated amplification). The HBcrAg concentration correlated positively with serum HBV-DNA (n=125, r = 0.860, p < 0.0001) regardless of HBeAg, although the HBcrAg levels were higher in HBeAg-positive group than in HBeAg-negative group. In the natural course of HBV infection, the HBcrAg concentration usually changed in accordance with HBV-DNA levels, however during lamivudine therapy the change of HBcrAg was more gradual than that of HBV-DNA. In conclusion, HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients.

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

    NASA Astrophysics Data System (ADS)

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

    2007-04-01

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

  8. HBsAg Positive Patient Characteristics in Hospital and Blood Donation Camps

    PubMed Central

    Varghese, Joy; Joseph, Jensingh; Srinivasan, Vijaya; Jayanthi, Venkataraman; Rela, Mohamed

    2013-01-01

    Background. Prevention of the residual risk of transfusion transmitted hepatitis B virus infection (HBV) is mostly dependant on serological screening of blood donors for HBsAg and antibody to hepatitis B core antigen (anti-HBc Ab). This study aimed to study the prevalence of HBsAg and anti-HBc Ab and to compare the profile of blood donors attending a blood donation camp and people attending a hospital based camp. Methods. In the blood donor camp, all the blood units were screened for HBV, (HBsAg and anti-HBc), and in the hospital based camp, screening was done for HBsAg alone. Baseline demographic characteristics were noted. Results. The number of blood bank donors was 363 (47.5%) and hospital camp attendees was 402 (52.5%). Prevalence of HBsAg positivity was similar in both the groups at 1.7% and 1.9%, respectively. Anti-HBc Ab positivity (Total) was 6% among the blood donors; Overall prevalence of HBV infection in this group was 3.2%. Conclusion. Policy for checking the collected blood unit by 3 tests for anti-HBc, anti-HBsAb, and HBsAg should be reconsidered to possibly achieve the zero risk goal of transfusion transmitted HBV infection. Blood obtained from a vaccinated donor may give an added protection to the recipient. PMID:24083029

  9. Development of Ss-NIE-1 recombinant antigen based assays for immunodiagnosis of strongyloidiasis.

    PubMed

    Rascoe, Lisa N; Price, Courtney; Shin, Sun Hee; McAuliffe, Isabel; Priest, Jeffrey W; Handali, Sukwan

    2015-04-01

    Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG4 specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG4. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG4 ELISA were 95% and 93%, respectively. For the IgG4 Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG4 ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.

  10. Determination of HBsAg subtypes in Western Siberian part of Russia.

    PubMed

    Netesova, I G; Swenson, P D; Osipova, L P; Gubina, M A; Posukh, O L; Netesov, S V

    2003-10-01

    A set of monoclonal antibodies with specificity for hepatitis B surface antigen (HBsAg) was used for subtyping this antigen in sera from indigenous natives, blood donors, and drug users in Western Siberia with a modified commercial enzyme immunoassay kit for HBsAg detection. Three subtypes of HBsAg in a ratio of 36 (78%) ayw2:8 ayw3varB (18%):2 (4%) adw2 were found in 46 (100%) HBsAg-positive sera of different aboriginal populations of Western Siberia: the Tundra Nenets, Northern Khanty, Southern Altaians, and Kazakhs. Four subtypes of HBsAg in a ratio of 81 (57%) ayw2:58 (15 ayw3varA and 43 ayw3varB; 44%):2 (1%) adw2 were detected in 141 (100%) samples of blood donors from ten cities of Western Siberia. Three subtypes of HBsAg in a ratio of 34 ayw3:(both variants, 33 ayw3varA and 1 ayw3varB; 97.1%):1 (2.9%) ayw2 were found in blood of 35 injection drug users in Novosibirsk.

  11. Minor antigen graft-versus-host reactions revealed in irradiated spleen and popliteal lymph node assays

    SciTech Connect

    Claman, H.N.; Jaffee, B.D.

    1984-10-01

    The graft-versus-hot (GVH) reaction across minor (non-H-2) histocompatibility barriers was studied in mice, in vivo. To increase GVH potential and to mimic clinical bone marrow transplantation protocols, we modified the popliteal lymph node (PLN) and the splenomegaly assays by irradiating the recipients before they received allogeneic lymphoid cell suspensions. In several combinations across major (H-2), minor (non-H-2) and multiple minor (non-H-2 plus minor lymphocyte stimulation) barriers, increased recipient organ weight (a measure of GVH activity) was seen with irradiated F1 recipients of parental cells. The irradiated splenomegaly (x-splenomegaly) assay was more sensitive than the (x-PLN) assay, but both correlated with in vivo GVH experiments of the P----F1 variety. The x-splenomegaly test indicated histoincompatibility in a system (B10.D2----BALB/c) in which the primary in vitro mixed leukocyte reactions is nonreactive, but in which systemic GVH can be induced. The x-splenomegaly test should be useful in analyzing complex reactions involving minor histocompatibility antigens in vivo.

  12. Molecular Characterization of Cronobacter Lipopolysaccharide O-Antigen Gene Clusters and Development of Serotype-Specific PCR Assays

    PubMed Central

    Jarvis, K. G.; Grim, C. J.; Franco, A. A.; Gopinath, G.; Sathyamoorthy, V.; Hu, L.; Sadowski, J. A.; Lee, C. S.; Tall, B. D.

    2011-01-01

    Cronobacter (formerly Enterobacter sakazakii) is a recently defined genus consisting of six species, C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and Cronobacter genomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located between galF and gnd, were used to identify serotypes in Cronobacter spp. Seven O-antigen RFLP clusters were generated, including three C. sakazakii clusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including two C. sakazakii strains, two C. malonaticus strains, one C. turicensis strain, and one C. muytjensii strain, revealed three O-antigen gene clusters shared among Cronobacter species. PCR assays were developed, targeting the wzx O-antigen polymerase gene, and used to screen 231 Cronobacter strains to determine the frequency of these newly identified serotypes. PMID:21531829

  13. Improved cell mediated immune responses after successful re-vaccination of non-responders to the hepatitis B virus surface antigen (HBsAg) vaccine using the combined hepatitis A and B vaccine.

    PubMed

    Nyström, Jessica; Cardell, Kristina; Björnsdottir, Thora Björg; Fryden, Aril; Hultgren, Catharina; Sällberg, Matti

    2008-11-01

    We successfully re-vaccinated hepatitis B virus (HBV) vaccine non-responders using a double dose of the combined hepatitis A virus (HAV) and HBV vaccine. The hope was to improve priming of hepatitis B surface antigen (HBsAg)-specific cell mediated immune response (CMI) by an increased antigen dose and a theoretical adjuvant-effect from the local presence of a HAV-specific CMI. A few non-responders had a detectable HBsAg-specific CMI before re-vaccination. An in vitro detectable HBsAg-specific CMI was primed equally effective in non-responders (58%) as in first time vaccine recipients (68%). After the third dose a weak, albeit significant, association was observed between the magnitude of HBsAg-specific proliferation and anti-HBs levels. This regimen improves the priming of HBsAg-specific CMIs and antibodies.

  14. Detection of prostate cancer with a blood-based assay for early prostate cancer antigen.

    PubMed

    Paul, Barbara; Dhir, Rajiv; Landsittel, Douglas; Hitchens, Moira R; Getzenberg, Robert H

    2005-05-15

    Prostate-specific antigen lacks specificity for prostate cancer, so the identification and characterization of a unique blood-based marker for the disease would provide for a more accurate diagnosis, reducing both unnecessary biopsies and patient uncertainty. We previously identified a novel biomarker for prostate cancer, early prostate cancer antigen (EPCA). EPCA antibodies positively stained the negative biopsies of men who, as much as 5 years later, were diagnosed with prostate cancer. The goal of this study was to determine whether EPCA antibodies could be used in a clinically applicable plasma-based immunoassay to specifically detect prostate cancer. Using an EPCA-based ELISA, the protein was measured in the plasma of 46 individuals, including prostate cancer patients, healthy individuals, other cancer patients, spinal cord injury victims, and patients with prostatitis. With a predetermined cutoff value of 1.7 absorbance at 450 nm, only the prostate cancer population, as a whole, expressed plasma-EPCA levels above the cutoff. Statistical analysis showed a significant difference in EPCA levels between the prostate cancer population and each of the other groups, specifically the healthy donors (P < 0.0001), bladder cancer patients (P = 0.03), and spinal cord injury patients (P = 0.001). Sensitivity of the EPCA assay for prostate cancer patients was 92% whereas the overall specificity was 94%. Specificity for the healthy donors was 100%. Although larger trials are required, this initial study shows the potential of EPCA to serve as a highly specific blood-based marker for prostate cancer. EPCA, when coupled with prostate-specific antigen, may help reduce the number of both unnecessary biopsies and undetected prostate tumors.

  15. Detection of non-jejuni and -coli Campylobacter species from stool specimens with an immunochromatographic antigen detection assay.

    PubMed

    Couturier, Brianne A; Couturier, Marc Roger; Kalp, Kim J; Fisher, Mark A

    2013-06-01

    The STAT! Campy immunochromatographic assay for Campylobacter antigen was compared to culture for 500 clinical stool specimens. Antigen was detected in six culture-negative, PCR-positive specimens. C. upsaliensis, a pathogenic species that is traditionally difficult to recover in routine stool cultures, was detected in two of these culture-negative specimens. This study provides evidence that antigen testing may cross-react with at least one additional non-jejuni and -coli Campylobacter species that may be missed by routine culture for campylobacteriosis.

  16. Virus-based assay for antigen detection using infective growth as signal transduction mechanism.

    PubMed

    Cheok, Hui Shan; Jaworski, Justyn

    2016-03-15

    Viruses have the ability to infect and thereby confer new phenotypes on host cells. E. coli, for example, if infected by viruses containing antibiotic resistance genes, can benefit by surviving in the presence of the corresponding antibiotics to grow into colonies observable by the naked eye. Using this concept as a signal transduction mechanism for our immunoassay, we have engineered ampicillin resistant virions to display a dimer of the z domain from Protein A. This zz-domain selectively binds to the conserved heavy domain of IgG across various species. As commercially available antibodies are in no short supply, this engineered virion can be used modularly with existing antibodies for converting the presence of target antigen into a visually detectable colony forming unit. Here we demonstrate that this scheme for zz-phage transfection and selective growth of infected E. coli can facilitate sub-nanomolar detection limits for target antigen. Moreover, this phage infectivity assay works over a range of concentrations competitive with existing ELISA techniques. Because this system is derived from self-regenerating components (i.e., virus and bacteria) and furthermore obviates the need for chromogenic substrates or spectroscopic equipment, we find it particularly suitable for use in regions where cost effective detection is a necessity.

  17. Assessment of Dextran Antigenicity of Intravenous Iron Preparations with Enzyme-Linked Immunosorbent Assay (ELISA)

    PubMed Central

    Neiser, Susann; Koskenkorva, Taija S.; Schwarz, Katrin; Wilhelm, Maria; Burckhardt, Susanna

    2016-01-01

    Intravenous iron preparations are typically classified as non-dextran-based or dextran/dextran-based complexes. The carbohydrate shell for each of these preparations is unique and is key in determining the various physicochemical properties, the metabolic pathway, and the immunogenicity of the iron-carbohydrate complex. As intravenous dextran can cause severe, antibody-mediated dextran-induced anaphylactic reactions (DIAR), the purpose of this study was to explore the potential of various intravenous iron preparations, non-dextran-based or dextran/dextran-based, to induce these reactions. An IgG-isotype mouse monoclonal anti-dextran antibody (5E7H3) and an enzyme-linked immunosorbent assay (ELISA) were developed to investigate the dextran antigenicity of low molecular weight iron dextran, ferumoxytol, iron isomaltoside 1000, ferric gluconate, iron sucrose and ferric carboxymaltose, as well as isomaltoside 1000, the isolated carbohydrate component of iron isomaltoside 1000. Low molecular weight iron dextran, as well as dextran-based ferumoxytol and iron isomaltoside 1000, reacted with 5E7H3, whereas ferric carboxymaltose, iron sucrose, sodium ferric gluconate, and isolated isomaltoside 1000 did not. Consistent results were obtained with reverse single radial immunodiffusion assay. The results strongly support the hypothesis that, while the carbohydrate alone (isomaltoside 1000) does not form immune complexes with anti-dextran antibodies, iron isomaltoside 1000 complex reacts with anti-dextran antibodies by forming multivalent immune complexes. Moreover, non-dextran based preparations, such as iron sucrose and ferric carboxymaltose, do not react with anti-dextran antibodies. This assay allows to assess the theoretical possibility of a substance to induce antibody-mediated DIARs. Nevertheless, as this is only one possible mechanism that may cause a hypersensitivity reaction, a broader set of assays will be required to get an understanding of the mechanisms that may

  18. Evaluating the Use of Commercial West Nile Virus Antigens as Positive Controls in the Rapid Analyte Measurement Platform West Nile Virus Assay.

    PubMed

    Burkhalter, Kristen L; Savage, Harry M

    2015-12-01

    We evaluated the utility of 2 types of commercially available antigens as positive controls in the Rapid Analyte Measurement Platform (RAMP®) West Nile virus (WNV) assay. Purified recombinant WNV envelope antigens and whole killed virus antigens produced positive RAMP results and either type would be useful as a positive control. Killed virus antigens provide operational and economic advantages and we recommend their use over purified recombinant antigens. We also offer practical applications for RAMP positive controls and recommendations for preparing them. PMID:26675461

  19. Evaluating the Use of Commercial West Nile Virus Antigens as Positive Controls in the Rapid Analyte Measurement Platform West Nile Virus Assay.

    PubMed

    Burkhalter, Kristen L; Savage, Harry M

    2015-12-01

    We evaluated the utility of 2 types of commercially available antigens as positive controls in the Rapid Analyte Measurement Platform (RAMP®) West Nile virus (WNV) assay. Purified recombinant WNV envelope antigens and whole killed virus antigens produced positive RAMP results and either type would be useful as a positive control. Killed virus antigens provide operational and economic advantages and we recommend their use over purified recombinant antigens. We also offer practical applications for RAMP positive controls and recommendations for preparing them.

  20. Mixed enzyme-linked immunosorbent assay (MELISA) for HLA class I antigen: a plasma membrane marker.

    PubMed

    Bjerrum, O W; Borregaard, N

    1990-03-01

    This study introduces a simple, reproducible assay for HLA class I antigen using antibodies against beta 2-microglobulin and the heavy chain on HLA. The sandwich technique was named mixed enzyme-linked immunosorbent assay (MELISA), and was designed for identification of plasma membranes in neutrophil subcellular fractions. The subcellular localization of HLA was identical to that of other plasma membrane markers, [3H]concanavalin A and detergent-independent alkaline phosphatase, and was unchanged by stimulation of cells by weak and strong secretagogues. In addition to the presence as part of the HLA complex in the plasma membrane uncomplexed beta 2-microglobulin is present in the specific granules of neutrophils. However, the release of beta 2-microglobulin from intact neutrophils stimulated with formyl-methionylleucylphenylalanine was much higher than could be explained by exocytosis of specific granules. Subcellular fractionation studies demonstrated that beta 2-microglobulin is localized in fractions characterized by latent alkaline phosphatase and released from this novel secretory compartment in response to stimulation with formyl-methionylleucylphenylalanine. PMID:2181625

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

    USGS Publications Warehouse

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

    2000-01-01

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

  2. Single chip SPR and fluorescent ELISA assay of prostate specific antigen.

    PubMed

    Breault-Turcot, J; Poirier-Richard, H-P; Couture, M; Pelechacz, D; Masson, J-F

    2015-12-01

    A multi-channel system combining fluidics and micropatterned plasmonic materials with wavelength interrogation surface plasmon resonance (SPR) and fluorescence detection was integrated from the combination of a small and motorized fluorescence microscope mounted on a portable 4-channel SPR instrument. The SPR and fluorescent measurements were performed based on the same detection area in a multi-channel fluidic, with a sensing scheme for prostate-specific antigen (PSA) consisting of a sandwich assay with a capture anti-PSA immobilized onto the SPR sensor and a detection anti-PSA modified with horseradish peroxidase (HRP). In this dual-detection instrument, fluorescence was measured from the solution side of the micropatterned gold film, while the interface between the glass prism and the gold film served to interrogate the SPR response. The SPR sensors were comprised of microhole arrays fabricated by photolithography to enhance the instrumental response for PSA detection by approximately a factor of 2 to 3 and they were coated with a self-assembled monolayer of a peptide (3-MPA-HHHDD-OH) to minimize nonspecific adsorption. PSA was successfully detected at clinical concentrations from 10 pM to 50 nM with this integrated system in a single assay lasting 12 minutes, almost centering on the desired range for PSA diagnostic tests (>4 ng mL(-1) or >150 pM). The combination of two robust techniques in a single chip and instrument has led to a simple and effective assay that can be carried out on a small and portable instrument providing rapid biodetection of an important cancer biomarker with a dynamic range of nearly 4 orders of magnitude in the clinical range.

  3. Comparison of different strains of Borrelia burgdorferi sensu lato used as antigens in enzyme-linked immunosorbent assays.

    PubMed Central

    Magnarelli, L A; Anderson, J F; Johnson, R C; Nadelman, R B; Wormser, G P

    1994-01-01

    Eight strains of Borrelia burgdorferi sensu lato were tested with serum samples from persons who had Lyme borreliosis or syphilis in class-specific enzyme-linked immunosorbent assays (ELISAs). Antigens of B. burgdorferi sensu stricto, of Borrelia garinii, and of Borrelia spirochetes in group VS461 were prepared from cultured bacteria isolated from ticks, a white-footed mouse (Peromyscus leucopus), or human tissues in North America, the former Soviet Union, and Japan. Nearly all of the serum specimens that contained immunoglobulins to strain 2591, a Connecticut isolate, were also positive in antibody tests with the other seven strains. In general, all eight strains reacted similarly and were suitable as coating antigens in class-specific ELISAs. Assay sensitivities ranged from 82.6 to 100% in analyses for immunoglobulin M and G antibodies. Compared with reference antigen strain 2591, strains 231 (a tick isolate from Canada) and NCH-1 (a human skin isolate from Wisconsin) resulted in higher antibody titers in an ELISA. Syphilitic sera cross-reacted in all tests regardless of the antigen used. Key immunodominant proteins are shared among the closely related strains of B. burgdorferi sensu lato tested, but it is suspected that variations in antigen compositions among these spirochetes may sometimes affect assay performance for detecting serum antibodies. PMID:8051239

  4. Development of Ss-NIE-1 recombinant antigen based assays for immunodiagnosis of strongyloidiasis.

    PubMed

    Rascoe, Lisa N; Price, Courtney; Shin, Sun Hee; McAuliffe, Isabel; Priest, Jeffrey W; Handali, Sukwan

    2015-04-01

    Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG4 specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG4. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG4 ELISA were 95% and 93%, respectively. For the IgG4 Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG4 ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States. PMID

  5. Additional Evaluation of the Point-of-Contact Circulating Cathodic Antigen Assay for Schistosoma mansoni Infection

    PubMed Central

    Mwinzi, Pauline N. M.; Kittur, Nupur; Ochola, Elizabeth; Cooper, Philip J.; Campbell, Carl H.; King, Charles H.; Colley, Daniel G.

    2015-01-01

    Studies of the urine-based point-of-contact cathodic circulating antigen test (POC-CCA) in Schistosoma mansoni-endemic settings in Africa indicate it has good sensitivity in detecting infections, but in areas of low prevalence, the POC-CCA can be positive for persons who are egg-negative by Kato-Katz stool assays. We examined the POC-CCA assay for: (a) batch-to-batch stability; (b) intra-reader and inter-reader variability; (c) day-to-day variability compared to Kato-Katz stool assays, and (d) to see if praziquantel (PZQ) treatment converted Kato-Katz-negative/POC-CCA positive individuals to POC-CCA negativity. We found essentially no batch-to-batch variation, negligible intra-reader variability (2%), and substantial agreement for inter-reader reliability. Some day-to-day variation was observed over 5 days of urine collection, but less than the variation in Kato-Katz stool assays over 3 days. To evaluate the effect of treatment on Kato-Katz(−)/POC-CCA(+) children, 149 children in an area of 10–15% prevalence who were Kato-Katz(−) based on 3 stool samples but POC-CCA(+) were enrolled. Seven days after treatment (PZQ 40 mg/kg) samples were again collected and tested. Almost half (47%) POC-CCA positive children turned negative. Those still POC-CCA positive received a second treatment, and 34% of them turned POC-CCA negative upon this second treatment. Most who remained POC-CCA positive shifted each time to a “lesser” POC-CCA “level of positivity.” The data suggest that most Kato-Katz-negative/POC-CCA positive individuals harbor low-intensity infections, and each treatment kills all or some of their adult worms. The data also suggest that when evaluated by a more sensitive assay, the effective cure rates for PZQ are significantly less than those inferred from fecal testing. These findings have public health significance for the mapping and monitoring of Schistosoma infections and in planning the transition from schistosomiasis morbidity control to

  6. An enzyme-linked immunosorbent assay, using an EDTA-extracted antigen for the serology of Haemophilus pleuropneumoniae.

    PubMed

    Nicolet, J; Paroz, P; Krawinkler, M; Baumgartner, A

    1981-12-01

    An enzyme-linked immunosorbent assay (ELISA) was proposed as an alternative to the complement-fixation test (CF) for the detection of antibodies of Haemophilus pleuropneumoniae, agent of the pleuropneumonia in pigs. In tests done with different antigen-extraction procedures (including Tween 20, sodium dodecyl sulfate, aqueous phenol, sonification, and heat treatment at 120 C), ethylenediaminetetraacetic acid (EDTA) provided a satisfactorily reactive antigen. Chromatography purification on Sephacryl S200 improved the specificity of this antigen. Using hyperimmune rabbit sera, we investigated the specificity and the sensitivity of the ELISA with the EDTA-purified antigen of the different serotypes of H pleuropneumoniae on selected swine sera in herds with confirmed H pleuropneumoniae infection, from specific-pathogen-free animals showing doubtful CF reactions. The ELISA proved to be highly specific and more sensitive than the CF test. Furthermore, evidence of cross-reactions with H parasuis, a common bacteria isolated in swine populations, was not found.

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

    PubMed Central

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

    2013-01-01

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

  8. Hantavirus antigen detection using human serum immunoglobulin M as the capturing antibody in an enzyme-linked immunosorbent assay.

    PubMed

    Alexeyev, O A; Elgh, F; Ahlm, C; Stigbrand, T; Settergren, B; Wadell, G; Juto, P

    1996-04-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to detect different hantavirus antigens in cell culture; i.e. Puumala (PUU), Hantaan (HTN), and Dobrava (DOB) viruses. The assay was based on binding human serum immunoglobulin M (IgM) antibodies to the solid phase by use of goat anti-IgM antibodies. The captured IgM antibodies were present in the acute phase serum from two patients: one infected in Sweden and the other in Bosnia. Antigens being bound to the solid phase by the human anti-PUU and anti-DOB/HTN IgM antibodies were detected by a broadly reacting polyclonal rabbit anti PUU-recombinant nucleocapsid protein antiserum. The IgM isotype was proven to be at least five times more efficient than IgG when used as the capturing antibody. The sensitivity of the PUU antigen ELISA was approximately 0.5 ng/ml, as measured by titration with a PUU recombinant nucleoprotein antigen. Cell-associated PUU antigen in tissue culture was seen after 48 hr by the PUU-ELISA and after 96 hr by immunofluorescent assay. When tested for capacity to discriminate between PUU, DOB, and HTN viruses, significant differences were found: the Swedish serum detected PUU antigen at high titers, whereas no reactivity was found against DOB and HTN; the Bosnian serum detected both DOB and HTN at high titers but had a low reactivity to PUU. The method was also tested for its usefulness in detecting PUU antigen in bank vole (clethrionomys glareolus) lungs. Of 59 animals captured from the surroundings of patients with nephropathia epidemica, three became positive with a high activity in the PUU-ELISA, but with low reactivity in the DOB/HTN-ELISA. It is concluded that a sensitive ELISA has been developed to detect different hantaviruses in cell culture and lungs of bank voles.

  9. Construction and immunological evaluation of truncated hepatitis B core particles carrying HBsAg amino acids 119–152 in the major immunodominant region (MIR)

    SciTech Connect

    Su, Qiudong; Yi, Yao; Guo, Minzhuo; Qiu, Feng; Jia, Zhiyuan; Lu, Xuexin; Meng, Qingling; Bi, Shengli

    2013-09-13

    Highlights: •The conformational HBV neutralization antigen domain was successfully displayed on the surface of truncated HBc particles. •Appropriate dialysis procedures to support the renaturing environment for the protein refolding. •Efficient purification procedures to obtain high purity and icosahedral particles of mosaic HBV antigen. •Strong immune responses not only including neutralization antibody response but also Th1 cell response were induced in mice. -- Abstract: Hepatitis B capsid protein expressed in Escherichia coli can reassemble into icosahedral particles, which could strongly enhance the immunogenicity of foreign epitopes, especially those inserted into its major immunodominant region. Herein, we inserted the entire ‘α’ antigenic determinant amino acids (aa) 119–152 of HBsAg into the truncated HBc (aa 1–144), between Asp{sup 78} and Pro{sup 79}. Prokaryotic expression showed that the mosaic HBc was mainly in the form of inclusion bodies. After denaturation with urea, it was dialyzed progressively for protein renaturation. We observed that before and after renaturation, mosaic HBc was antigenic as determined by HBsAg ELISA and a lot of viruslike particles were observed after renaturation. Thus, we further purified the mosaic viruslike particles by (NH{sub 4}){sub 2}SO{sub 4} precipitation, DEAE chromatography, and Sepharose 4FF chromatography. Negative staining electron microscopy demonstrated the morphology of the viruslike particles. Immunization of Balb/c mice with mosaic particles induced the production of anti-HBs antibody and Th1 cell immune response supported by ELISPOT and CD4/CD8 proportions assay. In conclusion, we constructed mosaic hepatitis core particles displaying the entire ‘α’ antigenic determinant on the surface and laid a foundation for researching therapeutic hepatits B vaccines.

  10. Serologic diagnosis of canine and equine borreliosis: use of recombinant antigens in enzyme-linked immunosorbent assays.

    PubMed Central

    Magnarelli, L A; Flavell, R A; Padula, S J; Anderson, J F; Fikrig, E

    1997-01-01

    Serum samples from dogs and equids suspected of having canine or equine borreliosis, respectively, were analyzed in polyvalent enzyme-linked immunosorbent assays (ELISAs) with whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Purified preparations of recombinant antigens included outer surface protein A (OspA), OspB, OspC, OspE, OspF, and p41-G (a fragment of flagellin). Of the 36 dog sera that reacted positively to whole-cell antigen, 32 (88.9%) contained antibodies to one or more recombinant antigens. Reactivities to OspF (88.9% positive) and p41-G (75% positive) were most prevalent. In analyses of 30 equid sera positive in an ELISA with whole cells, 24 (80%) contained antibodies to one or more recombinant antigens. Seropositivities in ELISAs with p41-G (50% positive) and OspF (46.7% positive) were more than twofold greater than in ELISAs with OspA, OspB, or OspC (10 to 20% positive). In parallel tests of eight canine and three equine sera, there was good agreement in results of Western blot (immunoblot) analyses and ELISAs. Although dog and equid sera with antibodies to whole-cell B. burgdorferi frequently reacted positively to one or more recombinant antigens, the inclusion of OspF and p41-G antigens in ELISAs was most useful in the serologic diagnosis of canine and equine borreliosis. PMID:8968901

  11. Rapid diagnosis of typhoid fever by enzyme-linked immunosorbent assay detection of Salmonella serotype typhi antigens in urine.

    PubMed

    Fadeel, Moustafa Abdel; Crump, John A; Mahoney, Frank J; Nakhla, Isabelle A; Mansour, Adel M; Reyad, Baheia; El Melegi, Dawlat; Sultan, Yehia; Mintz, Eric D; Bibb, William F

    2004-03-01

    We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies to capture somatic antigen 9 (O9), flagellar antigen d (Hd), and the Vi capsular polysaccharide antigen (Vi) from the urine of persons with and without typhoid fever. Sequential urine samples were collected from 44 patients with blood culture-confirmed typhoid fever and from two control groups. The first control group included patients with brucellosis (n = 12) and those with clinically diagnosed, non-typhoid, acute, febrile illness (n = 27). The second control group was a sample of healthy volunteer laboratory workers (n = 11). When assessed relative to date of fever onset, sensitivity was highest during the first week for all three antigens: Vi was detected in the urine of nine (100%) patients, O9 in 4 (44%) patients, and Hd in 4 (44%) patients. Sequential testing of two urine samples from the same patient improved test sensitivity. Combined testing for Vi with O9 and Hd produced a trend towards increased sensitivity without compromising specificity. The specificity for Vi exceeded 90% when assessed among both febrile and healthy control subjects, but was only 25% when assessed among patients with brucellosis. Detection of urinary Vi antigen with this ELISA shows promise for the diagnosis of typhoid fever, particularly when used within the first week after fever onset. However, positive reactions for Vi antigen in patients with brucellosis must be understood before urinary Vi antigen detection can be developed further as a useful rapid diagnostic test.

  12. Evaluation of five different antigens in enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

    PubMed

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

    2005-01-01

    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.

  13. Expression of Serum Sialic Acid, Early Antigen-IgA, and Viral Capsid Antigen-IgA in Nasopharynx Cancer Patients: The Diagnostic Implication of Combined Assays.

    PubMed

    Sun, Yuning; Sun, Caibo; Zhang, Endong

    2015-12-28

    BACKGROUND Ebstein-Barr virus (EBV) plays a critical role in nasopharynx cancer, which can be effectively monitored by serum levels of early antigen antibody (EA-IgA) and viral capsid antigen antibody (VCA-IgA). This study explored the diagnostic value of combined assays of sialic acid (SA), EA-IgA, and VCA-IgA via the expressional assay. MATERIAL AND METHODS A total of 42 nasopharynx cancer patients and 42 benign rhinitis and healthy controls were recruited in this study. Serum EA-IgA and VCA-IgA were tested by enzyme-linked immunosorbent assay (ELISA) and enzymatic assay of serum SA. Specificity and sensitivity of those 3 assays were compared. The diagnostic value of each parameter was evaluated by ROC curves. RESULTS All 3 indexes (SA, EA-IgA and VCA-IgA) showed elevated serum levels in nasopharynx cancer patients when compared to those with rhinitis, who had higher levels than healthy individuals. Concentrations of these factors were also positively correlated with the TNM staging of cancer. The sensitivity and specificity were 30.95% and 83.33% (in SA), 57.14% and 95.24% (in EA-IgA), and 76.19% and 92.86% (in VCA-IgA), respectively. VCA-IgA had the highest sensitivity among all 3 indexes. The combined assay increased the diagnostic sensitivity to 92.86% without compromising specificity. CONCLUSIONS SA, EA-IgA, and VCA-IgA levels were significantly elevated in nasopharynx patients' serum. The combined assay may have clinical value in diagnosis and monitoring.

  14. Diagnosis of Chikungunya Fever in an Indian Population by an Indirect Enzyme-Linked Immunosorbent Assay Protocol Based on an Antigen Detection Assay: a Prospective Cohort Study▿

    PubMed Central

    Kashyap, Rajpal Singh; Morey, Shweta H.; Ramteke, Sonali S.; Chandak, Nitin H.; Parida, Manmohan; Deshpande, Poonam S.; Purohit, Hemant J.; Taori, Girdhar M.; Daginawala, Hatim F.

    2010-01-01

    A Chikungunya virus (CHIKV) outbreak continues in India. Monitoring of the clinical features of CHIKV infection is an important component of assessing the disease process. Diagnosis is usually made by an immunoglobulin M (IgM)/IgG enzyme-linked immunosorbent assay (ELISA). However, these assays have extremely low sensitivities for the detection of infection in the majority of CHIKV patients during the acute stage of infection (during the 1 to 4 days after infection). In our laboratory, a sensitive ELISA protocol for antigen detection has been developed for the detection of CHIKV infection in the acute stage, and in the present study we assessed the usefulness of this ELISA-based system for the detection of CHIKV infection. We performed a prospective, double-blinded study of 205 Indian patients with suspected CHIKV infection in the Nagpur District. All patients underwent a full clinical assessment, and their serum samples were analyzed for the presence of antigens and of IgM and IgG by an ELISA protocol. In patients with CHIKV infection, the sensitivity of antigen detection was 85%, which was significantly higher (P < 0.001) than that of IgM (17%) or IgG (45%) detection. The sensitivity of IgM (20%) or IgG (25%) detection was significantly lower than that of the antigen assay (95%) for patients with acute infections (i.e., from day 1 to day 5 after infection). Antigen detection not only gives a positive confirmatory result in the early phase of the disease, but it is also useful in the prodromal and subclinical stage and may be useful for field applications for the rapid detection of CHIKV infection. PMID:20007365

  15. Development of a mixed antigen agar gel enzyme assay (AGEA) for the detection of antibodies to poxvirus in chicken and turkey sera.

    PubMed

    Tadese, Theodros; Potter, E A; Reed, W M

    2003-02-01

    A mixed-antigen agar gel enzyme assay (AGEA) was developed to detect antibodies to poxviruses in chicken and turkey sera. The assay combines the principles of immunodiffusion and enzyme assay. For the detection of antibodies to fowl poxvirus (FP), pigeon poxvirus (PP) and turkey poxvirus (TP) in turkey serum samples, the three antigens were combined to form a mixed-antigen assay. To screen for antibodies to FP and PP in chicken serum samples, the two antigens were combined. When FP and PP viruses were combined as antigens, the sensitivity for chicken sera was 64% but the sensitivity of the agar gel precipitation test (AGPT) was 34% (P<0.001). When antibodies were detected in turkey sera using the mixed antigens, the AGEA had a sensitivity of 66.4% while that of AGPT was 25% (P<0.001). PMID:12655123

  16. Aptamer-based microchip electrophoresis assays for amplification detection of carcinoembryonia antigen

    PubMed Central

    Pan, Li; Zhao, Jingjin; Huang, Yong; Zhao, Shulin; Liu, Yi-Ming

    2015-01-01

    Background Carcinoembryonic antigen (CEA) as one of the most widely used tumor marker is used in the clinical diagnosis of colorectal, pancreatic, gastric, and cervical carcinomas. We developed an aptamer-based microchip electrophoresis assay technique for assaying CEA in human serum for cancer diagnosis. Methods The magnetic beads (MBs) are employed as carriers of double strand DNA that is formed by an aptamer of target and a complementary DNA of aptamer. After the aptamer in MB-dsDNA conjugate binds with target, the complementary DNA was released from MB-dsDNA conjugate. The released complementary DNA hybridizes with a fluorescein amidite (FAM) labeled DNA, and forms DNA duplex, which triggers the selective cleavage of FAM labeled DNA by nicking endonuclease Nb.BbvCI, and generating FAM labeled DNA segment. The released complementary DNA hybridizes with another FAM labeled DNA, resulting in a continuous cleavage of FAM labeled DNA, and the generation of large numbers of FAM labeled DNA segments. In MCE laser induced fluorescence detection (LIF), FAM labeled DNA segment is separated and detected. Results The linear range for CEA was 130 pg/ml~8.0 ng/ml with a correlation coefficient of 0.9916 and a detection limit of 68 pg/ml. The CEA concentration in the serum samples from healthy subjects was found be in the range 1.3 ng/ml to 3.2 ng/ml. The CEA concentration in the samples from cancer patients was found to be >15 ng/ml. Conclusions This method may become a useful tool for rapid analysis of CEA and other tumor markers in biomedical analysis and clinical diagnosis. PMID:26344338

  17. Analytical assessment of the novel Maglumi squamous cell carcinoma antigen (SCCA) immunoluminometric assay

    PubMed Central

    Dipalo, Mariella; Gnocchi, Cecilia; Aloe, Rosalia

    2015-01-01

    Background The demand for routine measurement of squamous cell carcinoma antigen (SCCA) is rapidly increasing in clinical laboratories, due to the central role that this biomarker plays in staging and monitoring patients with various forms of squamous cell carcinomas (SCCs). Methods The present analytical evaluation of Maglumi SCCA was aimed to assess the imprecision, linearity and comparability against a widely used technique. Results The intra- and inter-assay imprecision was comprised between 2.6-4.2% and between 5.0-7.3%, respectively. The linearity of the test was excellent in the range of SCC values comprised between 1.0 and 18.0 ng/mL (r=0.998; P<0.001). A highly significant correlation was observed between Maglumi SCCA and BRAHMS Kryptor SCC in the range of values comprised between 0.44 and 15.18 ng/mL (r=0.960; P<0.001). The mean bias was 0.79 ng/mL (95% CI, 0.61-0.97) and the diagnostic agreement at the respective diagnostic cut-offs was 95%. Conclusions The results of this study confirm that Maglumi SCCA may be regarded as a suitable alternative to Kryptor SCC for routine and fully-automated assessment of SCCA in clinical laboratories. PMID:26807406

  18. Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

    PubMed

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

    2015-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

  19. Localization of immunodominant epitopes within the "a" determinant of hepatitis B surface antigen using monoclonal antibodies.

    PubMed

    Golsaz-Shirazi, Forough; Mohammadi, Hamed; Amiri, Mohammad Mehdi; Khoshnoodi, Jalal; Kardar, Gholam Ali; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2016-10-01

    The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There is an increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG). PMID:27439498

  20. Systemic and mucosal immune response induced by transcutaneous immunization using Hepatitis B surface antigen-loaded modified liposomes.

    PubMed

    Mishra, Dinesh; Mishra, Pradyumna Kumar; Dubey, Vaibhav; Nahar, Manoj; Dabadghao, Sunil; Jain, N K

    2008-04-23

    We have evaluated the efficiency of novel modified liposomes (ethosomes) for transcutaneous immunization (TCI) against Hepatitis B. Antigen-loaded ethosomes were prepared and characterized for shape, lamellarity, fluidity, size distribution, and entrapment efficiency. Spectral bio-imaging and flow cytometric studies showed efficient uptake of Hepatitis B surface antigen (HBsAg)-loaded ethosomes by murine dendritic cells (DCs) in vitro, reaching a peak by 180 min. Transcutaneous delivery potential of the antigen-loaded system using human cadaver skin demonstrated a much higher skin permeation of the antigen in comparison to conventional liposomes and soluble antigen preparation. Topically applied HBsAg-loaded ethosomes in experimental mice showed a robust systemic and mucosal humoral immune response compared to intramuscularly administered alum-adsorbed HBsAg suspension, topically applied plain HBsAg solution and hydroethanolic (25%) HBsAg solution. The ability of the antigen-pulsed DCs to stimulate autologous peripheral blood lymphocytes was demonstrated by BrdU assay and a predominantly TH1 type of immune response was observed by multiplex cytometric bead array analysis. HBsAg-loaded ethosomes are able to generate a protective immune response and their ability to traverse and target the immunological milieu of the skin may find a potential application in the development of a transcutaneous vaccine against Hepatitis B virus (HBV).

  1. Systemic and mucosal immune response induced by transcutaneous immunization using Hepatitis B surface antigen-loaded modified liposomes.

    PubMed

    Mishra, Dinesh; Mishra, Pradyumna Kumar; Dubey, Vaibhav; Nahar, Manoj; Dabadghao, Sunil; Jain, N K

    2008-04-23

    We have evaluated the efficiency of novel modified liposomes (ethosomes) for transcutaneous immunization (TCI) against Hepatitis B. Antigen-loaded ethosomes were prepared and characterized for shape, lamellarity, fluidity, size distribution, and entrapment efficiency. Spectral bio-imaging and flow cytometric studies showed efficient uptake of Hepatitis B surface antigen (HBsAg)-loaded ethosomes by murine dendritic cells (DCs) in vitro, reaching a peak by 180 min. Transcutaneous delivery potential of the antigen-loaded system using human cadaver skin demonstrated a much higher skin permeation of the antigen in comparison to conventional liposomes and soluble antigen preparation. Topically applied HBsAg-loaded ethosomes in experimental mice showed a robust systemic and mucosal humoral immune response compared to intramuscularly administered alum-adsorbed HBsAg suspension, topically applied plain HBsAg solution and hydroethanolic (25%) HBsAg solution. The ability of the antigen-pulsed DCs to stimulate autologous peripheral blood lymphocytes was demonstrated by BrdU assay and a predominantly TH1 type of immune response was observed by multiplex cytometric bead array analysis. HBsAg-loaded ethosomes are able to generate a protective immune response and their ability to traverse and target the immunological milieu of the skin may find a potential application in the development of a transcutaneous vaccine against Hepatitis B virus (HBV). PMID:18359615

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

    HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000 μg/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380 μg/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections. PMID:23691296

  4. Evaluation of single-round infectious, chimeric dengue type 1 virus as an antigen for dengue functional antibody assays.

    PubMed

    Yamanaka, Atsushi; Suzuki, Ryosuke; Konishi, Eiji

    2014-07-23

    Dengue fever and dengue hemorrhagic fever are endemic throughout tropical and subtropical countries. Four serotypes of dengue viruses (DENV-1 to DENV-4), each with several genotypes including various subclades, are co-distributed in most endemic areas. Infection-neutralizing and -enhancing antibodies are believed to play protective and pathogenic roles, respectively. Measurement of these functional antibodies against a variety of viral strains is thus important for evaluating coverage and safety of dengue vaccine candidates. Although transportation of live virus materials beyond national borders is increasingly limited, this difficulty may be overcome using biotechnology that enables generation of an antibody-assay antigen equivalent to authentic virus based on viral sequence information. A rapid system to produce flavivirus single-round infectious particles (SRIPs) was recently developed using a Japanese encephalitis virus (JEV) subgenomic replicon plasmid. This system allows production of chimeric SRIPs that have surface proteins of other flaviviruses. In the present study, SRIPs of DENV-1 (D1-SRIPs) were evaluated as an antigen for functional antibody assays. Inclusion of the whole mature capsid gene of JEV into the replicon plasmid provided higher D1-SRIP yields than did its exclusion in cases where a DENV-1 surface-protein-expressing plasmid was used for co-transfection of 293T cells with the replicon plasmid. In an assay to measure the balance between neutralizing and enhancing activities, dose (antibody dilution)-dependent activity curves in dengue-immune human sera or mouse monoclonal antibodies obtained using D1-SRIP antigen were equivalent to those obtained using DENV-1 antigen. Similar results were obtained using additional DENV-2 and DENV-3 systems. In a conventional Vero-cell neutralization test, a significant correlation was shown between antibody titers obtained using D1-SRIP and DENV-1 antigens. These results demonstrate the utility of D1-SRIPs as

  5. Bovine viral diarrhea virus antigen detection across whole cattle hides using two antigen-capture enzyme-linked immunosorbent assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine viral diarrhea virus is a costly disease of cattle that can be controlled by vaccination, biosecurity, and removal of persistently infected cattle. Development and proficiency testing of assays to identify persistently infected cattle substantial quantities of known positive and negative samp...

  6. Evaluation of an antigen capture enzyme-linked immunosorbent assay for detection of Cryptosporidium oocysts.

    PubMed Central

    Newman, R D; Jaeger, K L; Wuhib, T; Lima, A A; Guerrant, R L; Sears, C L

    1993-01-01

    The diagnosis of the small (4- to 6-microns) Cryptosporidium oocysts is labor intensive and relies on stool concentration, with subsequent staining and microscopy. The primary purpose of this study was to evaluate the clinical utility of an antigen capture enzyme-linked immunosorbent assay (ELISA) (LMD Laboratories, Carlsbad, Calif.) in detecting Cryptosporidium oocysts in human stools. A total of 591 specimens (76 diarrheal, 515 control) obtained from 213 inhabitants of an urban slum in northeastern Brazil were examined by both ELISA and conventional microscopic examination (CME) of formalin-ethyl acetate-concentrated stool samples stained with modified acid-fast and auramine stains. Forty-eight diarrheal stools (63.2%) were positive for Cryptosporidium oocysts by CME, with 40 of these positive by ELISA. Thirty-five control stools (6.8%) had Cryptosporidium oocysts detected by CME, with 15 of these also positive by ELISA. All of the 480 nondiarrheal stools and all but one of the diarrheal stools negative by CME were negative by ELISA. The test had an overall sensitivity of 66.3% and a specificity of 99.8% (positive predictive value, 98.2%; negative predictive value, 94.8%). In the evaluation of human diarrheal stool samples, the test sensitivity increased to 83.3%, with a specificity of 96.4%, and, in analysis of samples from individual patients with diarrhea, the sensitivity was 87.9%, with a specificity of 100%. These results indicate that this stool ELISA is sensitive and specific for the detection of Cryptosporidium oocysts in human diarrheal stool specimens but has limited use in epidemiologic studies for the diagnosis of asymptomatic Cryptosporidium infection. PMID:8370732

  7. Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay

    PubMed Central

    Fan, Peihu; Li, Xiaojun; Su, Weiheng; Kong, Wei; Kong, Xianggui; Wang, Zhenxin; Wang, Youchun; Jiang, Chunlai; Gao, Feng

    2015-01-01

    The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens. PMID:25915630

  8. Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay.

    PubMed

    Fan, Peihu; Li, Xiaojun; Su, Weiheng; Kong, Wei; Kong, Xianggui; Wang, Zhenxin; Wang, Youchun; Jiang, Chunlai; Gao, Feng

    2015-01-01

    The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens. PMID:25915630

  9. Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay.

    PubMed

    Fan, Peihu; Li, Xiaojun; Su, Weiheng; Kong, Wei; Kong, Xianggui; Wang, Zhenxin; Wang, Youchun; Jiang, Chunlai; Gao, Feng

    2015-01-01

    The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens.

  10. [Improvement of sensitivity in the second generation HCV core antigen assay by a novel concentration method using polyethylene glycol (PEG)].

    PubMed

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

    2007-11-01

    A HCV core antigen (Ag) detection assay system, Lumipulse Ortho HCV Ag has been developed and is commercially available in Japan with a lower detection level limit of 50 fmol/l, which is equivalent to 20 KIU/ml in PCR quantitative assay. HCV core Ag assay has an advantage of broader dynamic range compared with PCR assay, however the sensitivity is lower than PCR. We developed a novel HCV core Ag concentration method using polyethylene glycol (PEG), which can improve the sensitivity five times better than the original assay. The reproducibility was examined by consecutive five-time measurement of HCV patients serum, in which the results of HCV core Ag original and concentrated method were 56.8 +/- 8.1 fmol/l (mean +/- SD), CV 14.2% and 322.9 +/- 45.5 fmol/l CV 14.0%, respectively. The assay results of HCV negative samples in original HCV core Ag were all 0.1 fmol/l and the results were same even in the concentration method. The results of concentration method were 5.7 times higher than original assay, which was almost equal to theoretical rate as expected. The assay results of serially diluted samples were also as same as expected data in both original and concentration assay. We confirmed that the sensitivity of HCV core Ag concentration method had almost as same sensitivity as PCR high range assay in the competitive assay study using the serially monitored samples of five HCV patients during interferon therapy. A novel concentration method using PEG in HCV core Ag assay system seems to be useful for assessing and monitoring interferon treatment for HCV.

  11. Immunoblot Assay Using Recombinant Antigens as a Supplemental Test To Confirm the Presence of Antibodies to Trypanosoma cruzi▿

    PubMed Central

    Cheng, Kevin Y.; Chang, Chi-Deu; Salbilla, Vince A.; Kirchhoff, Louis V.; Leiby, David A.; Schochetman, Gerald; Shah, Dinesh O.

    2007-01-01

    The diagnosis of chronic Chagas' disease is generally made by detecting antibodies to Trypanosoma cruzi. Most conventional serological tests are based on lysates of whole parasites or semipurified antigen fractions from T. cruzi epimastigotes grown in culture. The occurrence of inconclusive and false-positive results has been a persistent problem with the conventional assays, and there is no universally accepted gold standard for confirmation of positive test results. We describe here an immunoblot assay for detecting antibodies to T. cruzi in which four chimeric recombinant antigens (rAgs), designated FP3, FP6, FP10, and TcF, are used as target antigens. Each of these rAgs is composed of several antigenically distinct regions and includes repetitive as well as nonrepetitive sequences. Each rAg is coated as a discrete line on a nitrocellulose strip. Assay sensitivity was assessed by testing 345 specimens known to be positive for antibodies to T. cruzi. All 345 of these samples showed two to four reactive test bands in addition to the three on-board control bands that are on each strip. Assay specificity was determined by testing 500 specimens from random U.S. blood donors, all of which gave negative results. Based on the results obtained in this study, we propose the following scheme for interpretation of test results: (i) no bands or a single test band = a negative result; (ii) two or more test bands with at least one band showing intensity of 1+ or higher = a positive result; and (iii) multiple faint test bands (±) = indeterminate result. Based on this scheme, the prototype immunoblot assay showed sensitivity of 100% (n = 345) and specificity of 100% (n = 500). Additionally, all 269 potentially cross-reacting and T. cruzi antibody-negative specimens tested negative in our immunoblot assay. The rAg-based immunoblot assay has potential as a supplemental test for confirming the presence of antibodies to T. cruzi in blood specimens and for identifying false

  12. The diagnostic value of the fibrinogen/fibrin fragment E antigen assay in clinically suspected deep vein thrombosis

    SciTech Connect

    Zielinsky, A.; Hirsh, J.; Straumanis, G.; Carter, C.J.; Gent, M.; Sackett, D.L.; Hull, R.; Kelton, J.G.; Powers, P.; Turpie, A.G.

    1982-02-01

    We have evaluated the fibrinogen/fibrin fragment E antigen assay as a diagnostic test in patients with clinically suspected venous thrombosis by comparing the results of this assay with venography in 272 patients. The result of the fragment E antigen assay was elevated in 79 of 80 patients with positive venograms for recent venous thrombosis (sensitivity 99%) and within the normal range in 161 of 192 patients with normal venograms (specificity 84%). The fragment E assay was also evaluated in 130 medical and surgical controls without evidence of venous thrombosis by leg scanning and the test was found to be relatively nonspecific. However, in the patient group under study, a correct clinical diagnosis of no thrombosis, based on a normal fragment E result, was made in 161 of 162 cases (negative predictive value of 99%). Therefore, a normal test result effectively excludes a diagnosis of venous thrombosis in clinically symptomatic patients. The assay, as currently performed, is technically demanding and takes 24 hr to complete. Therefore, it will have to be simplified before it can be applied to clinical practice.

  13. The diagnostic value of the fibrinogen/fibrin fragment E antigen assay in clinically suspected deep vein thrombosis

    SciTech Connect

    Zielinsky, A.; Hirsh, J.; Straumanis, G.; Carter, C.J.; Gent, M.; Sackett, D.L.; Hull, R.; Kelton, J.G.; Powers, P.

    1982-02-01

    We have evaluated the fibrinogen/fibrin fragment E antigen assay as a diagnostic test in patients with clinically suspected venous thrombosis by comparing the results of this assay with venography in 272 patients. The result of the fragment E antigen assay was elevated in 79 of 80 patients with positive venograms for recent venous thrombosis (sensitivity 99%) and within the normal range in 161 of 192 patients with normal venograms (specificity 84%). The fragment E assay was also evaluated in 130 medical and surgical controls without evidence of venous thrombosis by leg scanning and the test was found to be relatively nonspecific. However, in the patient group under study, a correct clinical diagnosis of no thrombosis, based on a normal fragment E result, was made in 161 of 162 cases (negative predictive value 99%). Therefore, a normal test result effectively excludes a diagnosis of venous thrombosis in clinically symptomatic patients. The assay, as currently performed, is technically demanding and takes 24 hr to complete. Therefore, it will have to be simplified before it can be applied to clinical practice.

  14. Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Mackett, Michael; Moss, Bernard

    1983-04-01

    Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

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

    USGS Publications Warehouse

    Pascho, R.J.; Mulcahy, D.

    1987-01-01

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

  16. Serology of Neisseria gonorrhoeae: W-antigen serogrouping by coagglutination and protein I serotyping by enzyme-linked immunosorbent assay both detect protein I antigens.

    PubMed Central

    Sandstrom, E G; Knapp, J S; Buchanan, T B

    1982-01-01

    A total of 224 strains were serogrouped by coagglutination (COA) and serotyped by protein I enzyme-linked immunosorbent assay (ELISA). Of these strains, 61 were from patients with disseminated gonococcal infection, 21 were from patients with pelvic inflammatory disease, and 115 were from patients with uncomplicated gonococcal infection in Singapore, the Philippines, and Denmark. Twenty-seven were laboratory reference strains. Of the patient strains, 102 belonged to COA serogroup WI, and all of the 100 strains that typed with protein I serotypes 1, 2, or 3 were in this group. Most of the strains of gonococci from the 61 patients with disseminated gonococcal infection were within this group (COA WI, 53 or 87%; protein I serotypes 1, 2, or 3, 51 or 84%). All 46 strains that were protein I serotypes 4 through 7 were also COA serogroup WII. Protein I serotypes 8 and 9 accounted for 49 (25%) of the 197 patient strains. Twenty-eight of these strains typed as COA serogroup WII, 20 typed as serogroup WIII, and 1 typed as serogroups WII and WIII. COA W serogrouping and protein I ELISA both appeared to detect antigens on the protein I molecule of the outer membrane of Neisseria gonorrhoeae. Protein I serotyping, which uses unboiled organisms, may generally recognize more variable and surface-exposed antigenic determinants. In contrast, COA W serogrouping, which uses boiled organisms, may recognize less exposed shared antigenic determinants in addition to variable protein I antigenic determinants. Both methods may prove useful for further studies of the epidemiology and pathogenesis of gonorrhea. Images PMID:6172380

  17. Comparison of Seven Commercial Antigen and Antibody Enzyme-Linked Immunosorbent Assays for Detection of Acute Dengue Infection

    PubMed Central

    Jarman, Richard G.; Gibbons, Robert V.; Tanganuchitcharnchai, Ampai; Mammen, Mammen P.; Nisalak, Ananda; Kalayanarooj, Siripen; Bailey, Mark S.; Premaratna, Ranjan; de Silva, H. Janaka; Day, Nicholas P. J.; Lalloo, David G.

    2012-01-01

    Seven commercial assays were evaluated to determine their suitability for the diagnosis of acute dengue infection: (i) the Panbio dengue virus Pan-E NS1 early enzyme-linked immunosorbent assay (ELISA), second generation (Alere, Australia); (ii) the Panbio dengue virus IgM capture ELISA (Alere, Australia); (iii) the Panbio dengue virus IgG capture ELISA (Alere, Australia); (iv) the Standard Diagnostics dengue virus NS1 antigen ELISA (Standard Diagnostics, South Korea); (v) the Standard Diagnostics dengue virus IgM ELISA (Standard Diagnostics, South Korea); (vi) the Standard Diagnostics dengue virus IgG ELISA (Standard Diagnostics, South Korea); and (vii) the Platelia NS1 antigen ELISA (Bio-Rad, France). Samples from 239 Thai patients confirmed to be dengue virus positive and 98 Sri Lankan patients negative for dengue virus infection were tested. The sensitivities and specificities of the NS1 antigen ELISAs ranged from 45 to 57% and 93 to 100% and those of the IgM antibody ELISAs ranged from 85 to 89% and 88 to 100%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics ELISAs gave the best compromise between sensitivity and specificity (87 and 96%, respectively), as well as providing the best sensitivity for patients presenting at different times after fever onset. The Panbio IgG capture ELISA correctly classified 67% of secondary dengue infection cases. This study provides strong evidence of the value of combining dengue virus antigen- and antibody-based test results in the ELISA format for the diagnosis of acute dengue infection. PMID:22441389

  18. Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with Neurocryptococcosis

    PubMed Central

    2012-01-01

    Background Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC) as primary cause of the mycosis. Methods Healthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil). Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of 3H-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI) and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve). We applied unpaired Student t tests to compare data and considered significant differences for p<0.05. Results The lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis. Conclusion Response to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed. PMID:23110700

  19. Identification of Hog Cholera Viral Antigen by Immunofluorescence. Application as a Diagnostic and Assay Method.

    PubMed

    Mengeling, W L; Pirtle, E C; Torrey, J P

    1963-10-01

    In the absence of detectable cytologic changes in hog cholera virus-infected tissue culture cells, hog cholera viral antigen was readily detected by immunofluorescence. The ability to detect hog cholera viral antigen by this method allowed for determination of infectivity titers and also for titration of homologous antibody. Immunofluorescence made possible the identification, in tissue culture, of hog cholera virus from blood, serum, and spleen extracts of experimentally infected swine. Further applications of this method and its limitations are being investigated.

  20. A new fluorescent based screening system for high throughput screening of drugs targeting HBV-core and HBsAg interaction.

    PubMed

    Suresh, V; Krishnakumar, K A; Asha, V V

    2015-03-01

    The existing screening systems for anti-hepatitis B virus (anti-HBV) drug discovery is time-consuming mainly due to the laborious detection system it is using. A new fluorescence based screening system for high throughput anti-HBV drug discovery was created by tagging hepatitis B surface antigen (HBsAg) with monomeric red fluorescent protein and hepatitis B virus (HBV) core protein with enhanced green fluorescent protein. The two constructs were co-transfected on to Hep3B cells and the transfection was stabilized by fluorescent activated cell sorter (FACS). The fusion proteins expressed through the secretory protein pathway as evidenced by localization with ER-Tracker and tubulin tracker. The new system has given analogues results like that of conventional enzyme-linked immunosorbent assay (ELISA). Hence it can be of very high potential for large scale drug screening systems.

  1. Enzyme-linked immunosorbent assay with a monoclonal antibody for detecting group A meningococcal antigens in cerebrospinal fluid.

    PubMed

    Sugasawara, R J; Prato, C M; Sippel, J E

    1984-02-01

    Hybridomas were produced from spleen cells of BALB/c mice immunized with a membrane preparation from Neisseria meningitidis group A strain 4402 and S194/5.XXOBU.14 myeloma cells. The hybridomas were screened for secretion of antibodies suitable for an enzyme-linked immunosorbent assay (ELISA) diagnostic for group A meningococcal meningitis. One hybridoma antibody, 3G7, was directed against the pilus protein. This antibody bound to all six lipopolysaccharide and protein group A meningococcal serotyping strains, as well as to meningococcal strains from serogroups C, W135, and Y, but not to a strain of Escherichia coli, Haemophilus influenzae type b, or to two or more strains of Streptococcus pneumoniae, Neisseria gonorrhoeae, and Salmonella typhi. The ELISA used on antibody, antigen, antibody-conjugate sandwich. Rabbit anti-meningococcal serum was the coating antibody for the antibody sandwich, cerebrospinal fluids contained the bacterial antigens, and 3G7-alkaline phosphatase conjugate was the detecting antibody. The monoclonal antibody conjugate ELISA system was able to detect group A meningococcal antigens in 21 of 25 cerebrospinal fluid specimens that were positive in an immune rabbit serum conjugate ELISA; cerebrospinal fluid samples from patients with Haemophilus meningitis served as the controls. Counterimmunoelectrophoresis detected meningococcal antigens in 16 of the same 25 cerebrospinal fluid samples.

  2. Prevalence of HBsAg, knowledge, and vaccination practice against viral hepatitis B infection among doctors and nurses in a secondary health care facility in Lagos state, South-western Nigeria

    PubMed Central

    Abiola, Abdul-Hakeem Olatunji; Agunbiade, Adebukola Bola; Badmos, Kabir Bolarinwa; Lesi, Adenike Olufunmilayo; Lawal, Abdulrazzaq Oluwagbemiga; Alli, Quadri Olatunji

    2016-01-01

    Introduction Hepatitis B Virus, a highly infectious blood-borne virus poses a major threat to public health globally due to its high prevalence rate and grave consequence in causing liver cirrhosis and hepatocelullar carcinoma, the third cause of cancer death worldwide. The aim is determine the prevalence of HBsAg, knowledge, and vaccination practices against viral hepatitis B infection among doctors and nurses in a health care facility. Methods Study design was a descriptive cross-sectional study among all the doctors and nurses in the health care facility. Data was collected using pre-tested, structured, self-administered questionnaire and blood samples were taken from respondents and tested using commercial enzyme-linked immunosorbent assay (ELIZA) test kit to determine prevalence of hepatitis B surface antigen after informed consent. Ethical approval was obtained from Health Research and Ethics Committee of the Lagos University Teaching Hospital. Responses of the respondents to the knowledge and vaccination practices against viral hepatitis B infection were scored and graded as poor (<50%), fair (50-74%) and good (≥75%). The study was carried out in January, 2014. Results A total of 134 out of the 143 recruited respondents participated in the study. Prevalence of HBsAg was 1.5%. Among the respondents, 56.7% had good knowledge and 94.8% reported poor practice of vaccination against viral hepatitis B infection. Mean knowledge and vaccination practices scores (%) were 72.54+7.60 and 29.44+14.37 respectively. Only 29% of the respondents did post vaccination testing for anti HBsAg. Conclusion Prevalence of HBsAg was low. Knowledge of viral hepatitis B was fair, and practice of post hepatitis B vaccination testing was poor. It is therefore recommended that the state ministry of health should organise further health education programme, institute compulsory occupational hepatitis B vaccination programme and post vaccination anti-HBS testing to ensure adequate

  3. Comparison of different antigen preparations as substrates for use in passive hemagglutination and enzyme-linked immunosorbent assays for detection of antibody against bovine enteric coronavirus.

    PubMed Central

    Crouch, C F; Raybould, T J

    1983-01-01

    Purified coronavirus, detergent extracts of purified coronavirus, and virus-infected Madin-Darby bovine kidney cells were evaluated as antigen substrates in enzyme-linked immunosorbent assay (ELISA) and passive hemagglutination systems. Only detergent-extracted and -unextracted, purified viruses were reactive as antigen substrates in ELISA, whereas all three antigen preparations could be used for sensitization of erythrocytes in the passive hemagglutination assay. The passive hemagglutination system with infected cell extracts exhibited a similar level of sensitivity and specificity to the ELISA system employing purified coronavirus but enabled 300 times more tests to be performed per volume of virus-infected cell culture. PMID:6309897

  4. Spontaneous meningitis due to Streptococcus salivarius subsp. salivarius: cross-reaction in an assay with a rapid diagnostic kit that detected Streptococcus pneumoniae antigens.

    PubMed

    Shirokawa, Taijiro; Nakajima, Jun; Hirose, Kazuhito; Suzuki, Hiromichi; Nagaoka, Shoko; Suzuki, Masatsune

    2014-01-01

    Streptococcus salivarius subsp. salivarius occasionally causes meningitis associated with iatrogenic or traumatic events. We herein describe a case of meningitis caused by this organism in a patient without any apparent risk factors. In an assay of the patient's cerebrospinal fluid, cross-reaction occurred with Streptococcus pneumoniae antigen-coated latex particles in the Pastorex Meningitis Kit. In the in vitro assays, three of the five clinically isolated S. salivarius strains showed cross-reactions with the kit, indicating that these strains expressed pneumococcal antigen-like antigens. This case shows that meningitis caused by S. salivarius can occur spontaneously and it may sometimes be misdiagnosed as S. pneumoniae infection. PMID:24492701

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    PubMed

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

    2005-01-01

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

  7. Detection of antibody to hepatitis B core antigen (anti-HBc) using a direct (antiglobulin) format and development of a confirmatory assay for anti-HBc.

    PubMed

    Nelles, M J; Taylor, L; Filer, S; Wellerson, R; Haberzettl, C; Sito, A; Geltosky, J E

    1988-07-01

    A direct (antiglobulin) solid-phase enzyme immunoassay for the detection of antibody to hepatitis B core antigen (anti-HBc) is described. The assay utilizes recombinant hepatitis B core antigen as the solid-phase 'capture' reagent and a mixture of monoclonal antibodies specific for human IgG and IgM conjugated to horseradish peroxidase as the 'detector' reagent. The direct assay demonstrated excellent sensitivity and specificity when compared with a commercially available competitive enzyme immunoassay. The direct assay format lends itself to a confirmatory assay for anti-HBc by addition of monoclonal anti-HBc to the reaction mixture. Feasibility of the confirmatory assay for anti-HBc was demonstrated using specimens reactive for anti-HBc as documented by both the direct and competitive assays.

  8. A homogenous fluorescence quenching based assay for specific and sensitive detection of influenza virus A hemagglutinin antigen.

    PubMed

    Chen, Longyan; Neethirajan, Suresh

    2015-01-01

    Influenza pandemics cause millions of deaths worldwide. Effective surveillance is required to prevent their spread and facilitate the development of appropriate vaccines. In this study, we report the fabrication of a homogenous fluorescence-quenching-based assay for specific and sensitive detection of influenza virus surface antigen hemagglutinins (HAs). The core of the assay is composed of two nanoprobes namely the glycan-conjugated highly luminescent quantum dots (Gly-QDs), and the HA-specific antibody-modified gold nanoparticle (Ab-Au NPs). When exposed to strain-specific HA, a binding event between the HA and the two nanoprobes takes place, resulting in the formation of a sandwich complex which subsequently brings the two nanoprobes closer together. This causes a decrease in QDs fluorescence intensity due to a non-radiative energy transfer from QDs to Au NPs. A resulting correlation between the targets HA concentrations and fluorescence changes can be observed. Furthermore, by utilizing the specific interaction between HA and glycan with sialic acid residues, the assay is able to distinguish HAs originated from viral subtypes H1 (human) and H5 (avian). The detection limits in solution are found to be low nanomolar and picomolar level for sensing H1-HA and H5-HA, respectively. Slight increase in assay sensitivity was found in terms of detection limit while exposing the assay in the HA spiked in human sera solution. We believe that the developed assay could serve as a feasible and sensitive diagnostic tool for influenza virus detection and discrimination, with further improvement on the architectures. PMID:25884789

  9. A Homogenous Fluorescence Quenching Based Assay for Specific and Sensitive Detection of Influenza Virus A Hemagglutinin Antigen

    PubMed Central

    Chen, Longyan; Neethirajan, Suresh

    2015-01-01

    Influenza pandemics cause millions of deaths worldwide. Effective surveillance is required to prevent their spread and facilitate the development of appropriate vaccines. In this study, we report the fabrication of a homogenous fluorescence-quenching-based assay for specific and sensitive detection of influenza virus surface antigen hemagglutinins (HAs). The core of the assay is composed of two nanoprobes namely the glycan-conjugated highly luminescent quantum dots (Gly-QDs), and the HA-specific antibody-modified gold nanoparticle (Ab-Au NPs). When exposed to strain-specific HA, a binding event between the HA and the two nanoprobes takes place, resulting in the formation of a sandwich complex which subsequently brings the two nanoprobes closer together. This causes a decrease in QDs fluorescence intensity due to a non-radiative energy transfer from QDs to Au NPs. A resulting correlation between the targets HA concentrations and fluorescence changes can be observed. Furthermore, by utilizing the specific interaction between HA and glycan with sialic acid residues, the assay is able to distinguish HAs originated from viral subtypes H1 (human) and H5 (avian). The detection limits in solution are found to be low nanomolar and picomolar level for sensing H1-HA and H5-HA, respectively. Slight increase in assay sensitivity was found in terms of detection limit while exposing the assay in the HA spiked in human sera solution. We believe that the developed assay could serve as a feasible and sensitive diagnostic tool for influenza virus detection and discrimination, with further improvement on the architectures. PMID:25884789

  10. Mannan as an antigen in cell-mediated immunity (CMI) assays and as a modulator of mannan-specific CMI.

    PubMed

    Domer, J E; Garner, R E; Befidi-Mengue, R N

    1989-03-01

    Mannan (MAN) extracted from Candida albicans 20A was investigated for its potential as an antigen in the detection of cell-mediated immunity (CMI) in vivo and in vitro and for its ability to modulate CMI when administered intravenously (i.v.). CBA/J mice were either immunized as adults by the cutaneous inoculation of 10(6) viable blastoconidia or colonized as infants (primed) and then boosted cutaneously as adults. When immunized animals were footpad tested with MAN, highly significant delayed-type hypersensitivity (DH) responses were detected. The DH responses to MAN were of a greater magnitude than those noted with the same quantity of cell wall glycoprotein (GP), an ethylenediamine extract of the cell wall which contains both glucan and MAN. In contrast, GP was a better antigen for the detection of CMI responses in an in vitro lymphoproliferative assay with either spleen or lymph node cell suspensions. Mice treated with MAN i.v. prior to the initiation of immunization or between priming and secondary inoculations developed significantly suppressed DH reactions when tested with either MAN or GP. The lowest effective dose of MAN was 250 micrograms, maximum suppression occurred with 500 micrograms, and either dose given 1 week prior to immunization was suppressive. The suppression by MAN was specific for MAN or the MAN-containing GP. Responses to another unrelated candidal antigen, a membrane extract designated BEX, were relatively unaffected. MAN, therefore, was an effective antigen for the detection of CMI in vivo, and its administration i.v. created what appeared to be a MAN-specific suppression since it could be detected with both MAN and a MAN-containing extract from the cell wall. Caution must be exercised in the interpretation of these data, however, since the protein component of each of these extracts has not been characterized with respect to its potential role in the phenomena observed.

  11. Evaluation by enzyme-linked immunosorbent assay (ELISA) of Renibacterium salmoninarum bacterins affected by persistence of bacterial antigens

    USGS Publications Warehouse

    Pascho, R.J.; Goodrich, T.D.; McKibben, C.L.

    1997-01-01

    Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with a bacterin containing killed Renibacterium salmoninarum cells delivered alone or in an oil-based adjuvant. We evaluated the relative abilities of the batterins to prevent the initiation or progression of infection in fish challenged by waterborne exposure to R. salmoninarum. Sixty-one days after vaccination, fish were held for 24 h in water containing either no bacteria or approximately 1.7 x 103, 1.7 x 105, or 5.3 x 106 live R. salmoninarum cells/mL. An enzyme-linked immunosorbent assay (ELISA) was used to monitor changes in the levels of R. salmoninarum antigen in live fish before and after the immersion challenges. High levels of R. salmoninarum antigens were detected by ELISA in kidney-spleen tissue homogenates from vaccinated fish immediately before the challenges. Levels of those antigens remained high in the tissues of unchallenged fish throughout the study. We found that the ELISA used in this study may be unsuitable for evaluating the efficacy of batterins because it did not distinguish antigens produced by the challenge bacteria during an infection from those of the bacterins. Groups of control and vaccinated fish also were injected with either 1.7 x 104 or 1.7 x 106 R. salmoninarum cells and served as R. salmoninarum virulence controls. Relative survival among the various subgroups in the injection challenge suggests that adverse effects might have been associated with the adjuvant used in this study. The lowest survival at both injection challenge levels was among fish vaccinated with bacteria in adjuvant.

  12. Analysis of tuberculous meningitis cases by an immunoblotting assay based on a mycobacterial antigen complex.

    PubMed Central

    Zou, Y L; Van Antwerpen, M P; Shi, G Q; Chen, Q X; Sindic, C J; Cocito, C

    1994-01-01

    Tuberculous meningitis cases were analyzed by an immunoblotting test based on Mycobacterium bovis BCG antigen complex A60. Anti-A60 immunoglobulin G (IgG) in cerebrospinal fluid (CSF) allowed early diagnosis, and concentrations decreased after recovery. In primary meningitis forms, anti-A60 IgGs were intrathecally synthesized and specific oligoclonal IgGs were present in CSF. In meningeal complications of pulmonary tuberculosis, there were matching titers of anti-A60 IgG in blood and CSF (mirror pattern). Correlation between CSF-restricted patterns and CSF pleocytosis was shown. Images PMID:7496976

  13. Japanese Reference Panel of Blood Specimens for Evaluation of Hepatitis C Virus RNA and Core Antigen Quantitative Assays

    PubMed Central

    Murayama, Asako; Sugiyama, Nao; Watashi, Koichi; Masaki, Takahiro; Suzuki, Ryosuke; Aizaki, Hideki; Mizuochi, Toshiaki; Wakita, Takaji

    2012-01-01

    An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits. PMID:22495557

  14. Bovine Tuberculosis: Analyzing the Parameters of the Interferon Gamma Assay and Improved Diagnosis with New Antigens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine tuberculosis (TB), a zoonotic disease with a major economic impact, continues to be a significant problem with a global perspective. The BOVIGAM® interferon gamma (IFN-gamma) assay constitutes a laboratory-based tuberculosis test and is widely used complementary to the tuberculin skin test....

  15. Identification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay.

    PubMed

    Cryan, Lorna M; Habeshian, Kaiane A; Caldwell, Thomas P; Morris, Meredith T; Ackroyd, P Christine; Christensen, Kenneth A; Rogers, Michael S

    2013-07-01

    Tumor marker endothelial 8 (TEM8) is a receptor for the protective antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared with normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z' value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complementary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for antianthrax and antiangiogenic diseases.

  16. Partial analysis of hepatitis B virus DNA from hepatocellular carcinoma showing negative hepatitis B virus surface antigen: an analysis of two Japanese cases.

    PubMed

    Shiota, Goshi; Oyama, Kenji; Udagawa, Akihide; Nomi, Takahiro; Tanaka, Kiwamu; Tsutsumi, Atsushi; Noguchi, Noya; Okano, Jun-ichi; Kishimoto, Yosuke; Kanbe, Takamasa; Kishimoto, Hiroyuki; Kawasaki, Hironaka

    2009-01-01

    It has been reported that hepatitis B virus (HBV) DNA is detected in serum and/or liver in patients with hepatocellular carcinoma (HCC) without HBsAg. To adress this issue, we analyzed HBV genome in 2 HCC cases without HBsAg. The DNA from serum from patients with HCC was amplified with a nested PCR, and 'a' determinant of S region, core promoter region and precore region were sequenced. The first case, a 50 years-old male, was negative for HBsAg and HBeAg, and positive for anti-HBs, anti-HBe and anti-HBc. Viral load of HBV in serum was 4.0 log genome equivalent/ml by TMA assay, and was 1.1 X 105 copy/ml by real-time PCR system. A nucleotide analysis of the common 'a' determinant of S gene showed that the 5 first amino acids of 'a' determinant, CTIPA, were changed to CKTCTTPA. The second case, a 76 years-old male, was positive for anti-HBe, but negative for HBsAg, anti-HBs, HBeAg and anti-HBc. No missense or nonsense mutations were seen in 'a' determinant of S region. Viral load of serum HBV was < 3.7 log genome equivalent/ml by TMA assay, but was 2.4X103 copy/ml by real-time PCR system. The results of the present study suggest that the mechanisms of HBsAg loss are diverse among HCC patients without HBsAg, and that an analysis of HBV genome is a useful tool to dissolve molecular mechanisms losing HBs antigenicity. PMID:19950820

  17. Evaluation of Recombinant Leptospira Antigen-Based Enzyme-Linked Immunosorbent Assays for the Serodiagnosis of Leptospirosis

    PubMed Central

    Flannery, Brendan; Costa, Dirceu; Carvalho, Fernanda Pinheiro; Guerreiro, Hygia; Matsunaga, James; Da Silva, Emilson Domingos; Ferreira, Antonio Gomes Pinto; Riley, Lee W.; Reis, Mitermayer G.; Haake, David A.; Ko, Albert I.

    2001-01-01

    There is an urgent need for development of new serodiagnostic strategies for leptospirosis, an emerging zoonosis with worldwide distribution. We have evaluated the diagnostic utility of five recombinant antigens in enzyme-linked immunosorbent assays (ELISAs) for serodiagnosis of leptospirosis. Sera from 50 healthy residents of a high-incidence region were used to determine cutoff values for 96% specificity. In paired sera from 50 cases of leptospirosis confirmed by the microscopic agglutination test, immunoglobulin G (IgG) but not IgM reacted with the recombinant leptospiral proteins. The recombinant LipL32 IgG ELISA had the highest sensitivities in the acute (56%) and convalescent (94%) phases of leptospirosis. ELISAs based on recombinant OmpL1, LipL41, and Hsp58 had sensitivities of 16, 24, and 18% during the acute phase and 72, 44, and 32% during convalescence, respectively. Compared to sera from healthy individuals, patient sera did not react significantly with recombinant LipL36 (P > 0.05). Recombinant LipL32 IgG ELISA demonstrated 95% specificity among 100 healthy individuals, and specificities ranging from 90 to 97% among 30 dengue patients, 30 hepatitis patients, and 16 patients with diseases initially thought to be leptospirosis. Among 39 Venereal Disease Research Laboratory test-positive individuals and 30 Lyme disease patients, 13 and 23% of sera, respectively, reacted positively with the rLipL32 antigen. These findings indicate that rLipL32 may be an useful antigen for the serodiagnosis of leptospirosis. PMID:11526167

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

    PubMed

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

    2015-06-01

    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. Diagnostic value of the indirect immunofluorescence assay in cat scratch disease with Bartonella henselae and Afipia felis antigens.

    PubMed Central

    Amerein, M P; De Briel, D; Jaulhac, B; Meyer, P; Monteil, H; Piemont, Y

    1996-01-01

    Serum samples from 35 cat scratch disease (CSD) patients, 180 control patients (123 without lymph node enlargement and 57 with lymph node enlargement not evoking CSD), and 102 nonpatient subjects (35 with cat contact and 67 without cat contact) were tested by semiquantitative indirect immunofluorescence assay for the presence of antibodies directed to Afipia felis (ATCC 53690T) or Bartonella henselae (ATCC 49882T). The CSD group had statistically higher antibody titers against B. henselae than the control groups (P < 10(-5)), whereas no difference in A. felis antibody titers was evidenced among all groups tested. Among the 317 serum samples studied, the three with high A. felis antibody titers ( > or = 64) also had high antibody titers against other alpha-2 proteobacteria. The value of the indirect immunofluorescence assay with B. henselae antigen for the diagnosis of CSD was as follows: for a cutoff of 32, sensitivity was 0.80, specificity was 0.85, and the likelihood ratio was 5.1; for a cutoff of 64, the likelihood ratio was 12.1. In summary, in France, CSD is associated with high antibody titers against B. henselae, as previously described in the United States. However, the causes for B. henselae seronegativity in CSD patients and those for high antibody titers outside the typical nosological frame of CSD still have to be identified. PMID:8991636

  20. Evaluation of prostate-specific antigen (PSA) membrane test assays for the forensic identification of seminal fluid.

    PubMed

    Hochmeister, M N; Budowle, B; Rudin, O; Gehrig, C; Borer, U; Thali, M; Dirnhofer, R

    1999-09-01

    Prostate specific antigen (PSA, also known as p30), a glycoprotein produced by the prostatic gland and secreted into seminal plasma, is a marker used for demonstrating the presence of seminal fluid. Methods for the detection of PSA include Ouchterlony double diffusion, crossover electrophoresis, rocket immuno-electrophoresis, radial immunodiffusion, and ELISA. The extremely sensitive ELISA technique can detect PSA in concentrations as low as approximately 4 ng/mL. However, all these techniques are cumbersome and time consuming to perform in forensic laboratories, especially when only a few samples per week are processed. Various membrane tests are currently used in clinical settings to screen a patient's serum for the presence of PSA at levels greater than 4 ng/mL. In this study we evaluated three immunochromatographic PSA membrane tests by analyzing semen stains stored at room temperature for up to 30 years, post-coital vaginal swabs taken at different time after intercourse, semen-free vaginal swabs, and various female and male body fluids, including urine. The data demonstrate that PSA membrane test assays offer the same sensitivity as ELISA-based tests and provide a rapid approach for the forensic identification of seminal fluid. Furthermore, when the supernatant from a DNA extraction is used for the assay, there is essentially no DNA consumption for determining the presence of PSA in a forensic sample.

  1. Application of synthetic 8-kD and recombinant GP50 antigens in the diagnosis of neurocysticercosis by enzyme-linked immunosorbent assay.

    PubMed

    Bueno, Edneia C; Scheel, Christina M; Vaz, Adelaide J; Machado, Luis R; Livramento, José A; Takayanagui, Osvaldo M; Tsang, Victor C W; Hancock, Kathy

    2005-03-01

    The gold standard serodiagnostic assay for cysticercosis and neurocysticercosis, diseases caused by the metacestode of Taenia solium, uses lentil lectin-purified glycoprotein (LLGP) in a Western blot assay. We tested two antigens derived from LLGP, synthetic TS18var1 (sTS18var1) and recombinant GP50 antigen (rGP50), in an enzyme-linked immunosorbent assay (ELISA) using serum and cerebrospinal fluid (CSF) samples. The sensitivity for serum and CSF was 94.7% and 100% for rGP50 and 90.4% and 90.2% for sTS18var1, respectively. The specificity for serum and CSF samples was 93.8% and 100% for rGP50 and 90.3% and 98.0% for sTS18var1, respectively. The use of these antigens individually or combined as a diagnostic antigen cocktail eliminates the need for purification of antigens from parasite material and offers the advantage of using a simple and quantitative ELISA format.

  2. Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

    PubMed Central

    DebRoy, Chitrita; Fratamico, Pina M.; Roberts, Elisabeth; Davis, Michael A.; Liu, Yanhong

    2005-01-01

    The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 106 and 108 CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55. PMID:16085897

  3. Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses

    PubMed Central

    Westgeest, Kim; Bestebroer, Theo M.; Spronken, Monique I.J.; Gao, Jin; Couzens, Laura; Osterhaus, Albert D.M.E.; Eichelberger, Maryna; Fouchier, Ron A.M.; de Graaf, Miranda

    2015-01-01

    Antibodies to neuraminidase (NA), the second most abundant surface protein of the influenza virus, contribute to protection against influenza virus infection. Although traditional and miniaturized thiobarbituric acid (TBA) neuraminidase inhibition (NI) assays have been successfully used to characterize the antigenic properties of NA, these methods are cumbersome and not easily amendable to rapid screening. An additional difficulty of the NI assay is the interference by hemagglutinin (HA)-specific antibodies. To prevent interference of HA-specific antibodies, most NI assays are performed with recombinant viruses containing a mismatched HA. However, generation of these viruses is time consuming and unsuitable for large-scale surveillance. The feasibility of using the recently developed enzyme-linked lectin assay (ELLA) to evaluate the antigenic relatedness of NA of wild type A(H3N2) viruses was assessed. Rather than using recombinant viruses, wild type A(H3N2) viruses were used as antigen with ferret sera elicited against recombinant viruses with a mismatched HA. In this study, details of the critical steps that are needed to modify and optimize the NI ELLA in a format that is reproducible, highly sensitive, and useful for influenza virus surveillance to monitor antigenic drift of NA are provided. PMID:25712563

  4. Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses.

    PubMed

    Westgeest, Kim B; Bestebroer, Theo M; Spronken, Monique I J; Gao, Jin; Couzens, Laura; Osterhaus, Albert D M E; Eichelberger, Maryna; Fouchier, Ron A M; de Graaf, Miranda

    2015-06-01

    Antibodies to neuraminidase (NA), the second most abundant surface protein of the influenza virus, contribute to protection against influenza virus infection. Although traditional and miniaturized thiobarbituric acid (TBA) neuraminidase inhibition (NI) assays have been successfully used to characterize the antigenic properties of NA, these methods are cumbersome and not easily amendable to rapid screening. An additional difficulty of the NI assay is the interference by hemagglutinin (HA)-specific antibodies. To prevent interference of HA-specific antibodies, most NI assays are performed with recombinant viruses containing a mismatched HA. However, generation of these viruses is time consuming and unsuitable for large-scale surveillance. The feasibility of using the recently developed enzyme-linked lectin assay (ELLA) to evaluate the antigenic relatedness of NA of wild type A(H3N2) viruses was assessed. Rather than using recombinant viruses, wild type A(H3N2) viruses were used as antigen with ferret sera elicited against recombinant viruses with a mismatched HA. In this study, details of the critical steps that are needed to modify and optimize the NI ELLA in a format that is reproducible, highly sensitive, and useful for influenza virus surveillance to monitor antigenic drift of NA are provided.

  5. Hepatitis B surface antigen clearance in inactive hepatitis B surface antigen carriers treated with peginterferon alfa-2a

    PubMed Central

    Li, Ming-Hui; Xie, Yao; Zhang, Lu; Lu, Yao; Shen, Ge; Wu, Shu-Ling; Chang, Min; Mu, Cai-Qin; Hu, Lei-Ping; Hua, Wen-Hao; Song, Shu-Jing; Zhang, Shu-Feng; Cheng, Jun; Xu, Dao-Zhen

    2016-01-01

    AIM: To examine the association between interferon (IFN) therapy and loss of hepatitis B surface antigen (HBsAg) in inactive HBsAg carriers. METHODS: This was a retrospective cohort study in inactive HBsAg carriers, who were treatment-naive, with a serum HBsAg level < 100 IU/mL and an undetectable hepatitis B virus (HBV) DNA level (< 100 IU/mL). All the 20 treated patients received subcutaneous PEG-IFN alfa-2a 180 μg/wk for 72 wk and were then followed for 24 wk. There were 40 untreated controls matched with 96 wk of observation. Serum HBsAg, HBV DNA, and alanine aminotransferases were monitored every 3 mo in the treatment group and every 3-6 mo in the control group. RESULTS: Thirteen (65.0%) of 20 treated patients achieved HBsAg loss, 12 of whom achieved HBsAg seroconversion. Mean HBsAg level in treated patients decreased to 6.69 ± 13.04 IU/mL after 24 wk of treatment from a baseline level of 26.22 ± 33.00 IU/mL. Serum HBV DNA level remained undetectable (< 100 IU/mL) in all treated patients during the study. HBsAg level of the control group decreased from 25.72 ± 25.58 IU/mL at baseline to 17.11 ± 21.62 IU/mL at week 96 (P = 0.108). In the control group, no patient experienced HBsAg loss/seroconversion, and two (5.0%) developed HBV reactivation. CONCLUSION: IFN treatment results in HBsAg loss and seroconversion in a considerable proportion of inactive HBsAg carriers with low HBsAg concentrations. PMID:27239256

  6. Detection of flagellar antigen of Campylobacter jejuni and Campylobacter coli in canine faeces with an enzyme-linked immunosorbent assay (ELISA)--new prospects for diagnosis.

    PubMed

    Monfort, J D; Bech-Nielsen, S; Stills, H F

    1994-01-01

    A new diagnostic procedure was developed to detect the flagellar antigen of Campylobacter jejuni and Campylobacter coli in canine faecal specimens and was tested on faecal samples from random-source dogs obtained from the local dog pound. Extraction of acid-soluble proteins was performed on faecal specimens and the extracted material was evaluated using species-specific monoclonal antibodies in an enzyme-linked immunosorbent assay. The assay detected all C. jejuni or C. coli infected specimens compared with direct selective faecal culture. One of 18 faecal specimens culture-negative for C. jejuni was identified as positive by the assay, i.e. a false positive rate of 1 of 18 (5.6%) and a corresponding specificity of 94.4%. These results suggest that the screening procedure developed to detect flagellar antigens of C. jejuni and C. coli in canine faecal samples should be further investigated as a diagnostic alternative to culture.

  7. Sensitive non-isotopic DNA hybridisation assay or immediate-early antigen detection for rapid identification of human cytomegalovirus in urine.

    PubMed

    Kimpton, C P; Morris, D J; Corbitt, G

    1991-04-01

    A sensitive non-radioactive DNA hybridisation assay employing digoxigenin-labelled probes was compared with immediate-early antigen detection and conventional virus isolation for the identification of human cytomegalovirus (HCMV) in 249 urine samples. Of 44 specimens yielding HCMV by virus isolation, more were positive by DNA hybridisation (32; 73%) than by immediate-early antigen detection (25; 52%) (P = 0.05). The specificity of the hybridisation assay in 45 apparently falsely positive specimens was supported by detection of HCMV DNA in 40 of these specimens using the polymerase chain reaction. Many urine specimens may thus contain large amounts of non-viable virus or free viral DNA. Evaluation of various protocols for the extraction and denaturation of virus DNA prior to hybridisation showed that proteinase K digestion with phenol/chloroform extraction was the most sensitive and reliable procedure. We conclude that the non-radioactive DNA hybridisation assay described is a potentially valuable routine diagnostic test.

  8. The European Sero-Epidemiology Network 2: standardization of assay results for hepatitis B virus.

    PubMed

    Kafatos, G; Anastassopoulou, C; Nardone, A; Andrews, N; Barbara, C; Boot, H J; Butur, D; Davidkin, I; Gelb, D; Griskevicius, A; Hesketh, L; Icardi, G; Jones, L; Kra-Oz, Z; Miller, E; Mossong, J; Nemecek, V; de Ory, F; Sobotová, Z; Thierfelder, W; Van Damme, P; Hatzakis, A

    2007-04-01

    The aim of the European Sero-Epidemiology Network 2 was to coordinate and standardize the serological surveillance of vaccine-preventable diseases in Europe. In this study, the standardization of hepatitis B virus (HBV) results is described. The 15 participating national laboratories tested a unique panel of 172 sera established by the Greek reference centre for HBV surface antigen (HBsAg), antibodies to HBsAg (anti-HBs) and/or to the HBV core antigen (anti-HBc) by assay methods of their choice. Country-specific quantitative measurements for anti-HBs and anti-HBc were transformed into common units using standardization equations derived by regressing each country's panel results against the reference centre's results, thus adjusting for interassay and interlaboratory variability. For HBsAg, a qualitative analysis (positive/negative) showed at least 99% agreement with the reference laboratory for all countries. By combining these standardized and qualitative results for the markers mentioned earlier, it was possible to achieve comparable estimates of the proportion of the population susceptible to HBV, vaccinated against HBV, with a past HBV infection, and with a current infection or chronic carrier state. Standardization is a very important tool that allows for international serological comparisons to assess the current vaccination policies and the progress of HBV control in Europe.

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

    PubMed

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

    2012-09-01

    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.

  10. The use of enzyme-linked immunosorbent assay systems for serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in The Netherlands.

    PubMed

    Rimmelzwaan, G F; Groen, J; Egberink, H; Borst, G H; UytdeHaag, F G; Osterhaus, A D

    1991-01-01

    Complex trapping blocking (CTB) enzyme-linked immunosorbent assays (ELISAs) and indirect ELISAs for the detection of antibodies to canine parvovirus (CPV), canine coronavirus (CCV) and rotavirus in sera of dogs were established. Double antibody sandwich ELISAs for the detection of CPV-, CCV- and rotavirus antigens in fecal samples were also developed. Both the serological and antigen-detection ELISAs were used to screen samples from dogs in The Netherlands, with or without a history of acute diarrhea. It was shown that the results of the respective serological ELISAs correlated well and that CPV was the major cause of virus-induced acute diarrhea in dogs in The Netherlands. PMID:1850889

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

    PubMed

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

    2013-05-31

    Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) β3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsβ3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems. PMID:23454035

  12. Comparison of Schistosoma mansoni Prevalence and Intensity of Infection, as Determined by the Circulating Cathodic Antigen Urine Assay or by the Kato-Katz Fecal Assay: A Systematic Review

    PubMed Central

    Kittur, Nupur; Castleman, Jennifer D.; Campbell, Carl H.; King, Charles H.; Colley, Daniel G.

    2016-01-01

    The relationship between results from Kato-Katz (KK) fecal microscopy and urine-based point-of-care circulating cathodic antigen (POC-CCA) assays for Schistosoma mansoni infection remains a critical issue. This systematic literature review of 25 published papers compares prevalence of S. mansoni infection by KK with that by the POC-CCA assay. Nineteen published studies met our inclusion criteria for data extraction and analysis. Above a prevalence of 50% by KK, KK and POC-CCA results yielded essentially the same prevalence. Below 50% prevalence by KK, the prevalence by the POC-CCA assay was between 1.5- and 6-fold higher and increased as prevalence by KK decreased. Five of nine publications met inclusion criteria for extractable data on intensity of S. mansoni infection by KK assay and visual band density using the POC-CCA assay. A clear positive relationship exists between intensity by the KK and POC-CCA assays. This systematic review indicates that below 50% prevalence, the POC-CCA assay is much more sensitive than the KK assay. However, the existing data are inadequate to precisely define the relationship between POC-CCA and KK at lower levels of KK prevalence. More studies directly comparing the two assays in low-prevalence areas are essential to inform decision-making by national schistosomiasis control programs. PMID:26755565

  13. Serodiagnosis of Toxoplasma gondii infection in farm animals (horses, swine, and sheep) by enzyme-linked immunosorbent assay using chimeric antigens.

    PubMed

    Ferra, Bartłomiej; Holec-Gąsior, Lucyna; Kur, Józef

    2015-10-01

    Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the animal husbandry. Commonly used serological tests for diagnosis of toxoplasmosis involve preparation of whole Toxoplasma lysate antigen (TLA) from tachyzoites. The production of this antigen is associated with high costs and lengthy preparation and the possibility of staff infection. There are also some difficulties in the standardization of such tests. One approach in order to improve the diagnosis of T. gondii infection is to use recombinant chimeric antigens in place of the TLA, which was confirmed by studies in the serodiagnosis of toxoplasmosis in humans. In this paper, we assess, for the first time, the diagnostic utility of five T. gondii recombinant chimeric antigens (MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, and GRA1-GRA2-GRA6) in immunoglobulin G (IgG) enzyme-linked immunosorbent assays (IgG ELISAs) with sera from three different groups of livestock animals (horses, pigs, and sheep). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISAs based on a mixture of three antigens (M1: rSAG1+rMIC1+rMAG1, M2: rSAG2+rGRA1+rROP1, and M3: rGRA1+rGRA2+rGRA6) and referenced to TLA. All chimeric antigens were characterized by high specificity (100%), and the sensitivity of the IgG ELISAs based on chimeric antigens was variable (between 28.4% and 100%) and mainly dependent on the animal species. The chimeric antigens were generally more reactive than mixtures of three antigens. The most effective for the diagnosis of toxoplasmosis was SAG2-GRA1-ROP1L, which can detect specific anti-T. gondii antibodies in 100%, 93.8%, and 100% of positive serum samples from horses, pigs, and sheep, respectively. The present study shows that recombinant chimeric antigens can be successfully used to diagnose T. gondii infection in farm animals, and can replace the commonly

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

    PubMed

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

    2015-01-01

    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. PMID:26061673

  15. Rapid competitive enzyme-linked immunosorbent assay using a monoclonal antibody reacting with a 15-kilodalton tegumental antigen of Schistosoma mansoni for serodiagnosis of schistosomiasis.

    PubMed Central

    Da Silva, A J; Piuvezam, M R; de Moura, H; Maddison, S; Peralta, J M

    1993-01-01

    A competitive enzyme-linked immunosorbent assay (CELISA) for antibody detection was developed by using a monoclonal antibody which reacts with a 15-kDa tegumental antigen of the adult worm of Schistosoma mansoni. This monoclonal antibody was not able to react with antigens of Schistosoma japonicum or Schistosoma haematobium in enzyme-linked immunoelectrotransfer blot (EITB) and indirect immunofluorescence tests. The assay was performed in a period of 1 h using an adult worm crude extract antigen. To evaluate the CELISA, a total of 73 serum samples was analyzed: 35 were from S. mansoni-infected patients, 23 were from individuals with parasitic infections other than schistosomiasis, and 14 were from healthy individuals. All serum samples from healthy individuals and from patients infected with other parasites were negative, as were two (6%) samples from patients infected with S. mansoni. EITB analysis showed that 32 of 33 CELISA-positive samples were positive in the EITB but with different patterns of reactivity. A 15-kDa protein reacted with 60% of serum samples, and a 60-kDa protein showed the highest level of reactivity (85%). The two samples from patients infected with S. mansoni that were negative in the CELISA reacted with 70-, 60-, 50-, 47-, and 38-kDa proteins. One sample, positive in CELISA, did not react with proteins of the antigenic extract. Images PMID:8408548

  16. Diagnosis of amebic dysentery by detection of Entamoeba histolytica fecal antigen by an invasive strain-specific, monoclonal antibody-based enzyme-linked immunosorbent assay.

    PubMed Central

    Gonzalez-Ruiz, A; Haque, R; Rehman, T; Aguirre, A; Hall, A; Guhl, F; Warhurst, D C; Miles, M A

    1994-01-01

    An invasive strain-specific monoclonal antibody against Entamoeba histolytica has been used in a capture enzyme-linked immunosorbent assay (ELISA) for the detection of invasive E. histolytica fecal antigen in clinical specimens and for the diagnosis of amebic dysentery in patients from Bangladesh. The fecal antigen capture ELISA (FAC-ELISA) did not cross-react with other parasite species in the clinical specimens or with noninvasive E. histolytica present in those specimens and in experimentally seeded stools. The limit of detection of the assay for invasive E. histolytica crude antigen diluted in phosphate-buffered saline or in stools was 0.58 and 3.9 micrograms/ml, respectively, which is the equivalent of approximately 72 and 487 E. histolytica trophozoites per well, respectively. The sensitivity, specificity, and efficiency of the FAC-ELISA were 87, 100, and 98%, respectively, for the detection of invasive E. histolytica antigens and 100, 100, and 100%, respectively, for the diagnosis of amebic dysentery. The FAC-ELISA is a potential alternative for the field diagnosis of amebic dysentery and for epidemiological studies to define the distribution of invasive E. histolytica. PMID:8027351

  17. A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum.

    PubMed

    Ren, Zhi-Qi; Liu, Tian-Cai; Hou, Jing-Yuan; Chen, Mei-Jun; Chen, Zhen-Hua; Lin, Guan-Feng; Wu, Ying-Song

    2014-09-01

    Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02-700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7-8.7% and 3.8-7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X - 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus.

  18. Interspecies antigenic determinants of structural proteins of mammalian type-C viruses as detected by a competitive sepharose bead immunofluorescence assay.

    PubMed

    Micheel, B; Fiebach, H; Wunderlich, V

    1979-01-01

    The present paper describes a competitive immunoassay using antiserum to FeLV p 27 and SSV-conjugated Sepharose beads. The assay is applied to compare the interspecies-specific antigenic determinants of the major structural proteins of type-C viruses of different mammalian species. The test proves to be highly sensitive and specific and may be used for the demonstration of viral proteins in crude cellular extracts.

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

    PubMed

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

    2012-06-01

    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. Development of a Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay Using a Synthetic Peptide Antigen for Detection of Avian Metapneumovirus Antibodies in Turkey Sera

    PubMed Central

    Alvarez, Rene; Njenga, M. Kariuki; Scott, Melissa; Seal, Bruce S.

    2004-01-01

    Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes. PMID:15013970

  1. Ultrasensitive Prostate Specific Antigen Assay Following Laparoscopic Radical Prostatectomy – An Outcome Measure for Defining the Learning Curve

    PubMed Central

    Viney, R; Gommersall, L; Zeif, J; Hayne, D; Shah, ZH; Doherty, A

    2009-01-01

    INTRODUCTION Radical retropubic prostatectomy (RRP) performed laparoscopically is a popular treatment with curative intent for organ-confined prostate cancer. After surgery, prostate specific antigen (PSA) levels drop to low levels which can be measured with ultrasensitive assays. This has been described in the literature for open RRP but not for laparoscopic RRP. This paper describes PSA changes in the first 300 consecutive patients undergoing non-robotic laparoscopic RRP by a single surgeon. OBJECTIVES To use ultrasensitive PSA (uPSA) assays to measure a PSA nadir in patients having laparoscopic radical prostatectomy below levels recorded by standard assays. The aim was to use uPSA nadir at 3 months' post-prostatectomy as an early surrogate end-point of oncological outcome. In so doing, laparoscopic oncological outcomes could then be compared with published results from other open radical prostatectomy series with similar end-points. Furthermore, this end-point could be used in the assessment of the surgeon's learning curve. PATIENTS AND METHODS Prospective, comprehensive, demographic, clinical, biochemical and operative data were collected from all patients undergoing non-robotic laparoscopic RRP. We present data from the first 300 consecutive patients undergoing laparoscopic RRP by a single surgeon. uPSA was measured every 3 months post surgery. RESULTS Median follow-up was 29 months (minimum 3 months). The likelihood of reaching a uPSA of ≤ 0.01 ng/ml at 3 months is 73% for the first 100 patients. This is statistically lower when compared with 83% (P < 0.05) for the second 100 patients and 80% for the third 100 patients (P < 0.05). Overall, 84% of patients with pT2 disease and 66% patients with pT3 disease had a uPSA of ≤ 0.01 ng/ml at 3 months. Pre-operative PSA, PSA density and Gleason score were not correlated with outcome as determined by a uPSA of ≤ 0.01 ng/ml at 3 months. Positive margins correlate with outcome as determined by a uPSA of ≤ 0

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

    PubMed

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

    2009-06-01

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

  3. Quantitation of HBsAg predicts response to entecavir therapy in HBV genotype C patients

    PubMed Central

    Orito, Etsuro; Fujiwara, Kei; Kanie, Hiroshi; Ban, Tesshin; Yamada, Tomonori; Hayashi, Katsumi

    2012-01-01

    AIM: To analysis the factors that predict the response to entecavir therapy in chronic hepatitis patients with hepatitis B virus (HBV) genotype C. METHODS: Fifty patients [hepatitis B e antigen (HBeAg)-negative:HBeAg-positive = 26:24] with HBV genotype C, who received naïve entecavir therapy for > 2 years, were analyzed. Patients who showed HBV DNA levels ≥ 3.0 log viral copies/mL after 2 years of entecavir therapy were designated as slow-responders, while those that showed < 3.0 log copies/mL were termed rapid-responders. Quantitative hepatitis B surface antigen (HBsAg) levels (qHBsAg) were determined by the Architect HBsAg QT immunoassay. Hepatitis B core-related antigen was detected by enzyme immunoassay. Pre-C and Core promoter mutations were determined using by polymerase chain reaction (PCR). Drug-resistance mutations were detected by the PCR-Invader method. RESULTS: At year 2, HBV DNA levels in all patients in the HBeAg-negative group were < 3.0 log copies/mL. In contrast, in the HBeAg-positive group, 41.7% were slow-responders, while 58.3% were rapid-responders. No entecavir-resistant mutants were detected in the slow-responders. When the pretreatment factors were compared between the slow- and rapid-responders; the median qHBsAg in the slow-responders was 4.57 log IU/mL, compared with 3.63 log IU/mL in the rapid-responders (P < 0.01). When the pretreatment factors predictive of HBV DNA-negative status at year 2 in all 50 patients were analyzed, HBeAg-negative status, low HBV DNA levels, and low qHBsAg levels were significant (P < 0.01). Multivariate analysis revealed that the low qHBsAg level was the most significant predictive factor (P = 0.03). CONCLUSION: Quantitation of HBsAg could be a useful indicator to predict response to entecavir therapy. PMID:23112549

  4. Comparison of the antibody in lymphocyte supernatant (ALS) and ELISPOT assays for detection of mucosal immune responses to antigens of enterotoxigenic Escherichia coli in challenged and vaccinated volunteers.

    PubMed

    Carpenter, C M; Hall, E R; Randall, R; McKenzie, R; Cassels, F; Diaz, N; Thomas, N; Bedford, P; Darsley, M; Gewert, C; Howard, C; Sack, R B; Sack, D A; Chang, H S; Gomes, G; Bourgeois, A L

    2006-05-01

    In the present study we compared the ELISPOT and antibody in lymphocyte supernatants (ALS) assays as surrogate measures of mucosal immunity. In separate studies, 20 inpatient volunteers received oral doses of 6 x 10(8) or 4 x 10(9)cfu of ETEC strain E24377A (LT+, ST+, CS1+, CS3+) and 20 subjects received 1 (n = 9) or 2 (n = 11) oral doses of the attenuated ETEC vaccine, PTL-003 expressing CFA/II (CS1+ and CS3+) (2 x 10(9)cfu/dose). Peripheral blood mononuclear cells (PBMCs) from all subjects were assayed for anti-colonization factor or toxin-specific IgA antibody responses using the ALS and ELISPOT procedures. ALS responses were measured using a standard ELISA, as well as by time-resolved fluorescence (TRF). Following challenge with E24377A, significant anti-CS3, CS1 and LT ALS responses were detected in the lymphocyte supernatants of 75-95% of the subjects. A similar proportion (75%) of subjects mounted an ALS response to CFA/II antigen after vaccination with the PTL-003 vaccine. Inter-assay comparisons between ALS and ELISPOT methods also revealed a high degree of correlation in both immunization groups. ALS sensitivity versus the ELISPOT assay for LT, CS3 and CS1-specific responses following challenge were 95%, 94% and 78%, respectively and 83% for the ALS response to CFA/II antigen after vaccination with PTL-003. Correlation coefficients for the LT and CS3 antigens were 0.94 (p<0.001) and 0.82 (p<0.001), respectively after challenge and 0.78 (p<0.001) after vaccination. The association between ALS and ELISPOT for the CS1 antigen was however, significant only when ALS supernatants were tested by TRF (r = 0.91, p<0.001). These results demonstrate the value and flexibility of the ALS assay as an alternative to ELISPOT for the measurement of mucosal immune responses to ETEC antigens, particularly when the complexities of ELISPOT may make it impractical to perform.

  5. A fully human IgG1 anti-PD-L1 MAb in an in vitro assay enhances antigen-specific T-cell responses

    PubMed Central

    Grenga, Italia; Donahue, Renee N; Lepone, Lauren M; Richards, Jacob; Schlom, Jeffrey

    2016-01-01

    Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being investigated for the treatment of infectious diseases and cancer, with the aim of enhancing the function of an impaired immune system. Avelumab (MSB0010718C) is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which differs from other checkpoint-blocking antibodies in its ability to mediate antibody-dependent cell-mediated cytotoxicity. These studies were conducted to define whether avelumab could enhance the detection of antigen-specific immune response in in vitro assays. Peripheral blood mononuclear cells from 17 healthy donors were stimulated in vitro, with and without avelumab, with peptide pools encoding for cytomegalovirus, Epstein–Barr virus, influenza and tetanus toxin or the negative peptide control encoding for human leukocyte antigen. These studies show for the first time that the addition of avelumab to an antigen-specific IVS assay (a) increased the frequency of activated antigen-specific CD8+ T lymphocytes, and did so to a greater extent than that seen with commercially available PD-L1-blocking antibodies, (b) reduced CD4+ T-cell proliferation and (c) induced a switch in the production of Th2 to Th1 cytokines. Moreover, there was an inverse correlation between the enhancement of CD8+ T-cell activation and reduction in CD4+ T-cell proliferation induced by avelumab. These findings provide the rationale for the use of avelumab anti-PD-L1 in in vitro assays to monitor patient immune responses to immunotherapies. PMID:27350882

  6. A novel assay for the detection of anti-human platelet antigen antibodies (HPA-1a) based on peptide aptamer technology

    PubMed Central

    Thibaut, Julien; Mérieux, Yves; Rigal, Dominique; Gillet, Germain

    2012-01-01

    Background Neonatal alloimmune thrombocytopenia is mostly due to the presence of maternal antibodies against the fetal platelet antigen HPA-1a on the platelet integrin GPIIb-IIIa. Accurate detection of anti-HPA-1a antibodies in the mother is, therefore, critical. Current diagnostic assays rely on the availability of pools of human platelets that vary according to donors and blood centers. There is still no satisfactory standardization of these assays. Design and Methods Peptide aptamer was used to detect and identify HPA-1a-specific antibodies in human serum that do not require human platelets. A peptide aptamer library was screened using an anti-HPA-1a human monoclonal antibody as a bait to isolate an aptamer that mimics the human platelet antigen HPA-1a. Results This is the first report in platelet immunology of the use of a peptide aptamer for diagnostic purposes. This assay gives better results than the MAIPA currently in use, detecting around 90% of the expected alloantibodies. Conclusions This assay could help define a standard for the quantitation of anti-HPA antibodies. This report also demonstrates that peptide aptamers can potentially detect a variety of biomarkers in body fluids; this is of particular interest for diagnostic purposes. PMID:22133781

  7. Further probes into quantitative aspects of competitive binding assays: allowance for effects of antigen multivalency in immunoassays.

    PubMed

    Hogg, P J; Winzor, D J

    1987-04-01

    Effects of antigen multivalency on procedures for the analysis of immunoassays are examined on the basis of a theoretical expression developed in the context of quantitative affinity chromatography [Nichol, L. W., Ward, L. D., and Winzor, D. J. (1981) Biochemistry 20, 4856-4860] but which is also pertinent to antigen-antibody interactions that may be described in terms of a single intrinsic association constant. Quantitative relationships are generated which provide the basis for more rigorous logit-log analyses of radioimmunoassays in which the antigen is multivalent, and an additional, theoretically superior, linear transform of the basic expression is developed. Simulated binding data for a tetravalent antigen system are then used to demonstrate the curvilinearity of the conventional Scatchard plot for such a system despite the homogeneity of binding sites, and the application of the various linear transforms involving logarithmic functions. Of particular interest in that regard is the observation that the traditional logit-log analyses yield linear plots with the predicted slope of unity even though antigen univalence is an implicit assumption in their application. Results obtained in a solid-phase radioimmunoassay of triiodothyronine are then presented to provide, for that system at least, experimental justification of the above-mentioned assumption that the antibody-antigen interactions may be described in terms of a single intrinsic association constant. Finally, an enzyme-linked immunoassay of ferritin is used to illustrate the possibility that a linear Scatchard plot may be obtained with a multivalent antigen under conditions where steric factors restrict participation of an antigen molecule to a single interaction with immobilized antibody. PMID:3579309

  8. Two-dimensional periodic relief grating as a versatile platform for selective immunosorbent assay and visualizing of antigens.

    PubMed

    Chen, Jem-Kun; Zhou, Gang-Yan; Huang, Chih-Feng; Chang, Jia-Yaw

    2013-04-24

    In this study, we fabricated a nanopillar array of silicon oxide, involving very-large-scale integration (VLSI) and reactive ion etching (RIE), as two-dimensional periodic relief gratings (2DPRGs) on Si surfaces. Antihuman ALB was successively oriented on the pillar surface of 2DPRG modified protein G as an optical detector that is specific for targeted antigen. The antibody modified 2DPRG alone produces insignificant structure change, but upon immunocapture of antigens, the antigen filling in the 2DPRG leads to a dramatic change of the pillar scale. Binding of the antibodies to the 2DPRG occurs in a way that still allows them to function and selectively bind antigen. The performance of the sensor was evaluated by capturing HRP-human ALB on the antibody-modified 2DPRG and measuring the effective refractive index (neff) resulting from the attachment of antigens. The neff values of the 2DPRG are found to relate with the pillar scale of the 2DPRG, generated by antigen coupling, resulting in color change from pure green to orange, observed by the naked eye along an incident angle of 10-20°. Moreover, we calculated the filling factors inside the 2DPRG with effective-medium theory to verify the pillar structure changes. This technique eliminates much of the surface modifications and the secondary immunochemical or enzyme-linked steps that are common in immunoassays. Such films have potential applications as optical biosensors.

  9. A novel double-isotope technique for the enzymatic assay of plasma histamine: application to estimation of mast cell activation assessed by antigen challenge in asthmatics

    SciTech Connect

    Brown, M.J.; Ind, P.W.; Causon, R.; Lee, T.H.

    1982-01-01

    The concentration of plasma histamine may provide an index of mast cell activation (degranulation) and can be measured by a sensitive radioenzymatic assay based on its specific conversion to (/sup 3/H)-methylhistamine in the presence of histamine-N-methyltransferase and (/sup 3/H)-S-adenosyl-L-methionine. In this assay, the separation of excess (/sup 3/H)-S-adenosyl-L-methionine from (/sup 3/H)-methylhistamine requires several steps, for which a correction factors is necessary to maintain precision. In the present modification, duplicate 50-microliters aliquots of each plasma sample were incubated with histamine-N-methyltransferase and (/sup 3/H)-S-adenosyl-L-methionine. A further aliquot, with an added standard of 200 ng/ml histamine, was incubated with histamine-N-methyl-transferase and (/sup 14/C)-S-adenosyl-L-methionine. This standard was converted to (/sup 14/C)-methylhistamine, and its recovery at the end of the assay corrected both for varying efficiency of methylation among plasma samples and for losses during the subsequent extraction and separation stages. The sensitivity of the assay was 25 pg/ml. The intra-assay and interassay coefficients of variation were 7.2% and 11.6%, respectively. In five asthmatics, antigen challenge caused a 28% fall in FEV1, and this was associated with a twofold to threefold rise in plasma histamine concentration. This assay may thus prove a useful method for assessing the role of mast cell release of mediators in vivo.

  10. Characterization of C69R variant HBsAg: effect on binding to anti-HBs and the structure of virus-like particles.

    PubMed

    Hadiji-Abbes, Nadia; Mihoubi, Wafa; Martin, Marta; Karakasyan-Dia, Carole; Frikha, Fakher; Gergely, Csilla; Jouenne, Thierry; Gargouri, Ali; Mokdad-Gargouri, Raja

    2015-10-01

    Several variants of the major "a" determinant of the HBsAg, the main target of HBV neutralization by antibodies, have been described. However, mutations outside this region have not been as thoroughly investigated. During the genotyping of HBV from Tunisian patients with chronic hepatitis B, we identified a variant with a C69R substitution in the cytosolic loop of the S protein, resulting in a change in the hydrophobicity profile compared to the wild-type HBsAg. Wild-type and mutant HBsAgs were produced in Saccharomyces cerevisiae and recombinant proteins were tested for their ability to correctly self-assemble into virus-like particles (VLPs), and their ability to bind to HBs antibodies. The C69R substitution resulted in a decrease in binding to commercial anti-HBs antibodies, and although the variant appeared to assemble properly into VLPs, the average size of the particles was larger than that of the wild-type HBsAg. Prediction of the tertiary structure of the C69R mutant revealed a change in the first (aa 60-70) and the second loop (aa 110 to 120) compared to the wild-type protein. Furthermore, we showed by an isothermal titration calorimetry assay that the interaction between the wild-type HBsAg and the anti-HBs antibody was exothermic, whereas that with the mutant C69R was endothermic, indicating an effect on the binding affinity.

  11. Screening for hepatitis C virus infection in a high prevalence country by an antigen/antibody combination assay versus a rapid test

    PubMed Central

    Tagny, Claude Tayou; Mbanya, Dora; Murphy, Edward L.; Lefrère, Jean-Jacques; Laperche, Syria

    2016-01-01

    In low-income-countries, screening for hepatitis C virus (HCV) infection is often based on rapid tests (RT). Their lower sensitivity compared to enzyme immunoassay (EIA) suggests that newer HCV Antigen/Antibody (Ag/Ab) combination assays might have a role in such countries. To test this idea, 1998 blood donors were tested at the University Teaching Hospital blood bank in Yaoundé, Cameroon simultaneously with a RT (HCV rapid test, Human Diagnostics, Berlin, Germany) according to standard practice (S1) and with an Ag/Ab assay (Monolisa HCV Ag/Ab Ultra, Biorad, France) (S2). All discordant, borderline and reactive samples were submitted to confirmatory testing by immunoblot and/or HCV-RNA. Of the 86 (4.3%) samples positive with one or both strategies, 29 were confirmed negative, 37 positive and 20 were false positive or resolved infection. There was a significant difference in test sensitivity (p = 0.01) between S1 (70.3%) and S2 (91.9%) but not in test specificity (99.4% and 98.6%, respectively). The benefit of the Ag/Ab assay in the detection of recent HCV seronegative infections could not be evaluated since no Antigen-only donations were identified. However, better Ag/Ab test sensitivity compared to RT supports the implementation of these newer immunoassays for HCV screening in the African blood bank setting. PMID:24487098

  12. Evaluation of point-of-contact circulating cathodic antigen assays for the detection of Schistosoma mansoni infection in low-, moderate-, and high-prevalence schools in western Kenya.

    PubMed

    Foo, Karen T; Blackstock, Anna J; Ochola, Elizabeth A; Matete, Daniel O; Mwinzi, Pauline N M; Montgomery, Susan P; Karanja, Diana M S; Secor, W Evan

    2015-06-01

    We evaluated the performance of a point-of-contact circulating cathodic antigen assay (POC-CCA) to detect schistosome infections in primary school children (N = 1,801) living in areas with low, moderate, and high Schistosoma mansoni prevalence in western Kenya. The commercially available assay (CCA-1) and a second, experimental formulation (CCA-2) were compared against Kato-Katz stool examinations and an anti-schistosome enzyme-linked immunosorbent assay (ELISA). A latent class model based on the four tests was used to establish "true infection status" in three different zones based on their distance from Lake Victoria. As a screening tool for community treatment according to World Health Organization (WHO) guidelines, the Kato-Katz examination was in closest agreement with the latent class model, followed by the experimental CCA-2, soluble adult worm antigen preparation (SWAP) ELISA, and CCA-1, which had high sensitivity compared with the other tests but was consistently the least specific. Our experience suggests that POC-CCA tests offer a field-friendly alternative to Kato-Katz, but need further interpretation for appropriate field use. PMID:25870418

  13. Evaluation of point-of-contact circulating cathodic antigen assays for the detection of Schistosoma mansoni infection in low-, moderate-, and high-prevalence schools in western Kenya.

    PubMed

    Foo, Karen T; Blackstock, Anna J; Ochola, Elizabeth A; Matete, Daniel O; Mwinzi, Pauline N M; Montgomery, Susan P; Karanja, Diana M S; Secor, W Evan

    2015-06-01

    We evaluated the performance of a point-of-contact circulating cathodic antigen assay (POC-CCA) to detect schistosome infections in primary school children (N = 1,801) living in areas with low, moderate, and high Schistosoma mansoni prevalence in western Kenya. The commercially available assay (CCA-1) and a second, experimental formulation (CCA-2) were compared against Kato-Katz stool examinations and an anti-schistosome enzyme-linked immunosorbent assay (ELISA). A latent class model based on the four tests was used to establish "true infection status" in three different zones based on their distance from Lake Victoria. As a screening tool for community treatment according to World Health Organization (WHO) guidelines, the Kato-Katz examination was in closest agreement with the latent class model, followed by the experimental CCA-2, soluble adult worm antigen preparation (SWAP) ELISA, and CCA-1, which had high sensitivity compared with the other tests but was consistently the least specific. Our experience suggests that POC-CCA tests offer a field-friendly alternative to Kato-Katz, but need further interpretation for appropriate field use.

  14. Toxoplasma gondii: Recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay.

    PubMed

    Holec-Gasior, Lucyna; Kur, Józef

    2010-03-01

    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.

  15. Development & validation of a quantitative anti-protective antigen IgG enzyme linked immunosorbent assay for serodiagnosis of cutaneous anthrax

    PubMed Central

    Ghosh, N.; Gunti, D.; Lukka, H.; Reddy, B.R.; Padmaja, Jyothi; Goel, A.K.

    2015-01-01

    Background & objectives: Anthrax caused by Bacillus anthracis is primarily a disease of herbivorous animals, although several mammals are vulnerable to it. ELISA is the most widely accepted serodiagnostic assay for large scale surveillance of cutaneous anthrax. The aims of this study were to develop and evaluate a quantitative ELISA for determination of IgG antibodies against B. anthracis protective antigen (PA) in human cutaneous anthrax cases. Methods: Quantitative ELISA was developed using the recombinant PA for coating and standard reference serum AVR801 for quantification. A total of 116 human test and control serum samples were used in the study. The assay was evaluated for its precision, accuracy and linearity. Results: The minimum detection limit and lower limit of quantification of the assay for anti-PA IgG were 3.2 and 4 µg/ml, respectively. The serum samples collected from the anthrax infected patients were found to have anti-PA IgG concentrations of 5.2 to 166.3 µg/ml. The intra-assay precision per cent CV within an assay and within an operator ranged from 0.99 to 7.4 per cent and 1.7 to 3.9 per cent, respectively. The accuracy of the assay was high with a per cent error of 6.5 - 24.1 per cent. The described assay was found to be linear between the range of 4 to 80 ng/ml (R2=0.9982; slope=0.9186; intercept = 0.1108). Interpretation & conclusions: The results suggested that the developed assay could be a useful tool for quantification of anti-PA IgG response in human after anthrax infection or vaccination. PMID:26354217

  16. Carcinoembryonic antigen: assay following heat compared with perchloric acid extraction in patients with colon cancer, non-neoplastic gastrointestinal diseases, or chronic renal failure.

    PubMed

    Witherspoon, L R; Shuler, S E; Alyea, K; Husserl, F E

    1983-10-01

    Heat inactivation has been proposed as an alternative to perchloric acid (PCA) precipitation for the extraction of carcinoembryonic antigen (CEA) from human plasma. We examined a commercial RIA kit using heat inactivation, and compared results with those obtained with PCA precipitation. Adequate sensitivity (1.5 micrograms CEA/l plasma), satisfactory analytical recovery of CEA added to plasma, and dilutional linearity of samples found to have elevated CEA concentrations, were demonstrated for the heat-inactivation assay. Between-assay precision was better with the heat inactivation than with the PCA assay. Although the absolute concentration of CEA estimated after heat inactivation was consistently lower than that estimated after PCA extraction of plasma specimens, there was excellent correlation between results obtained with the two methods in colon cancer patients free of disease, colon cancer patients with residual or recurrent disease, patients with benign gastrointestinal disease, and in patients with chronic renal failure. We conclude that the heat-inactivation assay is an excellent alternative to the PCA assay.

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-02-01

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

  20. Standardisation and comparison of serial dilution and single dilution enzyme linked immunosorbent assay (ELISA) using different antigenic preparations of the Babesia (Theileria) equi parasite.

    PubMed

    Kumar, Sanjay; Kumar, Yogesh; Malhotra, Dharam V; Dhar, Shruti; Nichani, Anil K

    2003-01-01

    Serial dilution and single dilution enzyme linked immunosorbent assays (ELISA) were standardised and their sensitivity and specificity were compared for serodiagnosis of Babesia equi infection. The antibody titres of 24 donkey sera of known identity were determined separately by serial dilution ELISA using three different B. equi antigens namely whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS). The ratios of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as the positive/negative (P/N) ratio. The coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of sera and the log10 antibody titres of the same sera were ascertained by serial dilution ELISA. The highest value of 'r' was obtained at a serum dilution of 1:200. From log10 antibody titre of sera (y) and their P/N ratio at a dilution of 1:200 (x), regression equations (y = a + bx) were calculated separately for the three antigens. Test sera were diluted to 1:200, their OD were read in duplicate wells and were converted to the P/N ratio. Antibody titres were predicted from the P/N ratio using a regression equation separately for the three antigens. Titres obtained by both ELISAs were not significantly different from each other, thus confirming that single dilution ELISA could be successfully used to replace conventional serial dilution ELISA. The sensitivity, specificity and predictive value of single dilution ELISA was validated statistically using 42 B. equi disease-positive sera and 106 B. equi disease-negative sera. The WM antigen was found to be the most sensitive with a higher predictive value for negative test sera as compared to the CM or HSS antigens. Sera positive for other equine infections including Babesia caballi showed no cross-reaction with the three B. equi antigens in ELISA, thus the test was immunologically specific. Antibody titres of 109 unknown field donkey

  1. Development of a New Limiting-Antigen Avidity Dot Immuno-Gold Filtration Assay for HIV-1 Incidence.

    PubMed

    Gao, Zhiyun; Yan, Hao; Feng, Xia; Wu, Lijin; Qiu, Maofeng; Xing, Wenge; Zhang, Guiyun; Zhang, Zhi; Jiang, Yan

    2016-01-01

    Several laboratory assays on cross-sectional specimens for detecting recent HIV infections were developed, but these assays could not be applied in resource-limited and high HIV-incidence areas. This study describes the development of a rapid assay that can simultaneously detect the presence of HIV-1 antibodies of current and/or recent infection. The dot immuno-gold filtration assay (DIGFA) was used to detect recent infection on the principle of antibody avidity changes between recent and long-term infections. The dot immuno-gold silver staining filtration assay (DIGSSA) increases the sensitivity and accuracy of antibody detection by adding a silver staining step to the DIGFA. In the meantime the digital results were produced by the scanner for ambiguous specimens. Further, HIV-1 routine diagnostic antibody was detected simultaneously for improving practicability. The performance of the assays was then assessed through five serum panels with known serological statuses and seroconversion dates. The proportion of false recent infection (PFR) of the DIGSSA was obtained. Through the optimization of basic parameters for DIGSSA, six specimens were all classified correctly. DIGSSA demonstrated good repeatability and high sensitivity. The agreement of DIGSSA with the BED assay was 92.10% (κ = 0.65) and 95.36% with the LAg-Avidity assay (κ = 0.75). Moreover, the gray values of DIGSSA correlated well with BED ODn (R2 = 0.9397) and LAg-Avidity ODn (R2 = 0.9549). The PFR of DIGSSA was 2.73%, which was lower than that of the BED assay but higher than that of the LAg-Avidity assay. The DIGSSA can feasibly be applied to detect HIV infection and estimate HIV incidence. PMID:27513563

  2. Development of a New Limiting-Antigen Avidity Dot Immuno-Gold Filtration Assay for HIV-1 Incidence

    PubMed Central

    Feng, Xia; Wu, Lijin; Qiu, Maofeng; Xing, Wenge; Zhang, Guiyun; Zhang, Zhi; Jiang, Yan

    2016-01-01

    Several laboratory assays on cross-sectional specimens for detecting recent HIV infections were developed, but these assays could not be applied in resource-limited and high HIV-incidence areas. This study describes the development of a rapid assay that can simultaneously detect the presence of HIV-1 antibodies of current and/or recent infection. The dot immuno-gold filtration assay (DIGFA) was used to detect recent infection on the principle of antibody avidity changes between recent and long-term infections. The dot immuno-gold silver staining filtration assay (DIGSSA) increases the sensitivity and accuracy of antibody detection by adding a silver staining step to the DIGFA. In the meantime the digital results were produced by the scanner for ambiguous specimens. Further, HIV-1 routine diagnostic antibody was detected simultaneously for improving practicability. The performance of the assays was then assessed through five serum panels with known serological statuses and seroconversion dates. The proportion of false recent infection (PFR) of the DIGSSA was obtained. Through the optimization of basic parameters for DIGSSA, six specimens were all classified correctly. DIGSSA demonstrated good repeatability and high sensitivity. The agreement of DIGSSA with the BED assay was 92.10% (κ = 0.65) and 95.36% with the LAg-Avidity assay (κ = 0.75). Moreover, the gray values of DIGSSA correlated well with BED ODn (R2 = 0.9397) and LAg-Avidity ODn (R2 = 0.9549). The PFR of DIGSSA was 2.73%, which was lower than that of the BED assay but higher than that of the LAg-Avidity assay. The DIGSSA can feasibly be applied to detect HIV infection and estimate HIV incidence. PMID:27513563

  3. Detection and titration of bluetongue virus in Culicoides insect cell culture by an antigen-capture enzyme-linked immunosorbent assay.

    PubMed

    Mecham, James O

    2006-08-01

    Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. of biting midges. Virus is typically isolated and characterized by infection of susceptible vertebrate cells that undergo detectable and measurable cytopathic effects. Cell lines derived from C. sonorensis may be useful for virus isolation and studies to better understand BTV replication in the insect vector. However, their use is hampered because BTV infection does not produce significant cytopathic effects in these insect cell cultures. Detection of virus replication in these cells typically requires co-cultivation with susceptible vertebrate cell culture. This report describes the use of an antigen-capture enzyme-linked immunosorbent assay (Ag-Cap ELISA) for direct detection and titration of BTV in cultures of a Culicoides cell line. This assay should facilitate use of this cell line for virus isolation, titration and studies of BTV replication.

  4. Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26

    PubMed Central

    Anderson, John P.; Rascoe, Lisa N.; Levert, Keith; Chastain, Holly M.; Reed, Matthew S.; Rivera, Hilda N.; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E.; Hotez, Peter J.; Wilkins, Patricia P.; Pohl, Jan; Handali, Sukwan

    2015-01-01

    The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag. PMID:26485145

  5. Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26.

    PubMed

    Anderson, John P; Rascoe, Lisa N; Levert, Keith; Chastain, Holly M; Reed, Matthew S; Rivera, Hilda N; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E; Hotez, Peter J; Wilkins, Patricia P; Pohl, Jan; Handali, Sukwan

    2015-01-01

    The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag. PMID:26485145

  6. Harmonization of Glutamic Acid Decarboxylase and Islet Antigen-2 Autoantibody Assays for National Institute of Diabetes and Digestive and Kidney Diseases Consortia

    PubMed Central

    Bonifacio, Ezio; Yu, Liping; Williams, Alastair K.; Eisenbarth, George S.; Bingley, Polly J.; Marcovina, Santica M.; Adler, Kerstin; Ziegler, Anette G.; Mueller, Patricia W.; Schatz, Desmond A.; Krischer, Jeffrey P.; Steffes, Michael W.; Akolkar, Beena

    2010-01-01

    Background/Rationale: Autoantibodies to islet antigen-2 (IA-2A) and glutamic acid decarboxylase (GADA) are markers for diagnosis, screening, and measuring outcomes in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) consortia studies. A harmonization program was established to increase comparability of results within and among these studies. Methods: Large volumes of six working calibrators were prepared from pooled sera with GADA 4.8–493 World Health Organization (WHO) units/ml and IA-2A 2–235 WHO units/ml. Harmonized assay protocols for IA-2A and GADA using 35S-methionine-labelled in vitro transcribed and translated antigens were developed based on methods in use in three NIDDK laboratories. Antibody thresholds were defined using sera from patients with recent onset type 1 diabetes and healthy controls. To evaluate the impact of the harmonized assay protocol on concordance of IA-2A and GADA results, two laboratories retested stored TEDDY study sera using the harmonized assays. Results: The harmonized assays gave comparable but not identical results in the three laboratories. For IA-2A, using a common threshold of 5 DK units/ml, 549 of 550 control and patient samples were concordantly scored as positive or negative, specificity was greater than 99% with sensitivity 64% in all laboratories. For GADA, using thresholds equivalent to the 97th percentile of 974 control samples in each laboratory, 1051 (97.9%) of 1074 samples were concordant. On the retested TEDDY samples, discordance decreased from 4 to 1.8% for IA-2A (n = 604 samples; P = 0.02) and from 15.4 to 2.7% for GADA (n = 515 samples; P < 0.0001). Conclusion: Harmonization of GADA and IA-2A is feasible using large volume working calibrators and common protocols and is an effective approach to ensure consistency in autoantibody measurements. PMID:20444913

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

    PubMed

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

    2010-01-01

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

  8. Detection of antibodies against avian antigens in bronchoalveolar lavage from patients with pigeon breeder's disease: usefulness of enzyme-linked immunosorbent assay and enzyme immunotransfer blotting.

    PubMed

    Sandoval, J; Bañales, J L; Cortés, J J; Mendoza, F; Selman, M; Reyes, P A

    1990-01-01

    The study reported here evaluated the usefulness of the enzyme-linked immunosorbent assay (ELISA) in the detection of antibodies against pigeon antigens in the serum and bronchoalveolar lavage (BAL) of patients with clinical, radiological, and functional evidence of interstitial lung disease (ILD) with and without pigeon breeder's disease (PBD). The results were compared with those obtained by the simultaneous use of counterimmunoelectrophoresis (CIE) in the same patients. In PBD, ELISA detected antibodies against pigeon's sera in both serum and BAL in 100% of patients, while CIE failed to detect the antibodies in the serum of one patient and in most of the samples of BAL. In addition, we used enzyme immunotransfer blotting to determine the number of epitopes in pigeon serum recognized by antibodies present in serum and BAL. There was a heterogeneous response in both fluids, but the reaction pattern demonstrated that patient's sera recognize to-25 different pigeon epitopes. We conclude that ELISA is a highly sensitive and specific method for the detection of antibodies against pigeon antigens in the serum and BAL of patients with PBD and that the host response involves a great number of avian antigens.

  9. Enzyme-Linked Immunosorbent Assay for Serological Diagnosis of Chagas' Disease Employing a Trypanosoma cruzi Recombinant Antigen That Consists of Four Different Peptides

    PubMed Central

    Ferreira, A. W.; Belem, Z. R.; Lemos, E. A.; Reed, S. G.; Campos-Neto, A.

    2001-01-01

    Serological tests to detect Trypanosoma cruzi antibodies have been used for screening blood donors, for epidemic studies, and for diagnosis of probably infected persons. Among different tests, the enzyme-linked immunosorbent assay (ELISA) with total, semipurified, or synthetic antigens has been widely used, mainly due to its easy automation. Aiming to improve serological studies concerning Chagas' disease, we have developed and evaluated a new test, the TcF-ELISA, using an artificially engineered recombinant antigen, which contains tandem sequences of different T. cruzi-specific peptides. The sensibility of the TcF-ELISA was determined with 101 serum samples from chagasic patients well-defined by clinical and epidemiological criteria. The specificity was determined with 39 serum samples from leishmaniasis or kala-azar patients and 150 serum samples from nonchagasic blood donors from Sao Paulo, Brazil. The TcF-ELISA showed 100% sensitivity and 98.94% of specificity. Compared with conventional ELISA (with semipurified T. cruzi epimastigote antigens), the TcF-ELISA showed advantages; for example, it distinguishes better between reagent and nonreagent serum and provides better precision and a lower occurrence of leishmaniasis cross-reactions. Our studies demonstrate high reproducibility between two different lots of the TcF ELISA and its applicability for the serological diagnosis of Chagas' disease. PMID:11724850

  10. Phage-displayed peptides as capture antigens in an innovative assay for Taenia saginata-infected cattle.

    PubMed

    Fogaça, Rafaela L; Capelli-Peixoto, Janaína; Yamanaka, Isabel B; de Almeida, Rodrigo P M; Muzzi, João Carlos D; Borges, Mariangela; Costa, Alvimar J; Chávez-Olortegui, Carlos; Thomaz-Soccol, Vanete; Alvarenga, Larissa M; de Moura, Juliana

    2014-11-01

    Bovine cysticercosis is detected during the routine post mortem examination of carcasses by visual inspection (knife and eye method). However, the sensitivity of this procedure is several times lower than immunoassays, even when it is performed by qualified professionals. In the present study, a new generation capture antigens were screened from a phage display peptide library using antibodies from Taenia saginata-infected animals. Eight phage clones were selected, and one, Tsag 3 (VHTSIRPRCQPRAITPR), produced similar results to the T. saginata metacestode crude antigen (TsCa) when used as a capture antigen in an ELISA. The phage-displayed peptides competed with TsCa for binding sites, reducing the reactivity by approximately 30 %. Alanine scanning indicated that proline, arginine, and serine are important residues for antibody binding. Tsag 1 (HFYQITWLPNTFPAR), the most frequent affinity-selected clone, and Tsag 6 (YRWPSTPSASRQATL) shared similarity with highly conserved proteins from the Taeniidae family with known immunogenicity. Due to their epitopic or mimotopic properties, these affinity-selected phages could contribute to the rational design of an ante mortem immunodiagnosis method for bovine cysticercosis, as well as an epitope-based vaccine to interrupt the taeniosis/cysticercosis complex. PMID:25081558

  11. Clinical Utility of a New Automated Hepatitis C Virus Core Antigen Assay for Prediction of Treatment Response in Patients with Chronic Hepatitis C

    PubMed Central

    2016-01-01

    Hepatitis C virus core antigen (HCV Ag) is a recently developed marker of hepatitis C virus (HCV) infection. We investigated the clinical utility of the new HCV Ag assay for prediction of treatment response in HCV infection. We analyzed serum from 92 patients with HCV infection who had been treated with pegylated interferon and ribavirin. HCV Ag levels were determined at baseline in all enrolled patients and at week 4 in 15 patients. Baseline HCV Ag levels showed good correlations with HCV RNA (r = 0.79, P < 0.001). Mean HCV Ag levels at baseline were significantly lower in patients with a sustained virologic response (SVR) than in those with a non SVR (relapse plus non responder) based on HCV RNA analysis (2.8 log10fmol/L vs. 3.27 log10fmol/L, P = 0.023). Monitoring of the viral kinetics by determination of HCV RNA and HCV Ag levels resulted in similarly shaped curves. Patients with undetectable HCV Ag levels at week 4 had a 92.3% probability of achieving SVR based on HCV RNA assay results. The HCV Ag assay may be used as a supplement for predicting treatment response in HCV infection, but not as an alternative to the HCV RNA assay. PMID:27510387

  12. A multicentric evaluation of the recombinant Leishmania infantum antigen-based immunochromatographic assay for the serodiagnosis of canine visceral leishmaniasis

    PubMed Central

    2014-01-01

    Background Visceral leishmaniasis (VL) is a serious public health challenge in Brazil and dogs are considered to be the main urban reservoir of the causative agent. The culling of animals to control VL in some countries makes the accurate diagnosis of canine VL (CVL) essential. Recombinant antigens rLci1A and rLci2B were selected from a cDNA library of Leishmania infantum amastigotes due to their strong potential as candidates in diagnostic testing for CVL. The present multicentric study aimed to evaluate the sensitivity of a prototype test using these antigens (DPP rLci1A/rLci2B) against 154 sera obtained from symptomatic dogs within three endemic areas of VL in Brazil. The specificity was evaluated using 40 serum samples from negative dogs and dogs infected with other pathogens. Sensitivity and specificity rates of DPP rLci1A/rLci2B prototype were compared to rates from other diagnostic tests currently in use by the Brazilian Ministry of Health, including DPP®LVC, EIE®LVC. Findings DPP rLci1A/rLci2B prototype offered similar performance to that offered by DPP®LVC rapid test, as follows: sensitivity of 87% (CI 81–91) and 88% (CI 82–93) and specificity of 100% (CI 91–100) and 97% (CI 87–100), respectively for DPP rLci1A/rLci2B and DPP®LVC. When results of these two tests were considered concomitantly, sensitivity increased to 93.5% (CI 89–96). Conclusions The recombinant antigens rLci1A and rLci2B represent promising candidates for use in a multi-antigen rapid test for CVL. The inclusion of novel antigens to the DPP rLci1A/rLci2B prototype model could offer additionally enhanced sensitivity to detect animals infected by L. infantum. PMID:24684857

  13. Reduced antibody reactivity to hepatitis C virus antigens in hemodialysis patients coinfected with hepatitis B virus.

    PubMed Central

    Devesa, M; Khudyakov, Y E; Capriles, F; Blitz, L; Fields, H A; Liprandi, F; Pujol, F H

    1997-01-01

    Antibody reactivities to hepatitis C virus (HCV) antigens and to synthetic peptides derived from different parts of the HCV genome (core, NS4, and NS5) were evaluated in HCV-infected hemodialysis patients. In the RIBA 3 assay, NS5 was significantly less recognizable by sera of hemodialysis patients compared to other HCV-infected subjects. Among hemodialysis patients, those coinfected with hepatitis B virus (HBV) (positive for hepatitis B surface antigen [HBsAg+]) showed a reduction in reactivity to C33 and C100. Sera of only 23% of the hemodialysis patients (37 of 161) reacted with more than three of eight peptides tested, significantly fewer than the 60% (12 of 20) of the sera of other HCV-infected patients tested (P = 0.001). This immunosuppression was also manifested by a reduced frequency of recognition of additional peptides on follow-up. An even more reduced reactivity was observed among the HBV-coinfected patients (HBsAg+). The low-responder hemodialysis patients were not infected with any particular genotype of HCV, and the same HCV genotypes observed in the whole group of hemodialysis patients (1a, 1b, 2a, and 3a) were found circulating in the low-responder group. Even in this low-responder population, the good performance of two peptides (peptide 716, corresponding to a portion of the core, and peptide 59, corresponding to a portion of NS4) corroborates the immunodominance of the conserved epitopes within these peptides. PMID:9384281

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

    PubMed

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

    2006-06-01

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

  15. Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

    PubMed

    Lang, Qiaolin; Wang, Fei; Yin, Long; Liu, Mingjun; Petrenko, Valery A; Liu, Aihua

    2014-03-01

    Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

  16. Comparison of Three Luminescent Immunoassays for Hepatitis B Virus Surface Antigen Quantification during the Natural History of Chronic Hepatitis B Virus Infection

    PubMed Central

    Cheng, Xiao-Dong; Song, Liu-Wei; Fang, Lin-Lin; Yang, Lin; Wu, Yong; Ge, Sheng-Xiang; Zhang, Jun; Xia, Ning-Shao

    2014-01-01

    Hepatitis B surface antigen (HBsAg) quantification has garnered attention because of its high predictive value in determining treatment responses. The HBsAg quantification assays, such as Architect and Elecsys, are commercially available, and more assays are in development. We aimed to compare the results of the Architect and Elecsys assays with those of a new assay, WTultra. The WTultra HBsAg assay is a sandwich chemiluminescent microplate enzyme immunoassay and provides an alternative choice which is more cost-effective and potentially applicable in developing or resource-constrained countries and areas. A total of 411 serum samples were collected from patients during various phases of chronic hepatitis B (CHB) infection. The samples were assessed using the three assays, and the results were compared and analyzed. The results for the Architect, Elecsys, and WTultra assays were well correlated according to the overall results for the samples (correlation coefficients, rArchitect versus WTultra = 0.936, rArchitect versus Elecsys = 0.952, and rWTultra versus Elecsys = 0.981) and the various infection phases (rArchitect versus WTultra ranging from 0.67 to 0.975, rArchitect versus Elecsys ranging from 0.695 to 0.982, and rWTultra versus Elecsys ranging from 0.877 to 0.99). Additionally, consistent results were observed according to genotype (genotype B: rArchitect versus WTultra = 0.976, rArchitect versus Elecsys = 0.978, and rWTultra versus Elecsys = 0.979; genotype C: rArchitect versus WTultra = 0.950, rArchitect versus Elecsys = 0.963, and rWTultra versus Elecsys = 0.981) and hepatitis B virus (HBV) DNA levels (rArchitect = 0.540, rWTultra = 0.553, and rElecsys = 0.580). In conclusion, the Elecsys and WTultra assays were well correlated with the Architect assay, irrespective of the CHB infection phase or genotype. All of these assays are reliable for HBsAg quantification. PMID:25209557

  17. The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays.

    PubMed

    Britten, C M; Gouttefangeas, C; Welters, M J P; Pawelec, G; Koch, S; Ottensmeier, C; Mander, A; Walter, S; Paschen, A; Müller-Berghaus, J; Haas, I; Mackensen, A; Køllgaard, T; thor Straten, P; Schmitt, M; Giannopoulos, K; Maier, R; Veelken, H; Bertinetti, C; Konur, A; Huber, C; Stevanović, S; Wölfel, T; van der Burg, S H

    2008-03-01

    The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8(+) T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols

  18. The specific detection of foot-and-mouth disease virus whole particle antigen (140S) by enzyme labelled immunosorbent assay.

    PubMed

    Elzein, E M; Crowther, J R

    1979-08-01

    A solid-phase micro-enzyme-labelled immunosorbent assay (ELISA) using guinea pig antiserum against purified (140S) inactivated foot-and-mouth disease (FMD) virus has been used in a sandwich technique to specifically measure 140S virus in the presence of 12S material. PMID:222837

  19. Immuno-Chromatographic Wicking Assay for the Rapid Detection of Chikungunya Viral Antigens in Mosquitoes (Diptera: Culicidae).

    PubMed

    Hinson, Juanita M; Davé, Sonia; McMenamy, Scott S; Davé, Kirti; Turell, Michael J

    2015-07-01

    The outbreak of disease caused by chikungunya virus (CHIKV) in 2006 and the recent spread of this virus to the Americas in 2013 indicate the potential for this virus to spread and cause significant disease. However, there are currently no accurate and reliable field-usable, diagnostic methods to provide critical, real-time information for early detection of CHIKV within the vector populations in order to implement appropriate vector control and personal protective measures. In this article, we report the ability of an immuno-chromatographic assay developed by VecTOR Test Systems Inc. to detect CHIKV in a pool of female Aedes mosquitoes containing a single CHIKV-infected mosquito. The CHIKV dipstick assay was simple to use, did not require a cold chain, and provided clear results within 1 h. It was highly specific and did not cross-react with samples spiked with a variety of other alpha, bunya, and flaviviruses. The CHIKV assay can provide real-time critical information on the presence of CHIKV in mosquitoes to public health personnel. Results from this assay will allow a rapid threat assessment and the focusing of vector control measures in high-risk areas.

  20. Immuno-chromatographic wicking assay for the rapid detection of dengue viral antigens in mosquitoes (Diptera: Culicidae).

    PubMed

    Wanja, Elizabeth; Parker, Zahra F; Odusami, Oluwakemi; Rowland, Tobin; Davé, Kirti; Davé, Sonia; Turell, Michael J

    2014-01-01

    There is a threat for dengue virus (DENV) reemergence in many regions of the world, particularly in areas where the DENV vectors, Aedes aegypti (L.) and Aedes albopictus (Skuse), are readily available. However, there are currently no accurate and reliable diagnostic methods to provide critical, real-time information for early detection of DENV within the vector populations to implement appropriate vector control and personal protective measures. In this article, we report the ability of an immuno-chromatographic assay developed by VecTOR Test Systems Inc. to detect DENV in a pool of female Aedes mosquitoes infected with any of the four viral serotypes. The DENV dipstick assay was simple to use, did not require a cold chain, and provided clear results within 30 min. It was highly specific and did not cross-react with samples spiked with West Nile, yellow fever, Japanese encephalitis, Rift Valley fever, chikungunya, Venezuelan equine encephalomyelitis, Ross River, LaCrosse, or Caraparu viruses. The DENV assay can provide real-time critical information on the presence of DENV in mosquitoes to public health personnel. Results from this assay will allow a rapid threat assessment and the focusing of vector control measures in high-risk areas. PMID:24605472

  1. Evaluating troponin C from Psoroptes cuniculi as a diagnostic antigen for a dot-ELISA assay to diagnose mite infestations in rabbits.

    PubMed

    Zheng, W; Zhang, R; Wu, X; Ren, Y; Nong, X; Gu, X; Wang, S; Peng, X; Yang, G

    2014-02-01

    The mite Psoroptes cuniculi is globally widespread and has a serious impact on commercial rabbit breeding. In China, diagnosis of P. cuniculi is currently based on conventional clinical methods that entail numerous disadvantages, including their failure to diagnose subclinical infections. Hence, alternative measures are required, and dot-ELISA is one of the most promising strategies. We cloned and expressed the recombinant P. cuniculi troponin C gene for use as a basis for novel dot-ELISA assay to detect P. cuniculi infections in rabbits. This amplified sequence encoded a 153 amino acid protein of 17·6 kDa and theoretical pI 4·18 without signal peptide. The recombinant troponin C of P. cuniculi is an outer membrane protein and may also be a new P. cuniculi allergen. Results of dot-ELISA test showed that this novel assay had more than 90% sensitivity but low specificity in distinguishing infections with P. cuniculi or Sarcoptic scabiei, despite very high agreement between observers (97-99%; κ values ranged from 0·95 to 0·98 for inter- and intra-observer variability test). This study showed that this novel method, at present, lacks diagnostic utility. Therefore, although simple serological assays such as dot-ELISA show great promise as diagnostic tools, we suggest that troponin C is not a suitable diagnostic antigen candidate. PMID:24102446

  2. Western Blot Assay Using Recombinant p26 Antigen for Detection of Equine Infectious Anemia Virus-Specific Antibodies▿

    PubMed Central

    Alvarez, I.; Gutierrez, G.; Ostlund, E.; Barrandeguy, M.; Trono, K.

    2007-01-01

    We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results. PMID:17959820

  3. Development of an antigen-capture enzyme-linked immunosorbent assay for Clostridium perfringens beta2-toxin in porcine feces and the neonatal piglet intestine.

    PubMed

    Kircanski, Jasmina; Hodgins, Douglas; Soltes, Glenn; Pei, Yanlong; Parreira, Valeria R; Songer, J Glenn; Prescott, John F

    2012-09-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for detection and quantitation of beta2-toxin in neonatal piglet intestinal contents. Polystyrene plates were coated with polyclonal capture antibodies prepared against consensus recombinant beta2-toxin. The ELISA was developed using consensus recombinant beta2-toxin, atypical recombinant beta2-toxin, purified consensus native beta2-toxin, and field samples of neonatal porcine intestinal contents. Captured antigen was detected using a horseradish peroxidase-labeled monoclonal antibody against consensus recombinant beta2-toxin. The limit of detection of the ELISA for consensus beta2-toxin was between 2.0 and 3.5 ng/ml. The ELISA detected atypical recombinant beta2-toxin only weakly. Optical density was protein concentration dependent. The test confirmed differences between consensus and atypical recombinant beta2-toxin, but similar results obtained when testing pure consensus recombinant beta2-toxin and native beta2-toxin. Results obtained from intestinal content samples, particularly from the small intestine, were highly inconsistent and suggested variable protease activity. Addition of protease inhibitors partially prevented degradation of the toxin; however, sample processing at low temperature, at a lower pH (citrate buffer with 5% of bovine serum albumin, pH 6.1), and "cold incubation" of applied antigens abolished protease activity. The recombinant toxin was preserved in spiked intestinal samples by freezing at -70°C, suggesting that necropsy samples can be stored frozen for periodic testing. With appropriate sample preparation, antigen-capture ELISA can detect beta2-toxin in the intestinal content and feces of neonatal piglets.

  4. Low frequency of anti-SLA/LP autoantibody in Japanese adult patients with autoimmune liver diseases: analysis with recombinant antigen assay.

    PubMed

    Miyakawa, Hiroshi; Kawashima, Yumi; Kitazawa, Eriko; Kawaguchi, Naomi; Kato, Takashi; Kikuchi, Kentaro; Imai, Erika; Fujikawa, Hirotoshi; Hashimoto, Etsuko; Schlumberger, Wolfgang

    2003-08-01

    Anti-soluble liver antigen/liver pancreas (SLA/LP) autoantibody has been proposed to be one of the autoantibodies characterizing autoimmune hepatitis (AIH). Recently, one of the autoantigens to anti-SLA/LP was identified as a UGA suppressor tRNA-associated protein. Although the function of this protein remains unknown, the recombinant protein has been prokaryotically expressed. Using this protein as an antigen, a recombinant immunoassay for anti-SLA/LP autoantibody has been established and the frequency and significance of this autoantibody have been discussed in European countries. So, in the present study, we investigated anti-SLA/LP autoantibodies in Japanese patients with autoimmune liver diseases using the recombinant antigen ELISA and Western blot assay. Seventy-five patients with AIH type 1, 5 with AIH type 2, 46 with primary biliary cirrhosis, 10 with primary sclerosing cholangitis, 47 with chronic hepatitis C, 48 with systemic lupus erythematosus, 3 with cryptogenic hepatitis, and 40 normal controls were the subjects of the present study. Anti-SLA/LP autoantibodies were detected in only 5 of 75 (6.7%) patients with AIH type 1, but in none of the other 159 patients or 40 normal controls. The clinicopathologic features of anti-SLA/LP-positive AIH type 1, including carriers of HLA DR locus variations, were not significantly different from anti-SLA/LP-negative patients except for the mortality rate. Anti-SLA/LP autoantibody was detected at a low frequency in Japanese patients with AIH type 1 and did not significantly influence clinical features. However, since it has high disease-specificity to AIH type 1, further analysis of SLA/LP may contribute to help clarify the pathogenesis of AIH type 1.

  5. Enzyme-Linked Immunosorbent Assay Using Recombinant SAG1 Antigen To Detect Toxoplasma gondii-Specific Immunoglobulin G Antibodies in Human Sera and Saliva

    PubMed Central

    Chahed Bel-Ochi, Nouha; Bouratbine, Aïda

    2013-01-01

    Serologic detection of Toxoplasma gondii IgG antibodies is widely accepted as a means to determine immune status and susceptibility to Toxoplasma infection during pregnancy. However, current commercial kits present some drawbacks, such as a requirement for whole-parasite antigen preparation or interassay variability. To address these problems, the purpose of this study was to produce a whole sequence of the recombinant T. gondii SAG1 antigen (rSAG1) to assess its diagnostic performance in Toxoplasma IgG screening and to explore a saliva-based method as a noninvasive alternative to serum-based testing. rSAG1 was expressed in recombinant bacteria as inclusion bodies, purified through one-step affinity chromatography, and refolded in native form by dialysis. A large amount was obtained, and the specific antigen immunoreactivity was confirmed by immunoblotting. Two rSAG1-based enzyme-linked immunosorbent assays (ELISAs) applied to paired serum and saliva samples were designed. The rSAG1-based ELISA evaluation consisted of testing intrinsic sensitivity and specificity of 49 serum samples from patients immune to toxoplasmosis and 42 serum samples from nonimmune controls identified by routinely used kits. To assess agreement between serum-based and saliva-based tests, the positive percent agreement (PPA) and negative percent agreement (NPA) between the 2 tests were estimated. The rSAG1 serum-based ELISA detected specific IgG with 100% sensitivity and specificity. The PPA and NPA between the serum-based and saliva-based tests varied according to the selected optical density threshold in saliva. Thus, for a selected cutoff of 0.14, the PPA was 100% and the NPA was 88.1%, whereas for a selected cutoff of 0.29, the PPA was 67.3% and the NPA was 100%. PMID:23345586

  6. The hybrid EIA test: a specific and sensitive assay for the detection of woodchuck antibody to hepatitis surface antigen (anti-WHs).

    PubMed

    Millman, I; Glass, R G

    1988-05-01

    'Ausria II' polystyrene beads (Abbott Labs, N. Chicago) are reacted with woodchuck serum positive for WHsAg in a dilution predetermined by titration. This modified bead is used in a blocking assay to detect the presence of antibody to the surface antigen of woodchuck hepatitis virus (anti-WHs). Serum containing woodchuck anti-WHs and commercial horseradish peroxidase (HRP) labeled anti-HBs are sequentially added. A drop in optical density at 492 nm of 50% or more due to the blocking of HRP conjugated anti-HBs by anti-WHs compared with a control (negative woodchuck serum) is a measure of anti-WHs. The ease and simplicity of converting readily available 'Ausria II' beads to specific reagents for detecting anti-WHs should be welcomed by investigators studying WHV. The method described is both sensitive and reproducible.

  7. Development and validation of an HPLC/UV assay for separation and quantification of peptide antigens from a liposomal vaccine delivery platform.

    PubMed

    Penwell, Andrea; Sharp, Kendall; Mansour, Marc; Sammatur, Leeladhar

    2012-07-01

    The development and validation of an HPLC method for the quantification of eight peptide antigens from the therapeutic cancer vaccine DPX-0907 is described. The antigens were formulated in DepoVax™, a patented liposomal vaccine delivery platform used in a phase 1 study for breast, ovarian, and prostate cancers. A gradient reversed-phase method with UV detection was optimized for separating and quantifying the peptide mixture. Several extraction methods investigated to extract the peptides from the lipids led to poor recovery of one or more of the peptides. A simple, reproducible, and high-recovery extraction procedure for the simultaneous quantification of hydrophilic and hydrophobic peptides was discovered using a liquid-liquid extraction with water-saturated n-butanol and sodium bicarbonate (0.1 M). The method was found to be specific, linear, accurate, precise, and reliable within the range of 50-150% of the nominal concentration for DPX-0907. The validated method was successfully applied to the assay of peptide content in pre-clinical and clinical batches of DPX-0907. PMID:22516680

  8. Chemopreventive effect of resveratrol, sesamol, sesame oil and sunflower oil in the Epstein-Barr virus early antigen activation assay and the mouse skin two-stage carcinogenesis.

    PubMed

    Kapadia, Govind J; Azuine, Magnus A; Tokuda, Harukuni; Takasaki, Midori; Mukainaka, Teruo; Konoshima, Takao; Nishino, Hoyoku

    2002-06-01

    Resveratrol, sesamol, sesame oil and sunflower oil are known natural dietary components with intrinsic cancer chemopreventive potentials. As a part of our study of dietary constituents as potential cancer chemopreventive agents, we have assessed the anti-cancer potentials of these products in the promotion stage of cancer development employing the in vitro Epstein-Barr virus early antigen activation assay induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Further, we studied the activities of these compounds in the brine shrimp cytotoxicity assay as well as on the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging bioassay with a view to comparing some of the mechanisms of their anti-cancer activity. Finally, we compared the observed chemoprotective capabilities of the four products in the in vivo 7,12 dimethylbenz(a)anthracene initiated and TPA-promoted mouse skin two-stage carcinogenesis protocols. All the products tested showed a profound inhibitory effect on the Epstein-Barr virus early antigen induction using Raji cells. Comparatively, sesame oil was the most potent followed by sesamol and then resveratrol. Only sesamol and resveratrol showed a remarkable cytotoxic activity in the brine shrimp lethality assays as well as profound free radical scavenging activity in the DPPH bioassay. In both test systems, sesamol exhibited a more remarkable activity than resveratrol while sesame oil and sunflower oil did not exhibit any appreciable activity even at the highest concentrations tested (4000 microg ml(-1) ). In our in vivo assay at a 50-fold molar ratio to TPA, sesamol offered 50% reduction in mouse skin papillomas at 20 weeks after promotion with TPA. Under an identical molar ratio to TPA, resveratrol offered a 60% reduction in the papillomas in mouse at 20 weeks. Thus sesamol seems to be an almost equally potent chemopreventive agent. Sesame oil and sunflower oil offered 20 and 40% protection, respectively, in the mouse

  9. Chemopreventive effect of resveratrol, sesamol, sesame oil and sunflower oil in the Epstein-Barr virus early antigen activation assay and the mouse skin two-stage carcinogenesis.

    PubMed

    Kapadia, Govind J; Azuine, Magnus A; Tokuda, Harukuni; Takasaki, Midori; Mukainaka, Teruo; Konoshima, Takao; Nishino, Hoyoku

    2002-06-01

    Resveratrol, sesamol, sesame oil and sunflower oil are known natural dietary components with intrinsic cancer chemopreventive potentials. As a part of our study of dietary constituents as potential cancer chemopreventive agents, we have assessed the anti-cancer potentials of these products in the promotion stage of cancer development employing the in vitro Epstein-Barr virus early antigen activation assay induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Further, we studied the activities of these compounds in the brine shrimp cytotoxicity assay as well as on the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging bioassay with a view to comparing some of the mechanisms of their anti-cancer activity. Finally, we compared the observed chemoprotective capabilities of the four products in the in vivo 7,12 dimethylbenz(a)anthracene initiated and TPA-promoted mouse skin two-stage carcinogenesis protocols. All the products tested showed a profound inhibitory effect on the Epstein-Barr virus early antigen induction using Raji cells. Comparatively, sesame oil was the most potent followed by sesamol and then resveratrol. Only sesamol and resveratrol showed a remarkable cytotoxic activity in the brine shrimp lethality assays as well as profound free radical scavenging activity in the DPPH bioassay. In both test systems, sesamol exhibited a more remarkable activity than resveratrol while sesame oil and sunflower oil did not exhibit any appreciable activity even at the highest concentrations tested (4000 microg ml(-1) ). In our in vivo assay at a 50-fold molar ratio to TPA, sesamol offered 50% reduction in mouse skin papillomas at 20 weeks after promotion with TPA. Under an identical molar ratio to TPA, resveratrol offered a 60% reduction in the papillomas in mouse at 20 weeks. Thus sesamol seems to be an almost equally potent chemopreventive agent. Sesame oil and sunflower oil offered 20 and 40% protection, respectively, in the mouse

  10. LATERAL FLOW ASSAY FOR CRYPTOCOCCAL ANTIGEN: AN IMPORTANT ADVANCE TO IMPROVE THE CONTINUUM OF HIV CARE AND REDUCE CRYPTOCOCCAL MENINGITIS-RELATED MORTALITY

    PubMed Central

    VIDAL, Jose E.; BOULWARE, David R.

    2015-01-01

    SUMMARY AIDS-related cryptococcal meningitis continues to cause a substantial burden of death in low and middle income countries. The diagnostic use for detection of cryptococcal capsular polysaccharide antigen (CrAg) in serum and cerebrospinal fluid by latex agglutination test (CrAg-latex) or enzyme-linked immunoassay (EIA) has been available for over decades. Better diagnostics in asymptomatic and symptomatic phases of cryptococcosis are key components to reduce mortality. Recently, the cryptococcal antigen lateral flow assay (CrAg LFA) was included in the armamentarium for diagnosis. Unlike the other tests, the CrAg LFA is a dipstick immunochromatographic assay, in a format similar to the home pregnancy test, and requires little or no lab infrastructure. This test meets all of the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User friendly, Rapid/robust, Equipment-free, and Delivered). CrAg LFA in serum, plasma, whole blood, or cerebrospinal fluid is useful for the diagnosis of disease caused by Cryptococcus species. The CrAg LFA has better analytical sensitivity for C. gattii than CrAg-latex or EIA. Prevention of cryptococcal disease is new application of CrAg LFA via screening of blood for subclinical infection in asymptomatic HIV-infected persons with CD4 counts < 100 cells/mL who are not receiving effective antiretroviral therapy. CrAg screening of leftover plasma specimens after CD4 testing can identify persons with asymptomatic infection who urgently require pre-emptive fluconazole, who will otherwise progress to symptomatic infection and/or die. PMID:26465368

  11. Improvement of antigen blotting in a tissue blot immunobinding assay for the detection of two chili pepper viruses.

    PubMed

    Han, Jung-Heon; Shin, Jun-Sung; Kim, Young Ho; Kim, Byung-Dong

    2007-11-01

    The tissue blot immunobinding assay (TBIA) is widely used for the detection and localization of plant viruses in various plant tissues. The basic experimental procedures of TBIA sampling and blotting were simplified using commercially available micropipette tips. This method was termed the ring-blot immunobinding assay (R-BIA), as the blot on the membrane forms a ring shape. The detection efficacy of R-BIA was tested for two chili pepper viruses, pepper mild mottle tobamovirus (PMMoV) and pepper mottle potyvirus (PepMoV), following the optimized serological procedures of TBIA (length of the incubation period and BSA concentration, and primary and secondary antibodies). Sensitivity of the R-BIA was about 1 ng/ml of purified PMMoV in pepper leaf sap from a healthy pepper plant. R-BIA also showed high specificity in the detection of PMMoV and PepMoV. Moreover, the modified sampling and blotting procedures were simpler and more reliable than other TBIA methods (such as whole-leaf blotting and crushed-leaf blotting), suggesting that the R-BIA may be used for medium- to large-scale detection of plant viruses in laboratories with minimal facilities.

  12. Biodegradable polylactide microspheres enhance specific immune response induced by Hepatitis B surface antigen.

    PubMed

    Qiu, Shaohui; Wei, Qiang; Liang, Zhenglun; Ma, Guanghui; Wang, Lianyan; An, Wenqi; Ma, Xiaowei; Fang, Xin; He, Peng; Li, Hemin; Hu, Zhongyu

    2014-01-01

    Hepatitis B (HB) infection caused by Hepatitis B virus (HBV) is the most common liver disease in the world. HB vaccine, when administered in conjunction with alum adjuvants, induces Th2 immunity that confers protection against HBV. However, currently available vaccine formulations and adjuvants do not elicit adequate Th1 and CTL responses that are important for prevention of maternal transmission of the virus. Microspheres synthesized from poly (D, L-lactide-co-glycolide) (PLGA) or poly (D, L-lactide) (PLA) polymers have been considered as promising tools for in vivo delivery of antigens and drugs. Here we describe PLA microspheres synthesized by premix membrane emulsification method and their application in formulating a new microsphere based HB vaccine. To evaluate the immunogenicity of this microsphere vaccine, BALB/c mice were immunized with microsphere vaccine and a series of immunological assays were conducted. Results of Enzyme-linked ImmunoSpot (ELISPOT) assays revealed that the number of interferon-gamma (IFN-γ)-producing splenocytes and CD8(+) T cells increased significantly in the microsphere vaccine group. Microsphere vaccine group showed enhanced specific cell lysis when compared with HB surface antigen (HBsAg) only group in (51)Cr cytotoxicity assays. Moreover, microsphere vaccine elicited a comparable level of antibody production as that of HB vaccine administered with alum adjuvant. We show that phagocytosis of HBsAg by dendritic cells is more pronounced in microsphere vaccine group when compared with other control groups. These results clearly demonstrate the potential of using PLA microspheres as effective HB vaccine adjuvants for an enhanced Th1 immune response.

  13. Biodegradable polylactide microspheres enhance specific immune response induced by Hepatitis B surface antigen

    PubMed Central

    Qiu, Shaohui; Wei, Qiang; Liang, Zhenglun; Ma, Guanghui; Wang, Lianyan; An, Wenqi; Ma, Xiaowei; Fang, Xin; He, Peng; Li, Hemin; Hu, Zhongyu

    2014-01-01

    Hepatitis B (HB) infection caused by Hepatitis B virus (HBV) is the most common liver disease in the world. HB vaccine, when administered in conjunction with alum adjuvants, induces Th2 immunity that confers protection against HBV. However, currently available vaccine formulations and adjuvants do not elicit adequate Th1 and CTL responses that are important for prevention of maternal transmission of the virus. Microspheres synthesized from poly (D, L-lactide-co-glycolide) (PLGA) or poly (D, L-lactide) (PLA) polymers have been considered as promising tools for in vivo delivery of antigens and drugs. Here we describe PLA microspheres synthesized by premix membrane emulsification method and their application in formulating a new microsphere based HB vaccine. To evaluate the immunogenicity of this microsphere vaccine, BALB/c mice were immunized with microsphere vaccine and a series of immunological assays were conducted. Results of Enzyme-linked ImmunoSpot (ELISPOT) assays revealed that the number of interferon-gamma (IFN-γ)-producing splenocytes and CD8+ T cells increased significantly in the microsphere vaccine group. Microsphere vaccine group showed enhanced specific cell lysis when compared with HB surface antigen (HBsAg) only group in 51Cr cytotoxicity assays. Moreover, microsphere vaccine elicited a comparable level of antibody production as that of HB vaccine administered with alum adjuvant. We show that phagocytosis of HBsAg by dendritic cells is more pronounced in microsphere vaccine group when compared with other control groups. These results clearly demonstrate the potential of using PLA microspheres as effective HB vaccine adjuvants for an enhanced Th1 immune response. PMID:25424942

  14. The novel panel assay to define tumor-associated antigen-binding antibodies in patients with metastatic melanomas may have diagnostic value.

    PubMed

    Kotlan, Beatrix; Liszkay, Gabriella; Blank, Miri; Csuka, Orsolya; Balatoni, Timea; Toth, Laszlo; Eles, Klara; Horvath, Szabolcs; Naszados, Gyorgy; Olasz, Judit; Banky, Balazs; Toth, Jozsef; Godeny, Maria; Marincola, Francesco M; Kasler, Miklos; Shoenfeld, Yehuda

    2015-02-01

    We aim to harness the natural humoral immune response by various technologies to get novel biomarkers. A complex antibody analysis in sera and in the tumor microenvironment leads to reveal tumor-specific antibodies. More strategies were introduced to select the most effective one to identify potential tumor antigen-binding capacity of the host. Epstein-Barr virus transformation and cloning with limiting dilution assay, magnetic cell sorting and antibody phage display with further methodological improvements were used in epithelial and neuroectodermal cancers. Column-purified sera of patient with melanoma were tested by immunofluorescence assay, while sera of further melanoma patients were processed for membrane-binding enzyme-linked immunosorbent assay. Some supernatants of selected B cell clones and purified antibodies showed considerable cancer cell binding capacity by immunofluorescence FACS analysis and confocal laser microscopy. Our native tumor cell membrane preparations helped to test soluble scFv and patients' sera for tumor binder antibodies. A complex tumor immunological study was introduced for patients with melanoma (ethical permission: ETT TUKEB 16462-02/2010); peripheral blood (n = 57) and surgically removed primary or metastatic tumors (n = 44) were gathered and processed at cellular immunological level. The technological developments proved to be important steps forward to the next antibody profile analyses at DNA sequence level. Cancer cell binding of patient-derived antibodies and natural immunoglobulin preparations of pooled plasma product intravenous immunoglobulins support the importance of natural human antibodies. Important cancer diagnostics and novel anticancer strategies are going to be built on these tools.

  15. Proteomic analysis of hepatitis B surface antigen positive transgenic mouse liver and decrease of cyclophilin A.

    PubMed

    Zhao, Chao; Fang, Cai-Yun; Tian, Xiao-Chen; Wang, Long; Yang, Peng-Yuan; Wen, Yu-Mei

    2007-10-01

    The small, 22-nm spherical particles associated with hepatitis B infection are composed of hepatitis B surface antigen (HBsAg) and usually outnumber the virions by a ratio of 10(2) or 10(3). To study the interactions and pathogenesis between liver cells and the expression of HBsAg, global protein profiles were compared by two dimensional gel-based differential proteomics between the livers of a lineage of HBsAg positive transgenic mice and their HBsAg negative control siblings. A total of 93 proteins were identified in the HBV transgenic mice. Around 45% of these differentially expressed proteins were enzymes associated with metabolism, suggesting that the processing of lipids, carbohydrates and certain amino acids were up- or down-regulated. Among these proteins, cyclophilin A (CypA), the major target for the potent immunosuppressive drug cyclosporin A, was found decreased in HBsAg positive transgenic mouse liver and in a stable cell line expressing HBsAg when compared to their controls. The decrease of intracellular CypA was accompanied by an increased secretion of this protein into the supernatant of HBsAg positive cells. Possible implications of HBsAg expression and the intracellular decrease of CypA are discussed.

  16. Presumptive diagnosis of subclinical infections utilizing computer-assisted analysis of sequential enzyme-linked immunosorbent assays against multiple antigens.

    PubMed

    Mallinson, E T; Snyder, D B; Marquardt, W W; Russek-Cohen, E; Savage, P K; Allen, D C; Yancey, F S

    1985-09-01

    One-hundred-seventy-two serum samples, collected sequentially from four flocks of egg- and meat-type chickens, were evaluated for antibodies to multiple infectious agents by enzyme-linked immunosorbent assay (MELISA). The MELISA system used provided simultaneous measurement of antibody titers against avian infectious bronchitis (IB), infectious bursal disease (BD), Newcastle disease, avian encephalomyelitis and reovirus infections, and Mycoplasma gallisepticum. The use of computer-generated graphic print outs of relative MELISA titers provided immediate visulization of over 740 data points and convenient detection of any temporal changes in median titer class (MTC). The temporally changing MTC, or flock profiles obtained, indicated that negligible or waning IB immunity may be a common occurrence in previously vaccinated commercial chickens. These profiles further suggested that, despite no IB revaccination, these same flocks experienced episodes of reexposure to IB which otherwise may have been difficult to detect by conventional clinical or diagnostic laboratory protocols. MELISA profiles and sequential histologic examinations of bursas of Fabricius also provided evidence of a possible BD vaccination problem in young chickens that also experienced excessive losses from coccidiosis, ulcerative enteritis, and Marek's disease. Short sampling intervals were found to foster the detection and definition of fluctuations in MTC which otherwise may have been missed. PMID:4048057

  17. Impact of pneumococcal vaccination in children on serotype distribution in adult community-acquired pneumonia using the serotype-specific multiplex urinary antigen detection assay.

    PubMed

    Pletz, Mathias W; Ewig, Santiago; Rohde, Gernot; Schuette, Hartwig; Rupp, Jan; Welte, Tobias; Suttorp, Norbert; Forstner, Christina

    2016-04-29

    The aim of the study was to compare the distribution of the vaccine-serotypes covered by pneumococcal conjugate vaccines (PCV7 and PCV13) in adult patients with pneumococcal community-acquired pneumonia in Germany between the periods 2002-2006 and 2007-2011 using a novel serotype-specific multiplex urinary antigen detection assay (SSUA). Vaccination of children started with PCV7 in 2007, which was replaced by PCV13 in 2010. Following confirmation of the accuracy of SSUA in long-term stored urine samples from 112 patients with confirmed pneumonia and known pneumococcal serotype, urine samples of 391 CAPNETZ patients with documented pneumococcal pneumonia (i.e. positive BinaxNOW) Streptococcus pneumoniae urine antigen test) but unknown serotype were tested for the 13 vaccine-serotypes using SSUA. The proportion of PCV7-serotypes significantly decreased in adult patients with pneumonia from 30.6% (2002-6) to 13.3% (2007-11, p < 0.001); in bacteremic pneumonia, PCV7-serotypes completely disappeared (3/14 versus 0/19, p = 0.058). Conversely, pneumococcal serotypes included by PCV13 remained stable during study period with a coverage of 61.5% (2002-06) and 59.7% (2007-11) in non-bacteremic pneumonia and 79% (for both periods) in bacteremic pneumonia, mainly due to an increase in pneumococcal serotypes 1, 3 and 7F during the second period. Thus, implementation of PCV7 in children in Germany in 2007 was associated with a significant decrease in vaccine-serotypes covered by PCV7 in adult patients with non-bacteremic pneumococcal pneumonia and with an elimination of PCV7 vaccine-serotypes in bacteremic pneumococcal pneumonia. PCV13 coverage remained high up to 2011, mainly due to an increase in serotypes 1, 3 and 7F.

  18. Validation of Rapid Point-of-Care (POC) Tests for Detection of Hepatitis B Surface Antigen in Field and Laboratory Settings in the Gambia, Western Africa

    PubMed Central

    Njai, Harr Freeya; Shimakawa, Yusuke; Sanneh, Bakary; Ferguson, Lynne; Ndow, Gibril; Mendy, Maimuna; Sow, Amina; Lo, Gora; Toure-Kane, Coumba; Tanaka, Junko; Taal, Makie; D'alessandro, Umberto; Njie, Ramou; Thursz, Mark

    2015-01-01

    Hepatitis B virus (HBV) infection is a leading cause of death in sub-Saharan Africa (SSA). Point-of-care tests for hepatitis B surface antigen (HBsAg) could be an ideal tool for a large-scale HBV screening/treatment program in SSA. Using data from the PROLIFICA (Prevention of Liver Fibrosis and Cancer in Africa) program, we conducted a cross-sectional study to assess the diagnostic accuracy of three point-of-care tests (Determine, Vikia, and Espline) for the detection of HBsAg in the field or a laboratory setting in the Gambia. In the field, we used finger-prick whole blood for the Determine and Vikia tests and dried blood spots for the reference standard test (AxSYM HBsAg enzyme-linked immunosorbent assay [ELISA]). In the laboratory we used serum for the Determine, Espline, and reference test (Architect chemiluminescent microparticle immunoassay). Of 773 participants recruited at the community and 227 known chronic HBV carriers (1,000 subjects in total), 293 were positive for HBsAg. The sensitivity and specificity of the Determine test were 88.5% and 100% in the field and 95.3% and 93.3% in the laboratory setting, respectively. The sensitivity and specificity were 90.0% and 99.8% for the Vikia test (in the field) and 93.9% and 94.7% for the Espline test (in the laboratory). There was no evidence that one kit was better than another. Most of the patients with false-negative results (18/19) were classified as inactive chronic carriers. In summary, the three point-of-care tests had acceptable ranges of diagnostic accuracy. These tests may represent accurate, rapid, and inexpensive alternatives to serology testing for the screening of HBV infection at field level in SSA. PMID:25631805

  19. Enzyme-linked immunosorbant assay (ELISA) of size-selected crotalid venom antigens by Wyeth's polyvalent antivenom.

    PubMed

    Schaeffer, R C; Randall, H; Resk, J; Carlson, R W

    1988-01-01

    The binding of Antivenom (Crotalidae) Polyvalent to fractions from crude venoms of eight crotalid and one viperid snake, obtained by high performance size-exclusion chromatography, was determined with an indirect enzyme-linked immunosorbent assay (ELISA). Most of the large (greater than 30,000 mol. wt) molecular mass crotalid venom fractions were associated with high (greater than 0.7 absorbance units) ELISA values. Similarly, the medium (13,000-30,000 mol. wt) and small (less than 14,000 mol. wt) molecular mass crotalid venom fractions were coincident with moderate (0.3-0.7 absorbance units) and low (less than 0.3 absorbance units) ELISA levels. Some variability in this pattern was seen with individual venom fractions. A distinctly different pattern of ELISA values were observed with two rattlesnake venoms: the South American (Crotalus durissus terrificus) and Mojave desert (Crotalus scutulatus scutulatus) rattlesnakes. The elution profile from these venoms showed a progression of low to moderate ELISA values within the large molecular mass fractions. This pattern was followed by a decline to low ELISA values throughout the remainder of the elution profile. When saw scaled viper (Echis carinatus leucogaster) venom fractions were tested, only background ELISA values were detected with antivenom. Similarly, background ELISA values were associated with the small molecular mass fractions of all venoms tested. In addition, the elution position for the basic peptides of southern Pacific (Crotalus viridis helleri) and timber (Crotalus h. horridus) rattlesnake venoms showed minimal ELISA values. These data support the view that except for the venom of C. durissus terrificus and C. s. scutulatus, most antivenom antibodies bind large (greater than 30,000 mol. wt) venom fractions. Thus, antivenom contains minimal levels of antibodies to the basic peptides in these venoms. PMID:3347932

  20. HIV Incidence Estimates Using the Limiting Antigen Avidity EIA Assay at Testing Sites in Kiev City, Ukraine: 2013-2014

    PubMed Central

    Kruglov, Yuri; Yurchenko, Alexander

    2016-01-01

    Objective To estimate HIV incidence and highlight the characteristics of persons at greatest risk of HIV in the Ukraine capital, Kiev. Method Residual samples from newly-diagnosed persons attending the Kiev City AIDS Centre were tested for evidence of recent HIV infection using an avidity assay. Questions on possible risk factors for HIV acquisition and testing history were introduced. All persons (≥16yrs) presenting for an HIV test April’13–March’14 were included. Rates per 100,000 population were calculated using region-specific denominators. Results During the study period 6370 individuals tested for HIV. Of the 467 individuals newly-diagnosed with HIV, 21 had insufficient samples for LAg testing. Of the remaining 446, 39 (8.7%) were classified as recent with an avidity index <1.5ODn, 10 were reclassified as long-standing as their viral load was <1000 copies/mL, resulting in 29 (6.5%) recent HIV infections. The only independent predictor for a recent infection was probable route of exposure, with MSM more likely to present with a recent infection compared with heterosexual contact [Odds Ratio 8.86; 95%CI 2.65–29.60]. We estimated HIV incidence at 21.5 per 100,000 population, corresponding to 466 new infections. Using population estimates for MSM and PWID, incidence was estimated to be between 2289.6 and 6868.7/100,000 MSM, and 350.4 for PWID. Conclusion A high proportion of persons newly-infected remain undiagnosed, with MSM disproportionally affected with one in four newly-HIV-diagnosed and one in three recently-HIV-infected. Our findings should be used for targeted public health interventions and health promotion. PMID:27276170

  1. A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays.

    PubMed Central

    Porrás, A; Bennett, J; Howe, A; Tokos, K; Bouck, N; Henglein, B; Sathyamangalam, S; Thimmapaya, B; Rundell, K

    1996-01-01

    At least three regions of the simian virus 40 small-t antigen (small-t) contribute to the protein's ability to enhance cellular transformation. As we showed previously for rat F111 cells, one region includes sequences from residues 97 to 103 that are involved in the binding and inhibition of protein phosphatase 2A. In the present study, the role of the protein phosphatase 2A binding region was confirmed in two additional small-t-dependent transformation systems. Second, small-t was found to provide a function previously identified as a large-T transformation domain. Mutations in residues 19 to 28 of large-T affected its transforming ability, but these mutations were complemented by a wild-type small-t. A third region of small-t was also required for efficient transformation. This region, the 42-47 region, is shared by large-T and small-t and contains a conserved HPDKGG hexapeptide. The 42-47 region function could be provided by either small-t or large-T in small-t-dependent systems. Mutations in the 42-47 region reduced the ability of small-t to transactivate the cyclin A promoter, of interest because small-t increased endogenous cyclin A mRNA levels in both human and monkey cells, as well as transactivating the promoter in transient assays. PMID:8794333

  2. Evolutionary analysis of HBV "S" antigen genetic diversity in Iranian blood donors: a nationwide study.

    PubMed

    Pourkarim, Mahmoud Reza; Sharifi, Zohre; Soleimani, Ali; Amini-Bavil-Olyaee, Samad; Elsadek Fakhr, Ahmed; Sijmons, Steven; Vercauteren, Jurgen; Karimi, Gharib; Lemey, Philippe; Maes, Piet; Alavian, Seyed Moayed; Van Ranst, Marc

    2014-01-01

    The genetic diversity of the HBV S gene has a significant impact on the prophylaxis and treatment of hepatitis B infection. The effect of selective pressure on this genetic alteration has not yet been studied in Iranian blood donors. To explore HBV evolution and to analyze the effects and patterns of hepatitis B surface antigen (HBsAg) mutations on blood screening assays, 358 Iranian blood donors diagnosed as asymptomatic HBV carriers were enrolled in this nationwide study. Large S and partial S genes were amplified and sequenced. HBV (sub) genotypes and synonymous and nonsynonymous mutations were investigated. The impact of naturally occurring mutations on HBsAg ELISA results was explored. Phylogenetic analyses revealed that isolated strains were of genotype D. The dominant subgenotype/subtype was D1/ayw2. Deletions and naturally occurring stop codons in the pre-S1 and major hydrophilic region (MHR) were identified. In total, 32.8% of the studied strains harbored 195 single or multiple mutations in the MHR, the majority of which were located at the first loop of the "a determinant" domain. The ayw2 subtype showed a significant effect on the ELISA signal/cut-off value and carried fewer mutations in the MHR. Nonsynonymous/synonymous substitution value indicated that negative selection was the dominant evolutionary force in the HBV S gene. This nationwide study revealed that mutation frequency of HBsAg among Iranian blood donors was much higher than previous reports from the different local regions. These findings regarding the significant differences in reactivity of ELISA among different subtypes of HBV and its correlation with the number of mutations at the MHR will be valuable to public health authorities. PMID:24150816

  3. Enhanced humoral and cell-mediated immune responses generated by cationic polymer-coated PLA microspheres with adsorbed HBsAg.

    PubMed

    Chen, Xiaoming; Liu, Yuying; Wang, Lianyan; Liu, Yuan; Zhang, Weifeng; Fan, Bei; Ma, Xiaowei; Yuan, Qipeng; Ma, Guanghui; Su, Zhiguo

    2014-06-01

    Surface-engineered particulate delivery systems for vaccine administration have been widely investigated in experimental and clinical studies. However, little is known about charge-coated microspheres as potential recombinant subunit protein antigen delivery systems in terms of adsorption and related immune responses. In the present study, cationic polymers, including chitosan (CS), chitosan chloride (CSC), and polyethylenimine (PEI), were used to coat PLA microspheres to build positively charged surfaces. Antigen adsorption capacity was enhanced with increased surface charge of coated microspheres. In macrophages, HBsAg adsorbed on the surface of cationic microspheres specifically enhanced antigen uptake and augmented CD86, MHC I, and MHC II expression and IL-1β, IL-6, TNF-α, and IL-12 release. Antigens were more likely to localize independent of lysosomes after phagocytosis in antigen-attached cationic microsphere formulations. After intraperitoneal immunization, cationic microsphere-based vaccine formulations generated a rapid and efficient humoral immune response and cytokine release as compared with aluminum-adsorbed vaccine and free antigens in vivo. Moreover, microspheres coated with cationic polymers with relatively high positive charges and higher antigen adsorption exhibited strong stimulation of the Th1 response. In conclusion, PLA microspheres coated with cationic polymers may be a potential recombinant antigen delivery system to induce strong cell and humoral immune responses.

  4. The fibrinogen antigenic turbidimetric assay (FIATA): the X2x test--the corrected chi-square comparison against the control-mean.

    PubMed

    Stief, Thomas W

    2007-01-01

    Vancomycin precipitates fibrinogen. The turbidity induced by this vancomycin-fibrinogen interaction is used to establish a simple standardized antigenic assay for plasmatic fibrinogen, the FIATA. 1 mM vancomycin or 2 mM chloramine-T inactivates 50% of fibrinogen in human plasma. In contrast to chloramine-T, vancomycin does not react in NaJ-based photometric assay for chloramines,vancomycin does not inactivate the singlet oxygen-sensible antithrombin III, and the vancomycin action against fibrinogen is not changed in spite of the presence of the 1O2 quenchers methionine or ascorbic acid. The FIATA is performed as follows: to 25 microL plasma 50 microL PBS are added and the absorbance (A) at 405 nm is read. Then 50 microL FIATA-reagent, consisting of 4.4 mM vancomycin in PBS, are added. After 2 minutes (RT) DeltaA is determined and standardized against a plasma pool of 100% of norm (2.8 g/L) fibrinogen. The FIATA is nearly linear up to a fibrinogen concentration of about 150% of norm (4.2 g/L), resulting in a DeltaA of about 600 mA. The lower detection limit is 4% of norm (0.1 g/L). The intra-assay and interessay CV values are < 4%. The normal range of FIATA is 100% +/-20% (x- +/- 1 SD). In = 321 or 344 unselected patient plasmas the FIATA (x- = 130%; SD = 52% or 43%) correlated with the functional fibrinogen assays a) modified Clauss-Method (x- = 4.1 g/L; SD =1.7 g/L) with r = 0.755 and b) FIFTA (x- = 124%; SD = 40%) with r = 0.813. The vancomycin/fibrinogen interaction (binding of about 16 molecules of vancomycin/molecule of fibrinogen) can be used to purify fibrinogen out of plasma. Vancomycin also clouds dysfunctional fibrinogen (fibrinogen in presence of EDTA or chloramine-T)or soluble fibrin. Vancomycin-reacted fibrinogen stimulates tissue type plasminogen activator (t-PA) up to about 20-fold. The experimental data are analyzed by a new significance test: the two foldYates-corrected chi-square comparison against the mean value ofthe control-collective, called

  5. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay

    PubMed Central

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

    2016-01-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

  6. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.

    PubMed

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

    2016-08-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

  7. Development of an artificial-antigen-presenting-cell-based assay for the detection of low-frequency virus-specific CD8(+) T cells in whole blood, with application for measles virus.

    PubMed

    Ndhlovu, Zaza M; Angenendt, Monika; Heckel, Diana; Schneck, Jonathan P; Griffin, Diane E; Oelke, Mathias

    2009-07-01

    Evaluation of the immune responses induced by childhood vaccines requires measurement of T-cell, as well as antibody, responses. However, cellular immune responses are often not analyzed because of technical hurdles and the volume of blood required. Therefore, a sensitive and specific assay for antigen-specific T cells that utilizes a small volume of blood would facilitate new vaccine evaluation. We developed a novel assay for quantifying virus-specific CD8(+) T cells that combines the use of HLA-A2 immunoglobulin-based artificial antigen-presenting cells (aAPCs) for stimulation of antigen-specific CD8(+) T cells in whole blood with quantitative real-time reverse transcription-PCR (qRT-PCR) to detect gamma interferon (IFN-gamma) mRNA. This assay was optimized using a well-established cytomegalovirus (CMV) CD8(+) T-cell system. The aAPC-qRT-PCR assay had comparable sensitivity to intracellular cytokine staining (ICS) in detecting CMV-specific CD8(+) T cells with a detection limit of less than 0.004%. The assay was applied to the detection of low-frequency measles virus (MV)-specific CD8(+) T cells by stimulating blood from five MV-immune HLA-A*0201 donors with four different MV-specific peptides (MV peptide aAPCs). Stimulation with three of the MV peptide aAPCs resulted in significant increases in IFN-gamma mRNA ranging from 3.3- to 13.5-fold. Our results show that the aAPC-qRT-PCR assay is highly sensitive and specific and can be standardized for screening MV-specific CD8(+) T cells in vaccine trials. The technology should be transferable to analysis of CD8(+) T-cell responses to other antigens. PMID:19494085

  8. Aggregation and antigenicity of virus like particle in salt solution--A case study with hepatitis B surface antigen.

    PubMed

    Chen, Yi; Zhang, Yan; Quan, Can; Luo, Jian; Yang, Yanli; Yu, Mengran; Kong, Yingjun; Ma, Guanghui; Su, Zhiguo

    2015-08-20

    The phenomenon of aggregation of virus-like particles (VLPs) in salt solution and the corresponding effect upon antigenicity was reported. Asymmetrical flow field-flow fractionation (AF4) combined with multi-angle laser light scattering (MALLS) was used to characterize the size and the aggregation behavior of hepatitis B surface antigen (HBsAg). The average diameter of HBsAg VLP was 22.8±0.4 nm and it tended to aggregate in salt solution to form large particles and the antigenicity changed accordingly. In 0-4 M NaCl solution, part of HBsAg molecules aggregated rapidly into oligomeric particles (OP), whose diameter distributed from 25 to 40 nm, and the antigenicity slightly decreased about 10%. The aggregation reaction is reversible. After removing NaCl, both size and antigenicity could recover to normal level (92-96%). By contrast, the aggregation process is more complicated in (NH4)2SO4 solution. Most of HBsAg particles aggregated into OP and further aggregated into polymeric particles (PP). The diameter of the PP could reach 40 to 140 nm. The concentration of (NH4)2SO4 had remarkable influence upon the rate of aggregation. When concentration of (NH4)2SO4 was below 1 M, most of HBsAg aggregated only into OP in 1 h. While with concentration of (NH4)2SO4 above 1 M, most of particles formed PP within 1 h. The aggregation process to PP was irreversible. After removing (NH4)2SO4, the large aggregates could not recover to normal particles and the remaining antigenicity was below 30%. PMID:25862298

  9. Comparison of the Raji cell line fluorescent antibody to membrane antigen test and the enzyme-linked immunosorbent assay for determination of immunity to varicella-zoster virus.

    PubMed Central

    Iltis, J P; Castellano, G A; Gerber, P; Le, C; Vujcic, L K; Quinnan, G V

    1982-01-01

    A prospective study was performed comparing the fluorescent antibody to membrane antigen (FAMA) test and the enzyme-linked immunosorbent assay (ELISA) for identifying susceptibility and seroconversion to varicella-zoster virus (VZV) infection. A total of 75 sera were collected from index cases and from sibling and parent contacts in 10 families. Varicella-zoster virus-infected human diploid embryonic fibroblasts and continuous lymphoblastoid cells (Raji cells) were compared as indicator cells in the FAMA test. Equivalent results were obtained with both types of cell. Results of the FAMA test and the ELISA were identical in two ways. (i) The same 11 individuals were initally defined as susceptible (seronegative), and 9 of them (82%) developed fourfold rises in antibody titers, clinical varicella, or both. (ii) Of 21 immune (seropositive) individuals, 4 developed fourfold antibody rises by FAMA tests, and 3 of these 4 responded by ELISA. Infection was asymptomatic in these individuals. The geometric mean titer by ELISA was significantly higher than by the FAMA test. The results indicated that the ELISA and the FAMA test have similar capacities to define susceptibility to varicella-zoster virus and that subclinical infection with varicella-zoster virus may be common. PMID:6759530

  10. A Novel Method of Safely Measuring Influenza Virus Aerosol Using Antigen-Capture Enzyme-Linked Immunosorbent Assay for the Performance Evaluation of Protective Clothing Materials.

    PubMed

    Shimasaki, Noriko; Nojima, Yasuhiro; Okaue, Akira; Takahashi, Hitoshi; Kageyama, Tsutomu; Hamamoto, Itsuki; Shinohara, Katsuaki

    2016-01-01

    Currently, threats caused by pathogens are serious public health problems worldwide. Protective clothing is essential when one is treating infected patients or dealing with unknown pathogens. Therefore, it is necessary to evaluate the performance of protective clothing against pathogens. In Japan, some methods for evaluating the performance of protective clothing have been established in the Japanese Industrial Standards (JIS). However, a test method against virus aerosols has not been established. Because there is a risk of infection from a live virus during the test, it is necessary to devise a safe method for the virus-aerosol-based test. Here, we propose a new method of safely measuring virus aerosols for the performance evaluation of protective clothing materials. To ensure safety, an inactivated virus was used. As a model virus, the influenza virus was selected owing to the proper small diameter of the virus particles. To quantitatively measure the particle-amount of the inactivated influenza virus, we developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) targeting the M1 protein. Furthermore, we evaluated two materials using our method. Significant differences in the protection performance against the virus aerosol were observed between different sample materials, thereby confirming the applicability of our new method for performance evaluation.

  11. Evaluation of skin cancer chemoprevention potential of sunscreen agents using the Epstein-Barr virus early antigen activation in vitro assay.

    PubMed

    Kapadia, G J; Rao, G S; Takayasu, J; Takasaki, M; Iida, A; Suzuki, N; Konoshima, T; Tokuda, H

    2013-04-01

    In our continuing search for novel cancer chemopreventive compounds of natural and synthetic origin, we have evaluated 14 commonly used ultraviolet (UV) sunscreen agents (designated UV-1 to UV-14) for their skin cancer chemoprevention potential. They belong to 8 different chemical categories: aminobenzoate (UV-5, UV-7, UV-8 and UV-14), benzophenone (UV-1, UV-2, UV-3 and UV-13), benzotriazole (UV-10), benzyloxyphenol (UV-9), cinnamate (UV-6), quinolone (UV-4), salicylate (UV-11) and xanthone (UV-12). In the in vitro assay employed, the sunscreens were assessed by their inhibition of the Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in human lymphoblastoid Raji cells. All sunscreens tested were found to exhibit anti-tumour promoting activity: listed in decreasing order, moderate (UV-11, UV-2, UV-7, UV-12, UV-3, UV-9 and UV-14) to weak (UV-1, UV-6, UV-8, UV-16, UV-5, UV-4 and UV-10) with octyl salicylate (UV-11) as the most potent and drometrizole (UV-10) as the least potent among the compounds evaluated. A plausible relationship between the antioxidant property of sunscreens and their ability to promote anti-tumour activity was noted. The results call for a comprehensive analysis of skin cancer chemoprevention potential of currently used UV sunscreen agents around the globe to identify those with the best clinical profile.

  12. Field applications of an immunoradiometric assay for the detection of Plasmodium falciparum antigen in a population in a malaria-endemic area in Thailand.

    PubMed

    Khusmith, S; Tharavanij, S; Chongsa-Nguan, M; Vejvongvarn, C; Kasemsuth, R

    1988-01-01

    Two surveys were made in people living in a malaria-endemic area in West Thailand in October 1985 (a transmission season) and in May 1986 (a nontransmission season) to detect Plasmodium falciparum antigen using the immunoradiometric assay (IRMA). In the first survey involving 101 people, the IRMA-positive rate was 56.4% and then significantly declined to 16.5% during the second survey involving 79 people of the same group. The parasitological-positive rates were likewise decreased from 11.9% to 1.3% (P = 0.015) during these two seasons. IRMA-positive rates were significantly higher than the corresponding parasitological-positive rates (P less than 0.0001 and 0.002 for the first and the second surveys, respectively). The geometric mean IRMA binding activity of samples collected in the first survey (1,726 cpm) was significantly higher than those collected during the second survey (920 cpm, P = 0.001). Regression analysis showed that IRMA activities were linearly correlated with the parasite counts by microscopic examination (r = 0.629, P = 0.022). IRMA was specific for P. falciparum since all 30 healthy controls and 6 of 7 vivax malaria cases were negative. PMID:3277461

  13. A five-country evaluation of a point-of-care circulating cathodic antigen urine assay for the prevalence of Schistosoma mansoni.

    PubMed

    Colley, Daniel G; Binder, Sue; Campbell, Carl; King, Charles H; Tchuem Tchuenté, Louis-Albert; N'Goran, Eliézer K; Erko, Berhanu; Karanja, Diana M S; Kabatereine, Narcis B; van Lieshout, Lisette; Rathbun, Stephen

    2013-03-01

    We evaluated a commercial point-of-care circulating cathodic antigen (POC-CCA) test for assessing Schistosoma mansoni infection prevalence in areas at risk. Overall, 4,405 school-age children in Cameroon, Côte d'Ivoire, Ethiopia, Kenya, and Uganda provided urine for POC-CCA testing and stool for Kato-Katz assays. By latent class analysis, one POC-CCA test was more sensitive (86% versus 62%) but less specific (72% versus ~100%) than multiple Kato-Katz smears from one stool. However, only 1% of POC-CCA tests in a non-endemic area were false positives, suggesting the latent class analysis underestimated the POC-CCA specificity. Multivariable modeling estimated POC-CCA as significantly more sensitive than Kato-Katz at low infection intensities (< 100 eggs/gram stool). By linear regression, 72% prevalence among 9-12 year olds by POC-CCA corresponded to 50% prevalence by Kato-Katz, whereas 46% POC-CCA prevalence corresponded to 10% Kato-Katz prevalence. We conclude that one urine POC-CCA test can replace Kato-Katz testing for community-level S. mansoni prevalence mapping.

  14. High throughput quantitative reverse transcription PCR assays revealing over-expression of cancer testis antigen genes in multiple myeloma stem cell-like side population cells.

    PubMed

    Wen, Jianguo; Li, Hangwen; Tao, Wenjing; Savoldo, Barbara; Foglesong, Jessica A; King, Lauren C; Zu, Youli; Chang, Chung-Che

    2014-09-01

    Multiple myeloma (MM) stem cells, proposed to be responsible for the tumourigenesis, drug resistance and recurrence of this disease, are enriched in the cancer stem cell-like side population (SP). Cancer testis antigens (CTA) are attractive targets for immunotherapy because they are widely expressed in cancers but only in limited types of normal tissues. We designed a high throughput assay, which allowed simultaneous relative quantifying expression of 90 CTA genes associated with MM. In the three MM cell lines tested, six CTA genes were over-expressed in two and LUZP4 and ODF1 were universally up-regulated in all three cell lines. Subsequent study of primary bone marrow (BM) from eight MM patients and four healthy donors revealed that 19 CTA genes were up-regulated in SP of MM compared with mature plasma cells. In contrast, only two CTA genes showed a moderate increase in SP cells of healthy BM. Furthermore, knockdown using small interfering RNA (siRNA) revealed that LUZP4 expression is required for colony-forming ability and drug resistance in MM cells. Our findings indicate that multiple CTA have unique expression profiles in MM SP, suggesting that CTA may serve as targets for immunotherapy that it specific for MM stem cells and which may lead to the long-term cure of MM.

  15. Immunotherapy of HCC metastases with autologous T cell receptor redirected T cells, targeting HBsAg in a liver transplant patient.

    PubMed

    Qasim, Waseem; Brunetto, Maurizia; Gehring, Adam J; Xue, Shao-An; Schurich, Anna; Khakpoor, Atefeh; Zhan, Hong; Ciccorossi, Pietro; Gilmour, Kimberly; Cavallone, Daniela; Moriconi, Francesco; Farzhenah, Farzin; Mazzoni, Alessandro; Chan, Lucas; Morris, Emma; Thrasher, Adrian; Maini, Mala K; Bonino, Ferruccio; Stauss, Hans; Bertoletti, Antonio

    2015-02-01

    HBV-DNA integration frequently occurs in HBV-related hepatocellular carcinoma (HCC), but whether HBV antigens are expressed in HCC cells and can be targeted by immune therapeutic strategies remains controversial. Here, we first characterized HBV antigen expression in HCC metastases, occurring in a patient who had undergone liver transplantation for HBV-related HCC. We then deployed for the first time in HCC autologous T cells, genetically modified to express an HBsAg specific T cell receptor, as therapy against chemoresistant extrahepatic metastases. We confirmed that HBV antigens were expressed in HCC metastases (but not in the donor liver) and demonstrated that tumour cells were recognized in vivo by lymphocytes, engineered to express an HBV-specific T cell receptor (TCR). Gene-modified T cells survived, expanded and mediated a reduction in HBsAg levels without exacerbation of liver inflammation or other toxicity. Whilst clinical efficacy was not established in this subject with end-stage metastatic disease, we confirm the feasibility of providing autologous TCR-redirected therapy against HCC and advocate this strategy as a novel therapeutic opportunity in hepatitis B-associated malignancies.

  16. Circulating Hepatitis B Surface Antigen Particles Carry Hepatocellular microRNAs

    PubMed Central

    Novellino, Luisa; Rossi, Riccardo L.; Bonino, Ferruccio; Cavallone, Daniela; Abrignani, Sergio; Pagani, Massimiliano; Brunetto, Maurizia R.

    2012-01-01

    Hepatitis B virus (HBV) produces high quantities of subviral surface antigen particles (HBsAg) which circulate in the blood outnumbering virions of about 1\\103–6 times. In individuals coinfected with the defective hepatitis Delta virus (HDV) the small HDV-RNA-genome and Delta antigen circulate as ribonucleoprotein complexes within HBsAg subviral particles. We addressed the question whether subviral HBsAg particles may carry in the same way cellular microRNAs (miRNAs) which are released into the bloodstream within different subcellular forms such as exosomes and microvescicles. Circulating HBsAg particles were isolated from sera of 11 HBsAg carriers by selective immunoprecipitation with monoclonal anti-HBs-IgG, total RNA was extracted and human miRNAs were screened by TaqMan real-time quantitative PCR Arrays. Thirty-nine human miRNAs were found to be significantly associated with the immunoprecipitated HBsAg, as determined by both comparative DDCT analysis and non-parametric tests (Mann-Whitney, p<0.05) with respect to controls. Moreover immunoprecipitated HBsAg particles contained Ago2 protein that could be revealed in ELISA only after 0.5% NP40. HBsAg associated miRNAs were liver-specific (most frequent = miR-27a, miR-30b, miR-122, miR-126 and miR-145) as well as immune regulatory (most frequent = miR-106b and miR-223). Computationally predicted target genes of HBsAg-associated miRNAs highlighted molecular pathways dealing with host-pathogen The finding that HBsAg particles carry selective pools of hepatocellular miRNAs opens new avenues of research to disentangle the complex interactions between host and HBV and provides a non invasive tool to study the physiopathology of liver epigenetics. PMID:22470417

  17. Circulating hepatitis B surface antigen particles carry hepatocellular microRNAs.

    PubMed

    Novellino, Luisa; Rossi, Riccardo L; Bonino, Ferruccio; Cavallone, Daniela; Abrignani, Sergio; Pagani, Massimiliano; Brunetto, Maurizia R

    2012-01-01

    Hepatitis B virus (HBV) produces high quantities of subviral surface antigen particles (HBsAg) which circulate in the blood outnumbering virions of about 1\\10(3-6) times. In individuals coinfected with the defective hepatitis Delta virus (HDV) the small HDV-RNA-genome and Delta antigen circulate as ribonucleoprotein complexes within HBsAg subviral particles. We addressed the question whether subviral HBsAg particles may carry in the same way cellular microRNAs (miRNAs) which are released into the bloodstream within different subcellular forms such as exosomes and microvescicles. Circulating HBsAg particles were isolated from sera of 11 HBsAg carriers by selective immunoprecipitation with monoclonal anti-HBs-IgG, total RNA was extracted and human miRNAs were screened by TaqMan real-time quantitative PCR Arrays. Thirty-nine human miRNAs were found to be significantly associated with the immunoprecipitated HBsAg, as determined by both comparative DDCT analysis and non-parametric tests (Mann-Whitney, p<0.05) with respect to controls. Moreover immunoprecipitated HBsAg particles contained Ago2 protein that could be revealed in ELISA only after 0.5% NP40. HBsAg associated miRNAs were liver-specific (most frequent = miR-27a, miR-30b, miR-122, miR-126 and miR-145) as well as immune regulatory (most frequent = miR-106b and miR-223). Computationally predicted target genes of HBsAg-associated miRNAs highlighted molecular pathways dealing with host-pathogen. The finding that HBsAg particles carry selective pools of hepatocellular miRNAs opens new avenues of research to disentangle the complex interactions between host and HBV and provides a non invasive tool to study the physiopathology of liver epigenetics.

  18. Investigating the impact of hepatitis B virus surface gene polymorphism on antigenicity using ex vivo phenotyping.

    PubMed

    Ijaz, Samreen; Szypulska, Renata; Andrews, Nick; Tedder, Richard S

    2012-11-01

    The hepatitis B virus (HBV) surface antigen (HBsAg) is a complex protein, and understanding accurately the impact of amino acid changes on the antigenicity of the immunodominant a determinant must take this complexity into consideration. Epitope mapping with four mAbs was used to phenotype HBsAg directly from patients' sera to investigate the effect of mutations in their native genetic backbone. The expected mAb reactivity was established initially for samples harbouring 'wild-type' HBsAg sequences across genotypes A-E. The alteration of HBsAg antigenicity, defined by mAb epitope loss, was demonstrated in a number of samples with sequence-inferred amino acid changes. Individual mutations within the mapped epitopes to which the mAbs were directed usually affected their binding. However, the loss of more than one epitope was observed as the number of mutations within a sequence increased. Conversely, not all mutations occurring in the a determinant altered the HBsAg conformation. The genotype backbone, the specific amino acid substitution and amino acid changes occurring outside the major antigenic region appeared to be important in determining expression of the predicted epitope loss. These data clearly demonstrate that sequence-based methods alone may not accurately define HBsAg phenotype. This phenotyping methodology allows for the rapid and accurate identification of antigenically altered viruses and will greatly enhance current HBV surveillance, research and diagnostic activities. The data generated can be used to inform on public health issues relating to prevalence, transmission and impact of HBsAg mutants in HBV-infected populations. PMID:22855781

  19. Hepatitis B virus-like particles access major histocompatibility class I and II antigen presentation pathways in primary dendritic cells.

    PubMed

    Moffat, Jessica M; Cheong, Wan-Shoo; Villadangos, José A; Mintern, Justine D; Netter, Hans J

    2013-04-26

    Virus-like particles (VLPs) represent high density displays of viral proteins that efficiently trigger immunity. VLPs composed of the small hepatitis B virus envelope protein (HBsAgS) are useful vaccine platforms that induce humoral and cellular immune responses. Notably, however, some studies suggest HBsAgS VLPs impair dendritic cell (DC) function. Here we investigated HBsAgS VLP interaction with DC subsets and antigen access to major histocompatibility complex (MHC) class I and II antigen presentation pathways in primary DCs. HBsAgS VLPs impaired plasmacytoid DC (pDC) interferon alpha (IFNα) production in response to CpG in vitro, but did not alter conventional DC (cDC) or pDC phenotype when administered in vivo. To assess cellular immune responses, HBsAgS VLPs were generated containing the ovalbumin (OVA) model epitopes OVA(257-264) and OVA(323-339) to access MHCI and MHCII antigen presentation pathways, respectively; both in vitro and following immunisation in vivo. HBsAgS VLP-OVA(257-264) elicited CTL responses in vivo that were not enhanced by inclusion of an additional MHCII helper epitope. HBsAgS VLP-OVA(257-264) administered in vivo was cross-presented by CD8(+) DCs, but not CD8(-) DCs. Therefore, HBsAgS VLPs can deliver antigen to both MHCI and MHCII antigen presentation pathways in primary DCs and promote cytotoxic and helper T cell priming despite their suppressive effect on pDCs.

  20. HBsAg positivity during pregnancy and adverse maternal outcomes: a retrospective cohort analysis.

    PubMed

    Tan, J; Liu, X; Mao, X; Yu, J; Chen, M; Li, Y; Sun, X

    2016-10-01

    Hepatitis B virus infection characterized by HBsAg positivity during pregnancy is a well-recognized issue in developing countries, but the association between HBsAg positivity and adverse maternal outcomes remains uncertain. To examine the association between HBsAg positivity during pregnancy and adverse maternal outcomes, a retrospective cohort study was conducted in Sichuan province, China. Deliveries were recorded from six hospitals between 1 January 2009 and 31 December 2010. Pre-eclampsia, gestational diabetes mellitus (GDM), postpartum haemorrhage (PPH), intrahepatic cholestasis, Caesarean section and placenta previa were prespecified adverse maternal outcomes. We used two multivariate logistic regression models to assess the association between HBsAg positivity and adverse maternal outcomes. In total, 948 (4.2%) pregnant women were HBsAg positive from 22 374 deliveries. Pregnant women with positive HBsAg had higher risk of GDM (aOR1.41, 95%CI 1.15-1.74), PPH (1.44, 1.13-1.83), intrahepatic cholestasis (1.74, 1.40-2.16) and Caesarean section (1.24, 1.06-1.45). No statistical associations were found between HBsAg positivity and pre-eclampsia (1.36, 0.94-1.97), and placenta previa (1.21, 0.87-1.67). HBsAg positivity during pregnancy was associated with higher risk of multiple adverse maternal outcomes. Although the causality has yet to be established, efforts may be warranted in routine care, particularly in those with high risk for adverse maternal outcomes, given the volume population infected with HBsAg. Future studies are needed to establish causality and examine the impact of HBeAg on the adverse outcomes. PMID:27167604

  1. Establishment of enzyme-linked immunosorbent assay using purified recombinant 83-kilodalton antigen of Borrelia burgdorferi sensu stricto and Borrelia afzelii for serodiagnosis of Lyme disease.

    PubMed

    Rauer, S; Kayser, M; Neubert, U; Rasiah, C; Vogt, A

    1995-10-01

    The 83-kDa antigen of Borrelia burgdorferi was expressed as a recombinant protein in Escherichia coli and purified for use in an enzyme-linked immunosorbent assay (p83-ELISA). Antibodies to the 83-kDa antigen of both the immunoglobulin G (IgG) and IgM isotypes could be detected in all stages of Lyme disease. Sensitivity varied, depending on the clinical stage of illness. In early stages, as defined for 118 patients with erythema migrans, it was found to be 20% (24 of 118 patients: 7 with IgM, 16 with IgG, and 1 with IgM and IgG). Of the patients with late-stage Lyme arthritis and acrodermatitis chronica atrophicans, 94% (16 of 17:2 with IgM and IgG and 14 with IgG) and 86% (36 of 42:2 with IgG and IgM and 34 with IgG) revealed positive results in the p83-ELISA, respectively. p83 displays sequence heterogeneity according to the genomospecies, but when the reactions of serum specimens from acrodermatitis chronica atrophicans patients and arthritis patients with p83 derived from representative strains of B. burgdorferi sensu stricto and Borrelia afzelii in ELISAs were compared, no differences in specificity and sensitivity were seen. When 82 serum specimens from healthy controls were tested, none had IgG and only 3 (4%) had IgM antibodies, indicating a high specificity. Positive reactions with antibodies against Treponema pallidum (1 of 37 patients; IgG) and Epstein-Barr virus (1 of 44 patients; IgM) and with autoantibodies of various specificities (1 of 53 patients; IgG) were seen with < 3% of the serum samples te11111111111111111111 high speficicity for B. burgdorferi.2+ 13% for IgM antibodies, the IgM p83-ELISA provided little diagnostic information for Lyme disease, whereas the IgG p83-ELISA appears to be a suita ;e test for serodiagnosis of advanced-stage Lyme disease.

  2. Prevalence of hepatitis B surface antigen & its subtypes in high risk group subjects & voluntary blood donors in Bombay.

    PubMed

    Elavia, A J; Banker, D D

    1991-09-01

    HBsAg positive subjects belonging to high risk groups and voluntary blood donors were analysed for prevalence of HBsAg among various groups of subjects for ascertaining the carrier status among the voluntary blood donors, HBsAg subtype distribution, and association of HBsAg with blood groups and caste or religion. The prevalence of HBsAg varied from 2.02 per cent in voluntary blood donors to 58.38 per cent in patients of acute viral hepatitis. 70.5 per cent subjects had subtype 'ay' while 23.9 per cent of the subjects had subtype 'ad'. We also found compound 'ady' subtype in 5.6 per cent of our subjects. HBsAg/adr, a subtype not usually prevalent in India, was found in 30 of the 90 'ad' sera. Co-occurrence of HBsAg and anti-HBs was noted in 9 subjects. Homotypic anti-HBs was found to occur together mainly in voluntary blood donors, while heterotypic anti-HBs was found to occur together mainly multi-transfused patients. There was no significant correlation between HBsAg and blood group antigens and a relatively higher incidence of HBsAg among the Jain community was observed.

  3. Commercial Assay for Detection of Giardia lamblia and Cryptosporidium parvum Antigens in Human Fecal Specimens by Rapid Solid-Phase Qualitative Immunochromatography

    PubMed Central

    Garcia, Lynne S.; Shimizu, Robyn Y.; Novak, Susan; Carroll, Marilyn; Chan, Frank

    2003-01-01

    The ImmunoCard STAT! Cryptosporidium/Giardia rapid assay (Meridian Bioscience, Inc.) is a solid-phase qualitative immunochromatographic assay that detects and distinguishes between Giardia lamblia and Cryptosporidium parvum in aqueous extracts of human fecal specimens (fresh, frozen, unfixed, or fixed in 5 or 10% formalin or sodium acetate-acetic acid-formalin). By using specific antibodies, antigens specific for these organisms are isolated and immobilized on a substrate. After the addition of appropriate reagents, a positive test is detected visually by the presence of a gray-black color bar (regardless of the intensity) next to the organism name printed on the test device. A control is included in the device. Steps include tube preparation (buffer, patient specimen, conjugates A and B), testing (addition of sample onto the test device), and visual reading (total time, 12 min). Test performance was evaluated with known positive and negative stool specimens (170 specimens positive for Giardia and 231 specimens negative for Giardia) (85 specimens positive for Cryptosporidium and 316 specimens negative for Cryptosporidium); they were tested with trichrome, iron-hematoxylin, or modified acid-fast stains or the Meridian Bioscience, Inc., Giardia/Cryptosporidium Merifluor combination reagent; specimens with discrepant results were retested by using the Merifluor combination reagent. On the basis of the results of the reference methods, the sensitivities, specificities, and positive and negative predictive values were as follows: for G. lamblia, 93.5, 100, 100, and 95.5%, respectively; for C. parvum, 98.8, 100, 100, and 99.7%, respectively. False-negative results for G. lamblia were obtained with specimens with low parasite numbers (n = 7) or specimens containing trophozoites only (n = 3); one specimen with a false-negative result contained numerous cysts. The one specimen false negative for C. parvum was confirmed to be positive by immunofluorescence. No cross

  4. Relationship of spermatoscopy, prostatic acid phosphatase activity and prostate-specific antigen (p30) assays with further DNA typing in forensic samples from rape cases.

    PubMed

    Romero-Montoya, Lydia; Martínez-Rodríguez, Hugo; Pérez, Miguel Antonio; Argüello-García, Raúl

    2011-03-20

    In the forensic laboratory the biological analyses for rape investigation commonly include vaginal swabs as sample material combined to biochemical tests including sperm cytology (SC) and detection of acid phosphatase activity (AP) and prostate-specific antigen (PSA, p30) for the conclusive identification of semen components. Most reports comparing these tests relied on analysis of semen samples or donor swabs taken under controlled conditions; however their individual or combined efficacy under real live sampling conditions in different laboratories is largely unknown. We carried out SC, APA and PSA analyses in vaginal swabs collected from casework rapes submitted to Mexican Forensic Laboratories at Texcoco and Toluca. On the basis of positive and negative results from each assay and sample, data were classified into eight categories (I-VIII) and compared with those obtained in the two only similar studies reported in Toronto, Canada and Hong Kong, China. SC and APA assays had the higher overall positivity in Toluca and Texcoco samples respectively and otherwise PSA had a lower but very similar positivity between these two laboratories. When compared to the previous studies some similarities were found, namely similar frequencies (at a ratio of approximately 1 out of 3) of samples being positive or negative by all techniques (Categories I and VI respectively) and a comparable overall positivity of APA and SC but higher than that of PSA. Indeed the combined results of using SC, APA and PSA tests was considered as conclusive for semen detection from approximately 1 out of 3 cases (Category I) to approximately 1 out of 2 cases in a scenario where at least SC is positive, strongly presumptive in 2 out of 3 cases (with at least one test positive) and the remainder 1 out of 3 cases (Category VI) suggested absence of semen. By determining Y-STR polymorphisms (12-loci) in additional samples obtained at Toluca laboratory, complete DNA profiles were determined from all

  5. [A simple ELISA method for the detection of HBsAg: Organon Teknika HBsAg Uniform II screening and confirmation test. Comparative study using the HBsAg Hapanostika method. A multicenter study].

    PubMed

    Pár, A; Mihály, I; Kömives, K

    1994-09-25

    An one-step enzyme-linked immunoabsorbent method, named as HBsAg Uniform II has been described for the detection of serum HBsAg, and a comparison was made with a widely used ELISA technique HBsAg Hepanostika test, to evaluate sensitivity, specificity and reproducibility of the method. A total of 531 serum samples from patients with liver disease and with renal failure, as well as 1065 samples from blood donors have been investigated. While the sensitivity of Uniform II vs. Hepanostika was 99.5% vs. 72.7%, the specificity was 99.2% of both methods. The positive predictive values did not differ (99.5% vs. 99.2%), however, the negative predictive values were 99.2% vs. 71.7%, respectively, in favour of Uniform II test. The Uniform II confirmatory test confirmed the positive HBsAg results in 94%, this rate was 74% using Hepanostika system. The new method proved to be a simple, quick, reliable test, which can be useful as a valuable tool in both the clinical diagnosis and blood donor screening.

  6. Evaluation of saliva specimens as an alternative sampling method to detect hepatitis B surface antigen.

    PubMed

    Cruz, Helena Medina; da Silva, Elisangela Ferreira; Villela-Nogueira, Cristiane A; Nabuco, Letícia C; do Ó, Kycia Maria Rodrigues; Lewis-Ximenez, Lia Laura; Yoshida, Clara Fumiko Tachibana; Lampe, Elisabeth; Villar, Livia Melo

    2011-01-01

    In this study, a modified enzyme immunoassay (EIA) was evaluated for the Hepatitis B surface antigen (HBsAg) among whole saliva and oral fluid samples. Specimens were collected from 115 individuals who gave serum and oral fluid using Salivette (Sarstedt, Nümbrecht, Germany) and whole saliva. Saliva specimens were tested following a modified ELISA, and the results were compared with paired serum specimens that were tested according to the supplier's instructions. Transport buffer for the oral fluids, sample volume for assay, incubation period of sample with conjugate as well as cut-off values were evaluated to optimize the assay. The highest sensitivity and specificity were obtained by increasing the incubation of sample and conjugate to 16  hr and using the area under the receiver operating characteristic curve to calculate cut-off values. HBsAg was detected in 40 oral fluids and 44 whole saliva samples out of 47 paired positive serum specimens and not detected in 64 oral fluids and 63 whole saliva samples out of 68 matched negative sera samples by the ELISA assay. There was excellent agreement between the results for the serum and saliva specimens kappa value (κ): 0.80 for oral fluid and κ: 0.87 for whole saliva and there was excellent reproducibility. Using an optimized protocol, the sensitivities of whole saliva and oral fluid were 93.6 and 85.1%, respectively, whereas specificities of whole saliva and oral fluid were 92.6 and 94.1%, respectively. Our data showed a significant promise for the use of whole saliva and oral fluid together with the modified commercial EIA for Hepatitis B virus infection surveillance.

  7. Detection and estimation of avian infectious bronchitis virus antigen by a novel indirect liquid-phase blocking enzyme-linked immunosorbent assay using chicken and rabbit affinity purified immunoglobulins.

    PubMed

    Lougovskaia, Natalia N; Lougovskoi, Andrei A; Bochkov, Yuri A; Batchenko, Galina V; Mudrak, Natalia S; Drygin, Vladimir V; Borisov, Alexander V; Borisov, Vladimir V; Gusev, Anatoly A

    2002-12-01

    An indirect liquid-phase blocking (LPB) enzyme-linked immunosorbent assay (ELISA) using chicken and rabbit affinity purified immunoglobulin G (IgG) has been developed to detect and estimate avian infectious bronchitis virus (IBV) antigen concentration directly in infected allantoic fluid. The method is based on the principle of binding of specific IgG to the test IBV antigen and the assay of unbound IgG on an antigen-coated ELISA plate. The immunoglobulins are chicken N-terminal S2 peplomeric protein-specific IgG isolated by immunoaffinity chromatography on synthetic peptide coupled to CNBr-activated Sepharose 4B or rabbit polyclonal IgG purified from the serum using Protein A Sepharose 4B. The assay detected all tested IBV strains and field isolates propagated in chicken embryos. Signal to noise ratios were calculated from LPB ELISA absorbance units and a diagnostic threshold was established from the signal to noise ratio frequency distribution of samples positive or negative for IBV by virus titration or reverse transcription polymerase chain reaction. The relative sensitivity of the test ranged between 10(5) and 10(6) median egg infectious doses (EID(50)) for chicken IgG and between 10(3) and 10(4) EID(50) for rabbit IgG, depending on the test strain. The assay is simple and takes less than 3 h to perform. It does not require expensive reagents and can be readily adapted to monitor the IBV antigen concentration in allantoic fluids during propagation of vaccine strains or in samples of freeze-dried, live-attenuated IBV vaccines.

  8. Invader Assisted Enzyme-Linked Immunosorbent Assay for Colorimetric Detection of Disease Biomarkers Using Oligonucleotide Probe-Modified Gold Nanoparticles.

    PubMed

    Song, Qinxin; Qi, Xiemin; Jia, Huning; He, Liang; Kumar, Shalen; Pitman, Janet L; Zou, Bingjie; Zhou, Guohua

    2016-04-01

    We successfully developed an invader assisted ELISA assay (iaELISA) for sensitive detection of disease biomarkers. The method includes three key steps as follows; biotinylated detection antibody was at first used to capture targeted antigen by sandwich ELISA. The biotinylated oligonucleotide was then attached to detection antibody via streptavidin. Finally, the cascade invader reactions were employed to amplify the biotinylated oligonucleotide specific to the antigen so that detection of the antigen was transformed into signal amplification of the antigen-specific DNA. To achieve colorimetric detection, oligonucleotide probe and modified gold nanoparticles (AuNPs) were coupled with the invader assay. Utilization of the hairpin probes in the invader reaction brought about free AuNPs, resulting in the positive read-out (red color). On the other hand, aggregation of the AuNPs occurred when the hairpin probes were not utilized in the reaction. This method was successfully used to detect as low as 2.4 x 10(-11) g/mL of HBsAg by both naked eye and spectrophotometer. This sensitivity was about 100 times higher than that of conventional ELISA method. The method was also used to assay 16 serum specimens from HBV-infected patients and 8 serum specimens from HBV-negative donors and results were in good agreement with those obtained from the conventional ELISA. As the invader assay is sensitive to one base sequence, a good specificity was also obtained by detecting other antigens like hepatitis A virus (HAV) and BSA. The method has therefore much potential for ultrasensitive and cost-effective detection of targeted proteins that have clinical importance.

  9. Invader Assisted Enzyme-Linked Immunosorbent Assay for Colorimetric Detection of Disease Biomarkers Using Oligonucleotide Probe-Modified Gold Nanoparticles.

    PubMed

    Song, Qinxin; Qi, Xiemin; Jia, Huning; He, Liang; Kumar, Shalen; Pitman, Janet L; Zou, Bingjie; Zhou, Guohua

    2016-04-01

    We successfully developed an invader assisted ELISA assay (iaELISA) for sensitive detection of disease biomarkers. The method includes three key steps as follows; biotinylated detection antibody was at first used to capture targeted antigen by sandwich ELISA. The biotinylated oligonucleotide was then attached to detection antibody via streptavidin. Finally, the cascade invader reactions were employed to amplify the biotinylated oligonucleotide specific to the antigen so that detection of the antigen was transformed into signal amplification of the antigen-specific DNA. To achieve colorimetric detection, oligonucleotide probe and modified gold nanoparticles (AuNPs) were coupled with the invader assay. Utilization of the hairpin probes in the invader reaction brought about free AuNPs, resulting in the positive read-out (red color). On the other hand, aggregation of the AuNPs occurred when the hairpin probes were not utilized in the reaction. This method was successfully used to detect as low as 2.4 x 10(-11) g/mL of HBsAg by both naked eye and spectrophotometer. This sensitivity was about 100 times higher than that of conventional ELISA method. The method was also used to assay 16 serum specimens from HBV-infected patients and 8 serum specimens from HBV-negative donors and results were in good agreement with those obtained from the conventional ELISA. As the invader assay is sensitive to one base sequence, a good specificity was also obtained by detecting other antigens like hepatitis A virus (HAV) and BSA. The method has therefore much potential for ultrasensitive and cost-effective detection of targeted proteins that have clinical importance. PMID:27301208

  10. Comparison of Dissociation-Enhanced Lanthanide Fluorescent Immunoassays to Enzyme-Linked Immunosorbent Assays for Detection of Staphylococcal Enterotoxin B, Yersinia pestis-Specific F1 Antigen, and Venezuelan Equine Encephalitis Virus

    PubMed Central

    Smith, Darci R.; Rossi, Cynthia A.; Kijek, Todd M.; Henchal, Erik A.; Ludwig, George V.

    2001-01-01

    The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log10 concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories. PMID:11687442

  11. The supercritical CO₂ extract from the skin of Bufo bufo gargarizans Cantor blocks hepatitis B virus antigen secretion in HepG2.2.15 cells.

    PubMed

    Cui, Xiaoyan; Inagaki, Yoshinori; Wang, Dongliang; Gao, Jianjun; Qi, Fanghua; Gao, Bo; Kokudo, Norihiro; Fang, Dingzhi; Tang, Wei

    2014-02-01

    The skin of Bufo bufo gargarizans Cantor has long been used for the treatment of hepatitis B in China and supercritical carbon dioxide extraction (SC-CO₂) is widely used in extracting active ingredients from natural products. The aim of present study was to assess the anti-hepatitis B virus (HBV) effect of the supercritical CO₂ extract from the skin of Bufo bufo gargarizans Cantor (SCE-BC). Cytotoxicity of SCE-BC was analyzed using an MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] assay in HepG2.2.15 cells. The hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core-related antigen (HBcrAg) concentrations in cell culture medium were determined by chemiluminescent enzyme immunoassay. HBV mRNA in cells was determined using real-time polymerase chain reaction. SCE-BC concentrations below 10(-2) μg/mL had no significant toxicity to HepG2.2.15 cells. SCE-BC at 10(-4) μg/mL effectively inhibited the secretion of HBeAg by 23.36% on day 6. It was more potent than the positive control lamivudine (100 μg/mL) in terms of the inhibition of HBeAg and HBcrAg secretion on day 6. Consistent with the HBV antigen reduction, HBV mRNA expression was markedly inhibited in comparison to the control when HepG2.2.15 cells were treated with SCE-BC. Moreover, SCE-BC had greater inhibitory activity with respect to HBeAg than to HBsAg. Since HBeAg promotes immune tolerance and persistent infection during HBV infection, the present results suggest that immune tolerance induced by HBeAg might be overcome by SCE-BC. Therefore, SCE-BC warrants further investigation.

  12. An investigation into the antigenic cross-reactivity of Ophiophagus hannah (king cobra) venom neurotoxin, phospholipase A2, hemorrhagin and L-amino acid oxidase using enzyme-linked immunosorbent assay.

    PubMed

    Tan, N H; Lim, K K; Jaafar, M I

    1993-07-01

    The antigenic cross-reactivity of four Ophiophagus hannah (king cobra) venom components, the neurotoxin (OH-NTX), phospholipase A2 (OH-PLA2), hemorrhagin (OH-HMG) and L-amino acid oxidase (OH-LAAO) were examined by indirect and double sandwich ELISAs. The indirect ELISAs for OH-NTX, OH-PLA2 and OH-HMG were very specific when assayed against the various heterologous snake venoms and O. hannah venom components, at 25 ng/ml antigen level. At higher antigen concentrations (100-400 ng/ml), there were moderate to strong indirect ELISA cross-reactions between anti-O. hannah neurotoxin and venoms from various species of cobra as well as two short neurotoxins. However, anti-O. hannah hemorrhagin did not cross-react with any of the venoms tested, even at these high antigen concentrations, indicating that O. hannah hemorrhagin is antigenically very different from other venom hemorrhagins. Examination of the indirect ELISA cross-reactions between anti-O. hannah PLA2 and several elapid PLA2 enzymes suggests that the elapid PLA2 antigenic class has more than two subgroups. The antibodies to O. hannah L-amino acid oxidase, however, yielded indirect ELISA cross-reactions with many venoms as well as with OH-NTX, OH-PLA2 and OH-HMG, indicating that OH-LAAO shares common epitopes even with unrelated proteins. The double sandwich ELISAs for the four anti-O. hannah venom components, on the other hand, generally exhibited a higher degree of selectivity than the indirect ELISA procedure.

  13. Antigen detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter.

    PubMed

    Vassileva, A; Chugh, D A; Swaminathan, S; Khanna, N

    2001-06-01

    High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter. However, the time taken to reach peak product concentration is usually very long ( approximately 240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in

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

    PubMed

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

    2014-11-01

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

  16. [Basic and clinical evaluation of an immunoradiometric competitive inhibition assay for 2----6 sialyl Lewis a antigen--2. Evaluation of clinical significance].

    PubMed

    Imura, H; Takahashi, T; Matsuda, T; Yoshida, O; Ohkura, H; Seitetsu, Y; Seino, Y; Ishii, M; Kuwabara, M; Ariyoshi, Y

    1989-06-01

    The clinical significance of serum 2----6 sialyl Lewisa antigen was evaluated using "SLA 2----6 Otsuka" kits. Results indicated that the antigen was frequently elevated in sera obtained from patients with various cancers, including pancreas (73%), liver (67%), bilialy tract (66%), uterus (35%), and stomach (33%). Among other tumor markers examined, CA 19-9 (2----3 sialyl Lewisa) had a very similar cancer spectrum as 2----6 sialyl Lewisa. In the sera of patients with malignant disorders of digestive and respiratory organs, including stomach, intestine, pancreas, biliary tract and lung, the serum levels of CA19-9 tended to be higher than those of 2----6 sialyl Lewisa, usually exceeded those of CA19-9. This suggests that the 2----6 sialylation of Lewisa antigen is equally observed in malignant and non-malignant diseases, while the 2----3 sialylation is relatively specific to cancers. As a result, the ratio of the two antigens, CA 19-9: 2----6 sialyl Lewisa antigen ratio, exceeded 2.0 in most of the sera obtained from patients with malignancy, whereas the ratio was below 2.0 in most of patients with corresponding non-malignant diseases of those organs. The determination of the ratio may be clinically useful in the differential diagnosis of the malignant and non-malignant diseases in those organs.

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

    PubMed

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

    2013-11-15

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

  18. The HBsAg Prevalence Among Blood Donors From Eastern Mediterranean and Middle Eastern Countries: A Systematic Review and Meta-Analysis

    PubMed Central

    Babanejad, Mehran; Izadi, Neda; Najafi, Farid; Alavian, Seyed Moayed

    2016-01-01

    Context The world health organization (WHO) recommends that all blood donations should be screened for evidence of infections, such as hepatitis B. The present study aimed to determine the prevalence of hepatitis B surface antigen (HBsAg) in blood donors at the eastern Mediterranean region office (EMRO) of the WHO and middle eastern countries. Evidence Acquisition A meta-analysis was carried out based on the results of an electronic literature search of PubMed, Ovid, Scopus, and Google Scholar for articles published from January 1, 2000, to August 31, 2015. In accordance with a significant homogeneity test and a large value of I2, the random effects model was used to aggregate data from the studies and produce the pooled estimates using the “Metan” command. Results We included 66 eligible studies. The pooled prevalence of HBsAg in blood donors of both EMRO and middle eastern (E and M) countries was 2.03% (95% confidence interval [CI]: 1.79 – 2.26). In addition, the prevalence rates in the EMRO countries was 1.99% (95% CI: 1.84 – 2.14) and 1.62% in the Middle Eastern countries (95% CI: 1.36 – 1.88). The prevalence among blood donors with more than one study was 1.58% in Egypt, 0.58% in Iran, 0.67% in Iraq, 2.84% in Pakistan, 3.02% in Saudi Arabia, 1.68% in Turkey, and 5.05% in Yemen. Conclusions Based on the WHO classification of hepatitis B virus (HBV) prevalence, the prevalence of HBsAg in blood donors from E and M countries reached an intermediate level. However, there were low prevalence levels in some E and M countries. PMID:27226804

  19. Identification and evaluation of new Mycobacterium bovis antigens in the in vitro interferon gamma release assay for bovine tuberculosis diagnosis.

    PubMed

    Eirin, María E; Macias, Analia; Magnano, Gabriel; Morsella, Claudia; Mendez, Laura; Blanco, Federico C; Bianco, María V; Severina, Walter; Alito, Alicia; Pando, Maria de Los Angeles; Singh, Mahavir; Spallek, Ralph; Paolicchi, Fernando A; Bigi, Fabiana; Cataldi, Angel A

    2015-12-01

    Bovine tuberculosis (bTB) is a common zoonotic disease, caused by Mycobacterium bovis (M. bovis), responsible for significant economic losses worldwide. Its diagnosis is based on the detection of cell mediated immunity under the exposure to protein purified derivative tuberculin (PPD), a complex and poorly characterized reagent. The cross-reactivity to non-tuberculous mycobacterium species (false-positive results) has been crucial to develop a more proper antigen. In the present study, we selected six M. bovis Open Reading Frames (Mb1992, Mb2031c, Mb2319, Mb2843c, Mb2845c and Mb3212c) by in-silico analysis and evaluated them in experimental and natural infection; none of these antigens had been previously assessed as diagnostic antigens for bTB. The reactivity performance was tested in animals with both positive and negative Tuberculin Skin Test (TST) results as well as in cattle infected with Mycobacterium avium subesp. paratuberculosis (MAP). The six recombinant antigens individually induced an IFN-γ response, with overall responder frequency ranging from 18.3 to 31%. Mb2845c was the most valuable antigen with the potential to discriminate TST-positive cattle from either TST-negative or MAP infected animals. Mb2845c showed similar performance to that observed with ESAT-6 and PPD-B among TST and MTC specific-PCR positive animals, although this result needs to be proven in further studies with a higher sample size. Our data confirm the feacibility to implement bioinformatic screening tools and suggest Mb2845c as a potential diagnostic antigen to be tested in protein cocktails to evaluate their contribution to bTB diagnosis.

  20. Preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by double-antigen sandwich enzyme-linked immunosorbent assay.

    PubMed

    Jiao, Yongjun; Zeng, Xiaoyan; Guo, Xiling; Qi, Xian; Zhang, Xiao; Shi, Zhiyang; Zhou, Minghao; Bao, Changjun; Zhang, Wenshuai; Xu, Yan; Wang, Hua

    2012-02-01

    The recent emergence of the human infection confirmed to be caused by severe fever with thrombocytopenia syndrome virus (SFTSV) in China is of global concern. Safe diagnostic immunoreagents for determination of human and animal seroprevalence in epidemiological investigations are urgently needed. This paper describes the cloning and expression of the nucleocapsid (N) protein of SFTSV. An N-protein-based double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) system was set up to detect the total antibodies in human and animal sera. We reasoned that as the double-antigen sandwich ELISA detected total antibodies with a higher sensitivity than traditional indirect ELISA, it could be used to detect SFTSV-specific antibodies from different animal species. The serum neutralization test was used to validate the performance of this ELISA system. All human and animal sera that tested positive in the neutralization test were also positive in the sandwich ELISA, and there was a high correlation between serum neutralizing titers and ELISA readings. Cross-reactivity was evaluated, and the system was found to be highly specific to SFTSV; all hantavirus- and dengue virus-confirmed patient samples were negative. SFTSV-confirmed human and animal sera from both Anhui and Hubei Provinces in China reacted with N protein in this ELISA, suggesting no major antigenic variation between geographically disparate virus isolates and the suitability of this assay in nationwide application. ELISA results showed that 3.6% of the human serum samples and 47.7% of the animal field serum samples were positive for SFTSV antibodies, indicating that SFTSV has circulated widely in China. This assay, which is simple to operate, poses no biohazard risk, does not require sophisticated equipment, and can be used in disease surveillance programs, particularly in the screening of large numbers of samples from various animal species.

  1. Prostate-specific antigen as a biomarker of condom failure: comparison of three laboratory assays and self-reported condom use problems in a randomized trial of female condom performance☆, ☆☆

    PubMed Central

    Walsh, Terri; Warner, Lee; Macaluso, Maurizio; Frezieres, Ron; Snead, Margaret; Wraxall, Brian

    2015-01-01

    Background Prostate-specific antigen (PSA), a biomarker for semen exposure, may provide a more objective measure of condom failure than subject self-reports. Methods for measuring PSA vary and their comparability with respect to assessing condom performance has not been adequately evaluated. This study compared results from three different PSA assays of vaginal samples collected by subjects in a randomized clinical trial which compared the performance of female condoms. Study Design We selected 30 pairs of pre- and post-coital vaginal samples from subjects who reported condom functionality problems or whose original PSA assay was positive. Samples were retested using three different PSA assays [quantitative enzyme-linked immunoassay (EIA), rocket immune-electrophoresis (RIE) and chromatographic immunoassay (CIA)]. We compared the proportion of condom uses where the post-coital PSA result indicated semen exposure for each of the three assays. Results Despite varying levels of sensitivity, the results from all three assays were remarkably consistent. Self-reported condom failures did not correlate well with positive PSA results, suggesting that exclusive reliance on either PSA or user self-report may be inadequate for assessing condom functionality. Conclusion In combination with user self-report of condom failure, PSA testing provides a reliable, objective marker of condom functionality. Studies based on PSA testing may improve on conventional contraceptive clinical trials by offering a more direct assessment of a condom product's ability to prevent semen exposure. PMID:22386229

  2. Comparative Evaluation of the Novel bioNexia Legionella Test with the BinaxNOW Legionella Card Assay and the Sofia Legionella FIA Assay for Detection of Legionella pneumophila (Serogroup 1) Antigen in Urine Samples.

    PubMed

    Congestrì, Francesco; Crepaldi, Elisabetta; Gagliardi, Marina; Pedna, Maria Federica; Sambri, Vittorio

    2016-04-01

    A new immunochromatographic test (bioNexiaLegionella; bioMérieux) for the detection ofLegionella pneumophilaurinary antigen was evaluated in 255 urine samples. The results were compared with those obtained by the BinaxNOW and SofiaLegionellatests. The novel test compared well with those currently in use. PMID:26865691

  3. Performance of Bio-Rad and Limiting Antigen Avidity Assays in Detecting Recent HIV Infections Using the Quebec Primary HIV-1 Infection Cohort

    PubMed Central

    Serhir, Bouchra; Routy, Jean Pierre; Legault, Mario; Fauvel, Micheline

    2016-01-01

    Background Accurate and practical biologic tools to estimate HIV incidence is crucial to better monitor the epidemic and evaluate the effectiveness of HIV prevention and treatment programs. Methods We evaluated two avidity assays to measure recent HIV infection: the Sedia HIV-1 LAg-Avidity EIA (Sedia Biosciences, Portland) and the Centers for Disease Control and Prevention (CDC)-modified Bio-Rad-Avidity assay (Bio-Rad Laboratories, Mississauga, ON). Longitudinal specimens (n = 473) obtained from 123 treatment-naive seroconverted individuals enrolled in the Primary HIV-1 Infection (PHI) cohort of Quebec were used to determine the average time an individual is considered to be recently infected (mean duration of recent infection; MDRI), for the two avidity assays alone and in combination using a nonparametric survival method analysis. A total of 420 specimens from individuals with established HIV infection (90 individuals from the PHI cohort of Quebec and 330 individuals from the Laboratoire de santé publique du Quebec (LSPQ) serobank) were also tested to investigate false recency rate (FRR). Results The CDC-modified Bio-Rad-Avidity gave an estimated MDRI of 234 days (95% CI 220–249) at the avidity index cutoff of 30% while the Sedia-LAg-Avidity assay gave an estimated MDRI of 120 days (95% CI 109–132) at the normalized optical density (ODn) cutoff of 1.5. The FRR among individuals with established HIV infection was 10.2% (7.5%-13.5%) with the CDC-modified Bio-Rad-Avidity assay as compared to 6.0% (3.9%-8.7%) with the Sedia-LAg-Avidity assay. When optimizing a multiassay algorithm (MAA) that includes sequentially the CDC-modified Bio-Rad-Avidity assay then the Sedia-LAg-Avidity assay EIA (avidity index/ODn: 30%/1.7), the MDRI was 136 days (95% CI 123–148) and the FRR, 3.3% (95% CI 1.8–5.6). Conclusion Multiassay algorithms that include the CDC-modified Bio-Rad-Avidity assay and the Sedia-LAg-Avidity assay performed better than each avidity assay alone. Such

  4. Detection of Giardia lamblia and Cryptosporidium parvum Antigens in Human Fecal Specimens Using the ColorPAC Combination Rapid Solid-Phase Qualitative Immunochromatographic Assay

    PubMed Central

    Garcia, Lynne S.; Shimizu, Robyn Y.

    2000-01-01

    The ColorPAC Giardia/Cryptosporidium (Becton Dickinson) is a solid-phase qualitative immunochromatographic assay that detects and distinguishes between Giardia lamblia and Cryptosporidium parvum in human stool. Agreement between the Alexon-Trend ProSpecT Giardia Rapid EIA and the ColorPAC assay was 166 of 172 (96.5%). Agreement between the Alexon-Trend ProSpecT Cryptosporidium Rapid EIA and the ColorPAC assay was 169 of 171 (98.8%). No cross-reactions were seen with other parasites or human cells. PMID:10699038

  5. Rough lipopolysaccharide of Brucella abortus RB51 as a common antigen for serological detection of B. ovis, B. canis, and B. abortus RB51 exposure using indirect enzyme immunoassay and fluorescence polarization assay.

    PubMed

    Nielsen, K; Smith, P; Conde, S; Draghi de Benitez, G; Gall, D; Halbert, G; Kenny, K; Massengill, C; Muenks, Q; Rojas, X; Perez, B; Samartino, L; Silva, P; Tollersrud, T; Jolley, M

    2004-01-01

    Rough lipopolysaccharide (RLPS) antigens were prepared from cultures of Brucella abortus RB51, B. ovis, and B. canis. The preparations were standardized by weight and tested with sera from cattle immunized with B. abortus RB51, sheep infected with B. ovis, and dogs infected with B. canis. Populations of unexposed animals of each species were also tested. The tests used were the indirect enzyme immunoassay (IELISA) using RLPS and the fluorescence polarization assay (FPA) using RLPS core fractions, labeled with fluorescein isothiocyanate. The IELISA using B. abortus RB51 RLPS antigen resulted in sensitivity and specificity values of 94.8% and 97.3%, respectively, when testing bovine sera, 98.5% and 97.8% when testing ovine sera, and 95.8% and 100% when testing dog sera. The IELISA using B. ovis RLPS antigen gave sensitivity and specificity values of 80.5% and 91.7%, respectively with bovine sera, 98.9% and 93.8% with sheep sera, and 70.8% and 79.8% with dog sera. The IELISA using B. canis RLPS antigen resulted in sensitivity and specificity values of 97.0% and 97.4%, respectively, with bovine sera, 96.2% and 96.3% with sheep sera, and 95.8% and 98.8% with dog sera. Labeling RLPS core from B. ovis and B. canis with fluorescein was not successful. B. abortus RB51 core labeled with fluorescein resulted in sensitivity and specificity values of 93.5% and 99.8%, respectively, with bovine sera and 78.1% and 99.0% with sheep sera. It was not possible to test the dog sera in the FPA.

  6. Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope.

    PubMed Central

    Bonino, F; Heermann, K H; Rizzetto, M; Gerlich, W H

    1986-01-01

    Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d. Images PMID:3701932

  7. Application of Elephant TB STAT-PAK assay and MAPIA (multi-antigen print immunoassay) for detection of tuberculosis and monitoring of treatment in black rhinoceros (Diceros bicornis).

    PubMed

    Duncan, Ann E; Lyashchenko, Konstantin; Greenwald, Rena; Miller, Michelle; Ball, Ray

    2009-12-01

    Many wildlife species including rhinos are susceptible to infection with Mycobacterium tuberculosis or M. bovis. Antemortem diagnostic testing in large exotic hoof stock species has been limited by challenges associated with test administration, sample collection, and interpretation. Hence, a simple, rapid, blood-based test is needed. Two confirmed M. tuberculosis-infected black rhinoceros and one exposed suspect were evaluated for antibody responses using a lateral-flow rapid test (ElephantTB STAT-PAK) and multi-antigen print immunoassay (MAPIA). All three animals were seropositive by both tests. MAPIA detected antibodies to ESAT-6, CFP10, and MPB83 antigens. When the rhinos were treated with antitubercular therapeutics, their antibody responses gradually declined. One rhinoceros died approximately 9 mo after initiation of treatment and showed an increase in antibody titer shortly before death. The other two rhinoceros, which were treated for 1 and 2 yr, respectively, had no clinical signs or positive culture for M. tuberculosis at the time of necropsy performed 2 or 6 yr later for unrelated reasons. The antibody levels in these rhinos continued to be significantly decreased. The findings suggest that the ElephantTB STAT-PAK and MAPIA may be useful tools to detect M. tuberculosis infection and monitor treatment in black rhinoceros. PMID:20063826

  8. Clinical significance of hepatitis B surface antigen mutants

    PubMed Central

    Coppola, Nicola; Onorato, Lorenzo; Minichini, Carmine; Di Caprio, Giovanni; Starace, Mario; Sagnelli, Caterina; Sagnelli, Evangelista

    2015-01-01

    Hepatitis B virus (HBV) infection is a major public health problem in many countries, with nearly 300 million people worldwide carrying HBV chronic infection and over 1 million deaths per year due to cirrhosis and liver cancer. Several hepatitis B surface antigen (HBsAg) mutations have been described, most frequently due to a single amino acid substitution and seldom to a nucleotide deletion. The majority of mutations are located in the S region, but they have also been found in the pre-S1 and pre-S2 regions. Single amino acid substitutions in the major hydrophilic region of HBsAg, called the “a” determinant, have been associated with immune escape and the consequent failure of HBV vaccination and HBsAg detection, whereas deletions in the pre-S1 or pre-S2 regions have been associated with the development of hepatocellular carcinoma. This review article will focus on the HBsAg mutants and their biological and clinical implications. PMID:26644816

  9. Practical guidelines for assessing patients positive for hepatitis B surface antigen.

    PubMed Central

    Feinman, S. V.; Berris, B.; Sinclair, J. C.; Wrobel, D. W.

    1976-01-01

    In assessing the patient the hepatitis B surface antigen (HBsAg) the physician must decide on the basis of physical findings, results of laboratory tests and biopsy, when indicated, whether the patient is an asymptomatic carrier or has acute or chronic hepatitis. Asymptomatic carriers of HBsAg must be educated in personal hygiene and the possibility of transmission, should not be allowed to donate blood or breast-feed and should not work with blood products for human use or pharmaceutical products designated for intravenous use. However, it is otherwise not necessary to advise these individuals to change their profession. PMID:1032589

  10. Analysis of Mycobacterium avium subsp. paratuberculosis Antigens Used in an In-house Enzyme-linked Immunosorbent Assay for Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed a novel enzyme-linked immunosorbent assay (ELISA), called EVELISA, for the detection of MAP infection in cattle which showed a higher sensitivity than current ELISA test. We previously reported that the use of heat-killed M. flavascens for pre-absorption of cross-reactive antibodies im...

  11. Relationship of factor VIII-like antigen (VIII AGN) and clot promoting acitivty (VIII AHF) as measured by one- and two-stage assays in patients with liver disease.

    PubMed

    Rogers, J S; Eyster, E

    1976-12-01

    We recently observed a increase in factor-VIII clot promoting activity as measured by a one-stage assay (VIII AHF) in a haemophiliac with hepatitis. However, VIII AHF as measured by a two-stage assay (VIII AHF) was 0.013 u/ml at a time when VIII AHF measured 0.38 u/ml. We then studied seven non-haemophiliacs with liver disease, and attempted to correlate the lvels of VIII AHF and VIII AHF with factor VIII-like antigen (VIII AGN) as measured by quantitative immunoelectrophoresis. In four of the seven patients, disproportionate elevations of VIII AHF compared to VIII AHF were found. Furthermore, VIII AHF values correlated well with VIII AGN vales . No such discrepancy was apparent in four normal control subjects. These findings emphasize the necessity for performing two-stage assays in haemophiliacs as well as non-haemophiliacs with liver disease to assess factor-VIII levels. In addition, they suggest that confirmation of the diagnosis of haemophilia may not be possible in the haemophiliac with hepatitis unless VIII AHF determinations are performed. The reason for the disparity between VIII ahf and VIII AHF levels is not apparent. However, the correlation of VIII AGN and VIII AHF levels in the non-haemophiliacs with liver disease provides further support for the concept that VIII AGN and VIII AHF are closely related or identical molecular entities.

  12. Recombinant Antigens rLipL21, rLoa22, rLipL32 and rLigACon4-8 for Serological Diagnosis of Leptospirosis by Enzyme-Linked Immunosorbent Assays in Dogs

    PubMed Central

    Ye, Cuilian; Yan, Weiwei; Xiang, Hua; He, Hongxuan; Yang, Maosheng; Ijaz, Muhammad; Useh, Nicodemus; Hsieh, Ching-Lin; McDonough, Patrick L.; McDonough, Sean P.; Mohamed, Hussni; Yang, Zhibang; Chang, Yung-Fu

    2014-01-01

    Animal leptospirosis is one of the most common zoonotic diseases in the United States and around the world. In a previous study, we applied four recombinant antigens, rLipL21, rLoa22, rLipL32 and rLigACon4-8 of Leptospira interrogans (L. interrogans) for the serological diagnosis of equine leptospirosis (Ye et al, Serodiagnosis of equine leptospirosis by ELISA using four recombinant protein markers, Clin. Vaccine. Immunol. 21:478–483). In this study, the same four recombinant antigens were evaluated for their potential to diagnose canine leptospirosis by ELISA. A total of 305 canine sera that were Leptospira microscopic agglutination test (MAT)-negative (n = 102) and MAT-positive (n = 203) to 5 serovars (Pomona, Grippotyphosa, Icterohaemorrhagiae, Canicola and Hardjo) were tested. When individual recombinant antigens were used, the sensitivity and specificity of ELISA were 97.5% and 84.3% for rLigACon4-8; 89.7% and 81.4% for rLoa22; 92.6% and 84.3% for rLipL32 and 99.5% and 84.3% for rLipL21, respectively compared to the MAT. The sensitivity and specificity of ELISA were, 92.6% and 91.2% for rLigACon4-8 and rLipL32, 97.5% and 84.3% for rLigACon4-8 and rLipL21, 89.7% and 87.3% for rLigACon4-8 and rLoa22, 89.7% and 87.3% to rLipL21 and rLoa22, 92.6% and 91.2% for rLipL21 and rLipL32 and 89.2% and 94.1% for rLoa22 and rLipL32 when one of the two antigens was test positive. The use of all four antigens in the ELISA assay was found to be sensitive and specific, easy to perform, and agreed with the results of the standard Leptospira Microscopic Agglutination test (MAT) for the diagnosis of canine leptospirosis. PMID:25526513

  13. Recombinant antigens rLipL21, rLoa22, rLipL32 and rLigACon4-8 for serological diagnosis of leptospirosis by enzyme-linked immunosorbent assays in dogs.

    PubMed

    Ye, Cuilian; Yan, Weiwei; Xiang, Hua; He, Hongxuan; Yang, Maosheng; Ijaz, Muhammad; Useh, Nicodemus; Hsieh, Ching-Lin; McDonough, Patrick L; McDonough, Sean P; Mohamed, Hussni; Yang, Zhibang; Chang, Yung-Fu

    2014-01-01

    Animal leptospirosis is one of the most common zoonotic diseases in the United States and around the world. In a previous study, we applied four recombinant antigens, rLipL21, rLoa22, rLipL32 and rLigACon4-8 of Leptospira interrogans (L. interrogans) for the serological diagnosis of equine leptospirosis (Ye et al, Serodiagnosis of equine leptospirosis by ELISA using four recombinant protein markers, Clin. Vaccine. Immunol. 21:478-483). In this study, the same four recombinant antigens were evaluated for their potential to diagnose canine leptospirosis by ELISA. A total of 305 canine sera that were Leptospira microscopic agglutination test (MAT)-negative (n = 102) and MAT-positive (n = 203) to 5 serovars (Pomona, Grippotyphosa, Icterohaemorrhagiae, Canicola and Hardjo) were tested. When individual recombinant antigens were used, the sensitivity and specificity of ELISA were 97.5% and 84.3% for rLigACon4-8; 89.7% and 81.4% for rLoa22; 92.6% and 84.3% for rLipL32 and 99.5% and 84.3% for rLipL21, respectively compared to the MAT. The sensitivity and specificity of ELISA were, 92.6% and 91.2% for rLigACon4-8 and rLipL32, 97.5% and 84.3% for rLigACon4-8 and rLipL21, 89.7% and 87.3% for rLigACon4-8 and rLoa22, 89.7% and 87.3% to rLipL21 and rLoa22, 92.6% and 91.2% for rLipL21 and rLipL32 and 89.2% and 94.1% for rLoa22 and rLipL32 when one of the two antigens was test positive. The use of all four antigens in the ELISA assay was found to be sensitive and specific, easy to perform, and agreed with the results of the standard Leptospira Microscopic Agglutination test (MAT) for the diagnosis of canine leptospirosis. PMID:25526513

  14. Antigenic properties of human and animal bloodstains studied by enzyme-linked immunosorbent assay (ELISA) using various antisera against specific plasma proteins.

    PubMed

    Tsutsumi, H; Htay, H H; Sato, K; Katsumata, Y

    1987-01-01

    Antigenic properties of bloodstains of human and non-human primates as well as other animal bloodstains were investigated by the inhibition ELISA using commercially available anti-human albumin (Alb), alpha 2-macroglobulin (alpha 2-M), fibrinogen, transferrin, and immunoglobulin G. In general, chimpanzee bloodstains showed strong cross-reactions with these antisera, and the extent of the cross-reactions of other animal bloodstains decreased largely with the phylogenic order, i.e., agile gibbon (ape), Old World monkeys (Japanese monkey and hamadryas baboon), New World monkeys (night monkey and tufted capuchin monkey), prosimians (grand galago and ring-tailed lemur) and other animals (rat, cattle, swine, goat, dog, cat, and chicken). Among these antisera, anti-human alpha 2-M showed the weakest cross-reaction with chimpanzee bloodstains, and anti-human Alb showed next. PMID:2448970

  15. Impacts of the G145R Mutation on the Structure and Immunogenic Activity of the Hepatitis B Surface Antigen: A Computational Analysis

    PubMed Central

    Rezaee, Reza; Poorebrahim, Mansour; Najafi, Saeideh; Sadeghi, Solmaz; Pourdast, Alieh; Alavian, Seyed Moayed; Alavian, Seyed Ehsan; Poortahmasebi, Vahdat

    2016-01-01

    Background Vaccine-escaped hepatitis B virus (HBV) mutations occur within the “a” determinant area, which is located in the major hydrophilic region (MHR) of the hepatitis B surface antigen (HBsAg) protein. It is now well established that the common G145R mutation is highly capable of escaping from HBsAg immune recognition. However, the impacts of this mutation on the structure and immunogenic activity of HBsAg have been poorly investigated. Objectives The present study analyzed the effects of the G145R mutation on the structure and immunogenic activity of the HBsAg. Materials and Methods Three-dimensional (3D) structure of HBsAg for both the wild-type and G145R mutant were predicted and refined using several web tools. After quantitative evaluations, the effects of the G145R mutation on the secondary and 3D structures of the HBsAg were investigated. In parallel, the immunogenic activity of the wild-type and mutant HBsAg was also analyzed using a ClusPro docking server as well as the IEDB web tool. Further analyses were performed via molecular dynamics (MD) simulations using the GROMACS v5.0.2 simulation package. Results The G145R mutation causes a considerable reduction in the immunogenic activity of the HBsAg through a conformational change in the HBsAg antigenic loops. This mutation inserts a new β-strand in the “a” determinant region of the HBsAg, leading to a reduced binding affinity to its monoclonal antibody, MAb12. The G145R mutation also increased the compactness and stability of the HBsAg by enhancing the rigidity of the “a” determinant. Conclusions These data will be beneficial for designing more advanced antibodies for the recognition of the HBsAg in diagnostics. In addition, the results of this study may assist in the design or development of more effective hepatitis B vaccines. PMID:27642350

  16. Monoclonal antibodies to surface antigens of Mycobacterium tuberculosis and their use in a modified enzyme-linked immunosorbent spot assay for detection of mycobacteria.

    PubMed Central

    Glatman-Freedman, A; Martin, J M; Riska, P F; Bloom, B R; Casadevall, A

    1996-01-01

    Three monoclonal antibodies (MAbs) were generated from splenocytes of a BALB/c mouse immunized with heat-killed Mycobacterium tuberculosis. All three MAbs bound to surface epitopes of M. tuberculosis as shown by whole-cell enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and immunoelectron microscopy. One immunoglobulin M (IgM) MAb bound to lipoarabinomannan, the second IgM MAb bound to mycolyl-arabinogalactan-peptidoglycan complex, and the third MAb, an IgG3, bound to a surface epitope of an uncertain nature. The MAbs demonstrated different cross-reactivity patterns with other mycobacteria. Two of the MAbs were used to develop a modified ELISA spot assay for the detection of mycobacteria. PMID:8897185

  17. Diagnostic performance of a cytokine and IFN-γ-induced chemokine mRNA assay after Mycobacterium tuberculosis-specific antigen stimulation in whole blood from infected individuals.

    PubMed

    Kim, Sunghyun; Lee, Hyejon; Kim, Hyunjung; Kim, Yeun; Cho, Jang-Eun; Jin, Hyunwoo; Kim, Dae Yeon; Ha, Sang-Jun; Kang, Young Ae; Cho, Sang-Nae; Lee, Hyeyoung

    2015-01-01

    Interferon (IFN)-γ release assays have limited sensitivity and cannot differentiate between active tuberculosis (TB) disease and latent TB infection (LTBI). Numerous cytokines and regulator factors have been implicated in the pathogenesis and control of Mycobacterium tuberculosis infection. Additional cytokines and chemokines associated with M. tuberculosis infection may improve the performance of IFN-γ release assays. We developed a real-time RT-PCR TaqMan assay for targeting levels of eight human targets [IFN-γ, tumor necrosis factor (TNF)-α, IL-2R, IL-4, IL-10, CXCL9, CXCL10, and CXCL11] and evaluated the assay with three different study groups. Results showed that the sensitivity of TNF-α, IL-2R, and CXCL10 in the active pulmonary tuberculosis (PTB) group was 96.43%, 96.43%, and 100%, respectively. The sensitivity of IL-2R and CXCL10 in the latent tuberculosis infection group was 86.36% and 81.82%, respectively. Statistical results showed that TNF-α and CXCL9 were the best individual markers for differentiating between the PTB, LTBI, and non-TB groups. For optimal sensitivity and differentiation of M. tuberculosis infection status, the simultaneous detection of multiple targets was attempted. The combination of IFN-γ, TNF-α, and IL-2R, and the combination of TNF-α, IL-2R, CXCL9, and CXCL10 showed the best performance for detecting active PTB (both 100% positivity) and LTBI (86.36% and 81.82% positivity, respectively). These results imply that the combination of suitable markers is useful in efficiently diagnosing TB and differentiating M. tuberculosis infection status.

  18. Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen.

    PubMed

    Bannai, Hiroshi; Nemoto, Manabu; Tsujimura, Koji; Yamanaka, Takashi; Maeda, Ken; Kondo, Takashi

    2016-02-01

    To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.

  19. Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen

    PubMed Central

    BANNAI, Hiroshi; NEMOTO, Manabu; TSUJIMURA, Koji; YAMANAKA, Takashi; MAEDA, Ken; KONDO, Takashi

    2015-01-01

    To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA. PMID:26424485

  20. Recombinant 46-kilodalton surface antigen (P46) of Mycoplasma hyopneumoniae expressed in Escherichia coli can be used for early specific diagnosis of mycoplasmal pneumonia of swine by enzyme-linked immunosorbent assay.

    PubMed

    Futo, S; Seto, Y; Okada, M; Sato, S; Suzuki, T; Kawai, K; Imada, Y; Mori, Y

    1995-03-01

    The 46-kDa surface antigen (P46) is the early and species-specific immunogenic protein of Mycoplasma hyopneumoniae. Three TGA codons encoding tryptophan in the P46 gene were replaced with TGG by an in vitro mutagenesis technique. The mutated P46 gene was expressed in Escherichia coli by using the chelating peptide tag system. The purified recombinant P46 was successfully used in an enzyme-linked immunosorbent assay for detection of antibodies against M. hyopneumoniae in swine serum. It did not cross-react with sera from swine infected with Mycoplasma flocculate, Mycoplasma hyorhinis, or Mycoplasma hyosynoviae. With this method, mycoplasmal pneumonia of swine was detectable within 2 weeks after infection. PMID:7751376

  1. Recombinant 46-kilodalton surface antigen (P46) of Mycoplasma hyopneumoniae expressed in Escherichia coli can be used for early specific diagnosis of mycoplasmal pneumonia of swine by enzyme-linked immunosorbent assay.

    PubMed Central

    Futo, S; Seto, Y; Okada, M; Sato, S; Suzuki, T; Kawai, K; Imada, Y; Mori, Y

    1995-01-01

    The 46-kDa surface antigen (P46) is the early and species-specific immunogenic protein of Mycoplasma hyopneumoniae. Three TGA codons encoding tryptophan in the P46 gene were replaced with TGG by an in vitro mutagenesis technique. The mutated P46 gene was expressed in Escherichia coli by using the chelating peptide tag system. The purified recombinant P46 was successfully used in an enzyme-linked immunosorbent assay for detection of antibodies against M. hyopneumoniae in swine serum. It did not cross-react with sera from swine infected with Mycoplasma flocculate, Mycoplasma hyorhinis, or Mycoplasma hyosynoviae. With this method, mycoplasmal pneumonia of swine was detectable within 2 weeks after infection. PMID:7751376

  2. Expression of the hepatitis B virus surface antigen in Drosophila S2 cells

    PubMed Central

    Santos, Alexandra S.; Spina, Ângela; Pereira, Carlos A.

    2008-01-01

    Drosophila melanogaster S2 cells were transfected with a plasmid vector (pAcHBsAgHy) containing the S gene, coding for the hepatitis B virus surface antigen (HBsAg), under control of the constitutive drosophila actin promoter (pAc), and the hygromycin B (Hy) selection gene. The vector was introduced into Schneider 2 (S2) Drosophila cells by DNA transfection and a cell population (S2AcHBsAgHy) was selected by its resistance to hygromycin B. The pAcHBsAgHy vector integrated in transfected S2 cell genome and approximately 1,000 copies per cell were found in a higher HBsAg producer cell subpopulation. The HBsAg production varied in different subpopulations, but did not when a given subpopulation was cultivated in different culture flasks. Higher HBsAg expression was found in S2AcHBsAgHy cells cultivated in Insect Xpress medium (13.5 μg/1E7 cells) and SFX medium (7 μg/1E7 cells) in comparison to SF900II medium (0.6 μg/1E7 cells). An increase of HBsAg was observed in culture maintained under hygromycin selection pressure. Data presented in the paper show that S2AcHBsAgHy cells produce efficiently the HBsAg which is mainly found in the cell supernatant, suggesting that HBsAg is secreted from the cells. The data also show that our approach using the Drosophila expression system is suitable for the preparation of other viral protein preparation. PMID:19003172

  3. Prevalence of hepatitis B surface antigen among refugees entering the United States between 2006 and 2008.

    PubMed

    Rein, David B; Lesesne, Sarah B; O'Fallon, Ann; Weinbaum, Cindy M

    2010-02-01

    The Centers for Disease Control and Prevention recommends hepatitis B surface antigen (HBsAg) testing to identify chronic hepatitis B virus infection for foreign-born persons from countries or regions with HBsAg prevalence of >or=2%. However, limited data exist to indicate which countries meet this definition. To address this data gap, we estimated the HBsAg prevalence among refugees entering the United States between 2006 and 2008. We contacted state refugee health coordinators and asked them to report the number of refugees, country of origin, and HBsAg prevalence among refugees screened in their jurisdiction during the most recently available 12-month period prior to August 2008. We pooled data across jurisdictions and calculated the prevalence for any country with more than 30 refugees entering the United States, and where this level of data was not available by country, continents were considered. Of the 47 jurisdictions contacted, we received basic information from 31, with nine jurisdictions reporting HBsAg prevalence by country of origin applicable to 31,980 refugees (approximately 42% of refugees entering the United States during the observation period). We estimated an HBsAg prevalence of 2.8% (95% confidence interval 2.6%-3.0%) for refugees overall. Of the 37 countries with 30 or more refugees entering the United States, 25 had a prevalence of >or=2%. Prevalence was highest among refugees from Africa and Southeast Asia, and lowest among refugees from the Middle East and South/Central America. In the eight countries for which we had comparison data, six had lower HBsAg prevalence than in 1991.

  4. Prostate Specific Antigen/Solvent Interaction Analysis (PSA/SIA): A Preliminary Evaluation of a New Assay Concept for Detecting Prostate Cancer Using Urinary Samples

    PubMed Central

    Stovsky, Mark; Ponsky, Lee; Vourganti, Srinivas; Stuhldreher, Peter; Siroky, Mike B.; Kipnis, Victor; Fedotoff, Olga; Mikheeva, Larissa; Zaslavsky, Boris; Chait, Arnon; Jones, J. Stephen

    2011-01-01

    Objectives To provide preliminary clinical performance evaluation of a novel CaP assay, PSA/SIA (Solvent Interaction Analysis) that focused on changes to the structure of PSA. Methods 222 men undergoing prostate biopsy for accepted clinical criteria at three sites (University Hospitals Case Medical Center in Cleveland, Cleveland Clinic, and Veterans Administration Boston Healthcare System) were enrolled in IRB approved study. Prior to TRUS guided biopsy, patients received DRE with systematic prostate massage followed by collection of urine. The PSA/SIA assay determined the relative partitioning of heterogeneous PSA isoform populations in urine between two aqueous phases. A structural index, K, whose numerical value is defined as the ratio of the concentration of all PSA isoforms, was determined by total PSA ELISA and used to set a diagnostic threshold for CaP. Performance was assessed using ROC analysis with biopsy as the gold standard. Results Biopsies were pathologically classified as case (malignant, n=100) or control (benign, n=122). ROC performance demonstrated AUC=0.90 for PSA/SIA and 0.58 for serum tPSA. At a cutoff value of K=1.73, PSA/SIA displayed sensitivity=100%, specificity=80.3%, PPV=80.6%, and NPV=100%. No attempt was made in this preliminary study to further control patient population or selection criteria for biopsy, nor did we analytically investigate the type of structural differences in PSA that led to changes in K value. Conclusions PSA/SIA provides ratiometric information independently of PSA concentration. In this preliminary study, analysis of the overall structurally heterogeneous PSA isoform population using the SIA assay showed promising results to be further evaluated in future studies. PMID:21783231

  5. Combining rapid diagnostic tests and dried blood spot assays for point-of-care testing of human immunodeficiency virus, hepatitis B and hepatitis C infections in Burkina Faso, West Africa.

    PubMed

    Kania, D; Bekalé, A M; Nagot, N; Mondain, A-M; Ottomani, L; Meda, N; Traoré, M; Ouédraogo, J B; Ducos, J; Van de Perre, P; Tuaillon, E

    2013-12-01

    People screened for human immunodeficiency virus (HIV) using rapid diagnostic tests (RDTs) in Africa remain generally unaware of their status for hepatitis B (HBV) and hepatitis C (HCV) infections. We evaluated a two-step screening strategy in Burkina Faso, using both HIV RDTs and Dried Blood Spot (DBS) assays to confirm an HIV-positive test, and to test for HBV and HCV infections. HIV counselling and point-of-care testing were performed at a voluntary counselling and testing centre with HBV, HCV status and HIV confirmation using DBS specimens, being assessed at a central laboratory. Serological testing on plasma was used as the reference standard assay to control for the performance of DBS assays. Nineteen out of 218 participants included in the study were positive for HIV using RDTs. A fourth-generation HIV ELISA and immunoblot assays on DBS confirmed HIV status. Twenty-four out of 25 participants infected with HBV were found positive for hepatitis B surface antigen (HBsAg) using DBS. One sample with a low HBsAg concentration on plasma was not detected on DBS. Five participants tested positive for HCV antibodies were confirmed positive with an immunoblot assay using DBS specimens. Laboratory results were communicated within 7 days to participants with no loss to follow up of participants between the first and second post-test counselling sessions. In conclusion, DBS collection during HIV point-of-care testing enables screening and confirmation of HBV, HCV and HIV infections. Diagnosis using DBS may assist with implementation of national programmes for HBV, HCV and HIV screening and clinical care in middle- to low-income countries. PMID:23902574

  6. HBsAg detection by passive hemagglutination (Hepanosticon--Organon). Advantages and disadvantages in comparison with other methods.

    PubMed

    Balş, M G; Hagiescu, L

    1976-01-01

    Investigated comparatively with immunodiffusion, electroimmunodiffusion, complement fixation and Latex agglutination, passive hemagglutination with the Hepanosticon--Organon reagent proved to be an easy, rapid, highly reproducible method for HBsAg detection.

  7. Preparation and characterization of human monoclonal antibodies directed against the hepatitis B virus surface antigen.

    PubMed

    Colucci, G; Kohtz, D S; Waksal, S D

    1986-06-01

    The hepatitis B surface antigen (HBsAg) is highly immunogenic and induces an antibody response which is protective in vivo against hepatitis B virus (HBV) infection. Human monoclonal antibodies specific for HBsAg were produced, which could have potential therapeutic applications. Lymphocytes obtained from a vaccinated donor were stimulated in vitro and fused with the human myeloma cell line GM 4672, and eight hybridomas were obtained. Three of these clones, which reacted in an ELISA against the HBsAg vaccine, were expanded, subcloned and further analyzed. The subclones E7C2, C4C10, and D5B2 were able to bind to different HBsAg preparations, which express various subtypes, and recognized the major HBsAg peptides in Western blot analysis. Cross-inhibition experiments showed that E7C2, C4C10 and D5B2 are directed against the same epitope and have an affinity constant ranging from 5 X 10(7) to 3.3 X 10(9) M. Furthermore, these antibodies stained the surface and cytoplasm of the HBsAg-secreting cell lines PLC/PRF/5 and 4.10. The production of immunoglobulins varies from 0.3 to 1.3 micrograms/ml/10(6) and has remained stable over a period of 8 months. These human monoclonal antibodies, which appear to be directed against an antigenic determinant common to all HBsAg subtypes, could be useful in the study of HBV-related liver diseases as well as in their diagnosis and experimental therapy.

  8. Liver grafts from hepatitis B surface antigen-positive donors: A review of the literature.

    PubMed

    Loggi, Elisabetta; Conti, Fabio; Cucchetti, Alessandro; Ercolani, Giorgio; Pinna, Antonio Daniele; Andreone, Pietro

    2016-09-21

    The scarcity of available organs and the gap between supply and demand continue to be the main limitations of liver transplantation. To relieve the organ shortage, current transplant strategies have implemented extended criteria, which include the use of liver from patients with signs of past or present hepatitis B virus (HBV) infection. While the use of liver grafts from donors with evidence of past HBV infection is quite limited, some data have been collected regarding the feasibility of transplanting a liver graft from a hepatitis B surface antigen (HBsAg) positive donor. The aim of the present work was to review the literature regarding liver transplants from HBsAg-positive donors. A total of 17 studies were identified by a search in Medline. To date, HBsAg positive grafts have preferentially been allocated to HBsAg positive recipients. The large majority of these patients continue to be HBsAg positive despite the use of immunoglobulin, and infection prevention can only be guaranteed by using antiviral prophylaxis. Although serological persistence is evident, no significant HBV-related disease has been observed, except in patients coinfected with delta virus. Consistently less data are available for HBsAg negative recipients, although they are mostly promising. HBsAg-positive grafts could be an additional organ source for liver transplantation, provided that the risk of reinfection/reactivation is properly prevented. PMID:27672295

  9. Liver grafts from hepatitis B surface antigen-positive donors: A review of the literature

    PubMed Central

    Loggi, Elisabetta; Conti, Fabio; Cucchetti, Alessandro; Ercolani, Giorgio; Pinna, Antonio Daniele; Andreone, Pietro

    2016-01-01

    The scarcity of available organs and the gap between supply and demand continue to be the main limitations of liver transplantation. To relieve the organ shortage, current transplant strategies have implemented extended criteria, which include the use of liver from patients with signs of past or present hepatitis B virus (HBV) infection. While the use of liver grafts from donors with evidence of past HBV infection is quite limited, some data have been collected regarding the feasibility of transplanting a liver graft from a hepatitis B surface antigen (HBsAg) positive donor. The aim of the present work was to review the literature regarding liver transplants from HBsAg-positive donors. A total of 17 studies were identified by a search in Medline. To date, HBsAg positive grafts have preferentially been allocated to HBsAg positive recipients. The large majority of these patients continue to be HBsAg positive despite the use of immunoglobulin, and infection prevention can only be guaranteed by using antiviral prophylaxis. Although serological persistence is evident, no significant HBV-related disease has been observed, except in patients coinfected with delta virus. Consistently less data are available for HBsAg negative recipients, although they are mostly promising. HBsAg-positive grafts could be an additional organ source for liver transplantation, provided that the risk of reinfection/reactivation is properly prevented.

  10. Liver grafts from hepatitis B surface antigen-positive donors: A review of the literature

    PubMed Central

    Loggi, Elisabetta; Conti, Fabio; Cucchetti, Alessandro; Ercolani, Giorgio; Pinna, Antonio Daniele; Andreone, Pietro

    2016-01-01

    The scarcity of available organs and the gap between supply and demand continue to be the main limitations of liver transplantation. To relieve the organ shortage, current transplant strategies have implemented extended criteria, which include the use of liver from patients with signs of past or present hepatitis B virus (HBV) infection. While the use of liver grafts from donors with evidence of past HBV infection is quite limited, some data have been collected regarding the feasibility of transplanting a liver graft from a hepatitis B surface antigen (HBsAg) positive donor. The aim of the present work was to review the literature regarding liver transplants from HBsAg-positive donors. A total of 17 studies were identified by a search in Medline. To date, HBsAg positive grafts have preferentially been allocated to HBsAg positive recipients. The large majority of these patients continue to be HBsAg positive despite the use of immunoglobulin, and infection prevention can only be guaranteed by using antiviral prophylaxis. Although serological persistence is evident, no significant HBV-related disease has been observed, except in patients coinfected with delta virus. Consistently less data are available for HBsAg negative recipients, although they are mostly promising. HBsAg-positive grafts could be an additional organ source for liver transplantation, provided that the risk of reinfection/reactivation is properly prevented. PMID:27672295

  11. Antibody-capture enzyme-linked immunosorbent assays that use enzyme-labelled antigen for detection of virus-specific immunoglobulin M, A and G in patients with varicella or herpes zoster.

    PubMed Central

    van Loon, A. M.; van der Logt, J. T.; Heessen, F. W.; Heeren, M. C.; Zoll, J.

    1992-01-01

    Antibody-capture enzyme-linked immunosorbent assays (AC-ELISA) which use enzyme-labelled antigen were developed for detection of varicella-zoster virus-(VZV) specific IgM, IgA and IgG antibody in patients with varicella or herpes zoster and in sera from healthy individuals. All 18 patients with varicella developed a VZV-IgM and a VZV-IgG response, 17 also a VZV-IgA response. In contrast, all 19 patients with herpes zoster were shown to be positive for VZV-IgA whereas only 13 of these reacted positively for VZV-IgM. A VZV-IgM response was detected in only two sera from 100 healthy individuals and an IgA response in only one. The presence of virus-specific IgA and IgG in the cerebrospinal fluid as determined by AC-ELISA was a useful indicator of VZV infection of the central nervous system. By AC-ELISA, VZV-IgG was detected predominantly in sera from patients with acute or recent VZV infection. Only 14 sera from 100 healthy individuals were positive for VZV-IgG by AC-ELISA, whereas all were positive by an indirect ELISA. These results indicate that AC-ELISA's may be useful assays for determination for acute or recurrent VZV infection, but are not suitable for determination of past infection with this virus. PMID:1312479

  12. Detection of infections with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus by analyses of dried blood spots - performance characteristics of the ARCHITECT system and two commercial assays for nucleic acid amplification

    PubMed Central

    2013-01-01

    Background Nowadays, dried blood spots (DBS) are primarily used to obtain diagnostic access to risk collectives such as intravenous drug users, who are prone to infections with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Before DBS analyses can be used in this diagnostic context, however, a comprehensive evaluation of its performance characteristics must be conducted. To the best of our knowledge, the current study presents for the first time such essential data for the Abbott ARCHITECT system, which is currently the worldwide leading platform in this field of infection diagnostics. Methods The investigation comprised 1,762 paired serum/DBS samples and a total of 3,524 determinations with the Abbott ARCHITECT HBsAg, anti-HBc, anti-HBs, anti-HCV and HIV-1-p24-antigen/anti-HIV 1/2 assays as well as with the artus HBV LC PCR and VERSANT HCV RNA qualitative (TMA) tests. Results In the context of DBS testing, a specificity of 100% was recorded for the seven serological and molecular biological assays. The analytical sensitivity of HBsAg, anti-HBc, anti-HBs, anti-HCV, HIV-1-p24-antigen/anti-HIV 1/2, HBV DNA, and HCV RNA detections in DBS eluates was 98.6%, 97.1%, 97.5%, 97.8%, 100%, 93%, and 100%, respectively. Discussion/conclusions The results obtained indicate that it is today possible to reliably detect HBsAg, anti-HBc, anti-HBs, anti-HCV and HIV-1-p24 antigen/anti-HIV 1/2 with state-of-the-art analytical systems such as the Abbott ARCHITECT in DBS eluates even when a comparatively high elution volume of 1,000 μl is used. They also provide evidence for the inherent analytical limits of DBS testing, which primarily concern the anti-HBc/anti-HBs system for individuals with HIV infections and nucleic acid tests with relatively low analytical sensitivity. PMID:23497102

  13. A solid phase enzyme-linked immunosorbent assay for the antigenic detection of Legionella pneumophila (serogroup 1): A compliment for the space station diagnostic capability

    NASA Technical Reports Server (NTRS)

    Hejtmancik, Kelly E.

    1987-01-01

    It is necessary that an adequate microbiology capability be provided as part of the Health Maintenance Facility (HMF) to support expected microbial disease events and environmental monitoring during long periods of space flight. The application of morphological and biochemical studies to confirm the presence of certain bacterial and fungal disease agents are currently available and under consideration. This confirmation would be facilitated through employment of serological methods to aid in the identification of bacterial, fungal, and viral agents. A number of serological approaches are currently being considered, including the use of Enzyme Linked Immunosorbent Assay (ELISA) technology, which could be utilized during microgravity conditions. A solid phase, membrane supported ELISA for the detection of Legionella pneumophila, an expected disease agent, was developed to show a potential model system that would meet the HMF requirements and specifications for the future space station. These studies demonstrate the capability of membrane supported ELISA systems for identification of expected microbial disease agents as part of the HMF.

  14. Immunocapture assay for quantification of human IgA antibodies to parasite antigenic enzymes. Application with the alkaline phosphatase of Schistosoma mansoni.

    PubMed

    Lien, D N; Cesari, I M; Bouty, I; Bout, D; Hoebeke, J

    1992-01-01

    Conditions are described for using solid phase adsorbed jacalins in an immunocapture assay for IgA antibodies to the alkaline phosphatase of Schistosoma mansoni. Microtiter plates were activated with polylysine and jacalins were covalently adsorbed by means of glutaraldehyde. From three different jacalins, the one purified from seeds of Artocarpus tonkinensis showed the lowest non-specific adsorption and was used for further studies. Comparing solutions of bovine serum albumin, ovalbumin and Tween 20, it was shown that the latter was most successful in blocking non-specific adsorption. Low serum dilutions resulted in a less efficient IgA capture by the adsorbed jacalin than higher dilutions. Under optimal working conditions, a high correlation could be shown between the presence of specific anti-alkaline phosphatase antibodies of IgA isotype and IgG isotype.

  15. The importance of platelets in the expression of monocyte tissue factor antigen measured by a new whole blood flow cytometric assay.

    PubMed

    Amirkhosravi, A; Alexander, M; May, K; Francis, D A; Warnes, G; Biggerstaff, J; Francis, J L

    1996-01-01

    Previous methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean +/- SD) expression of monocyte TF in normal subjects was very low (1.1 +/- 0.95%, Mean Fluorescence [Mean FL] 0.20 +/- 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 +/- 11.2% (Mean FL 0.32 +/- 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment.

  16. Pyridoxamine-5-phosphate enzyme-linked immune mass spectrometric assay substrate for linear absolute quantification of alkaline phosphatase to the yoctomole range applied to prostate specific antigen.

    PubMed

    Florentinus-Mefailoski, Angelique; Marshall, John G

    2014-11-01

    There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap.

  17. The importance of platelets in the expression of monocyte tissue factor antigen measured by a new whole blood flow cytometric assay.

    PubMed

    Amirkhosravi, A; Alexander, M; May, K; Francis, D A; Warnes, G; Biggerstaff, J; Francis, J L

    1996-01-01

    Previous methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean +/- SD) expression of monocyte TF in normal subjects was very low (1.1 +/- 0.95%, Mean Fluorescence [Mean FL] 0.20 +/- 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 +/- 11.2% (Mean FL 0.32 +/- 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment. PMID:8713785

  18. A single dose of oral DNA immunization delivered by attenuated Salmonella typhimurium down-regulates transgene expression in HBsAg transgenic mice.

    PubMed

    Zheng, Bo Jian; Ng, Mun Hon; Chan, Kwok Wah; Tam, Sidney; Woo, Patrick C Y; Ng, Sze Park; Yuen, Kwok Yung

    2002-11-01

    The efficacy of immunization with Salmonella typhimurium aroA to deliver the plasmid pRc/CMV-HBsAg (i.e. an oral DNA vaccine) was compared with that of intramuscular immunization with the same plasmid DNA, and with recombinant HBsAg protein, in a HBsAg transgenic mouse model. A single dose of oral DNA vaccine evoked vigorous Th1 cell and CTL responses and production of IgG2 subclass of anti-HBs after 2 weeks, and this was accompanied by a transient hepatitic flare with elevated alanine aminotransferase in the first 3 weeks. Concomitantly, the level of HBsAg-mRNA in liver tissues decreased by more than fourfold and viral-antigen expression was curtailed markedly in hepatocytes compared with controls. Hepatitic flare subsided after 3 weeks, but suppression of the transgene expression was continued in the absence of overt liver pathology for the remaining duration of the experiment (i.e. 12 weeks), and possibly beyond. The other vaccines could also break immune tolerance, but this was achieved only after repeated booster doses of the respective vaccines, and they did not affect transgene expression, or induce hepatic flare. We previously showed in non-transgenic mice that immunization by the oral DNA vaccine is achieved by an active intestinal infection with a bacterial carrier that is an adept intracellular parasite, and the immune response to the vaccination is orchestrated by phagocytic APC. Our present findings further implicated that the combined effects of an innate and a specific immune response induced by oral DNA vaccination are crucial in down-regulating HBsAg-transgene expression in hepatocytes.

  19. Competitive binding inhibition enzyme-linked immunosorbent assay that uses the secreted aspartyl proteinase of Candida albicans as an antigenic marker for diagnosis of disseminated candidiasis.

    PubMed

    Morrison, Christine J; Hurst, Steven F; Reiss, Errol

    2003-09-01

    The secreted aspartyl proteinases (Saps) of Candida albicans have been implicated as virulence factors associated with adherence and tissue invasion. The potential use of proteinases as markers of invasive candidiasis led us to develop a competitive binding inhibition enzyme-linked immunosorbent assay (ELISA) to detect Sap in clinical specimens. Daily serum and urine specimens were collected from rabbits that had been immunosuppressed with cyclophosphamide and cortisone acetate and infected intravenously with 10(7) C. albicans blastoconidia. Disseminated infection was confirmed by organ culture and histopathology. Although ELISA inhibition was observed when serum specimens from these rabbits were used, more significant inhibition, which correlated with disease progression, occurred when urine specimens were used. Urine collected as early as 1 day after infection resulted in significant ELISA inhibition (mean inhibition +/- standard error [SE] compared with preinfection control urine, 15.7% +/- 2.7% [P < 0.01]), and inhibition increased on days 2 through 5 (29.4% +/- 4.8% to 44.5% +/- 3.5% [P < 0.001]). Urine specimens from immunosuppressed rabbits infected intravenously with Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Aspergillus fumigatus, or Staphylococcus aureus were negative in the assay despite culture-proven dissemination. Nonimmunosuppressed rabbits receiving oral tetracycline and gentamicin treatment were given 2 x 10(8) C. albicans blastoconidia orally or intraurethrally to establish colonization of the gastrointestinal tract or bladder, respectively, without systemic dissemination; urine specimens from these rabbits also gave negative ELISA results. Dissemination to the kidney and spleen occurred in one rabbit challenged by intragastric inoculation, and urine from this rabbit demonstrated significant inhibition in the ELISA (mean inhibition +/- SE by day 3 after infection, 32.9% +/- 2.7% [P < 0.001]). The overall

  20. Effect of a polysaccharide from Poria cocos on humoral response in mice immunized by H1N1 influenza and HBsAg vaccines.

    PubMed

    Wu, Yajun; Li, Shuai; Li, Haixia; Zhao, Chunzhi; Ma, Hao; Zhao, Xiunan; Wu, Junhua; Liu, Kunlu; Shan, Junjie; Wang, Yuxia

    2016-10-01

    Poria cocos has a long history of medicinal use in China. Polysaccharides and their derivatives in the medicine exhibit many beneficial biological activities including anticancer, anti-inflammatory, antioxidant and antiviral activities. In this study, a new polysaccharide (PCP-II) was isolated from sclerotium of Poria cocos. Its physico-chemical characters were identified and its adjuvant activity was investigated in mice co-immunized with H1N1 influenza vaccine and hepatitis B surface antigen (HBsAg). The results revealed that PCP-II has a molecular weight of 29.0kDa. It was composed of fucose, mannose, glucose and galactose in molar ration of 1.00:1.63:0.16:6.29 respectively. Pharmacological data demonstrated that PCP-II increased antigen-specific antibody levels in mice immunized with influenza vaccine. PCP-II also elicited anti-HBsAg antibodies at significantly higher titers and generated robust and durable immunity compared to mice immunized with HBsAg-alum following two administrations. PCP-II improved proliferation of splenocytes, stimulated IL-12p70 and TNF-α productions in dendritic cells and macrophages respectively. These results suggested that PCP-II-adjuvanted vaccines enhanced humoral and cellular immunity. PCP-II could be developed as an efficacious adjuvant in human and animal vaccines. PMID:27185068

  1. Effect of a polysaccharide from Poria cocos on humoral response in mice immunized by H1N1 influenza and HBsAg vaccines.

    PubMed

    Wu, Yajun; Li, Shuai; Li, Haixia; Zhao, Chunzhi; Ma, Hao; Zhao, Xiunan; Wu, Junhua; Liu, Kunlu; Shan, Junjie; Wang, Yuxia

    2016-10-01

    Poria cocos has a long history of medicinal use in China. Polysaccharides and their derivatives in the medicine exhibit many beneficial biological activities including anticancer, anti-inflammatory, antioxidant and antiviral activities. In this study, a new polysaccharide (PCP-II) was isolated from sclerotium of Poria cocos. Its physico-chemical characters were identified and its adjuvant activity was investigated in mice co-immunized with H1N1 influenza vaccine and hepatitis B surface antigen (HBsAg). The results revealed that PCP-II has a molecular weight of 29.0kDa. It was composed of fucose, mannose, glucose and galactose in molar ration of 1.00:1.63:0.16:6.29 respectively. Pharmacological data demonstrated that PCP-II increased antigen-specific antibody levels in mice immunized with influenza vaccine. PCP-II also elicited anti-HBsAg antibodies at significantly higher titers and generated robust and durable immunity compared to mice immunized with HBsAg-alum following two administrations. PCP-II improved proliferation of splenocytes, stimulated IL-12p70 and TNF-α productions in dendritic cells and macrophages respectively. These results suggested that PCP-II-adjuvanted vaccines enhanced humoral and cellular immunity. PCP-II could be developed as an efficacious adjuvant in human and animal vaccines.

  2. Mass Spectrometry and Multiplex Antigen Assays to Assess Microbial Quality and Toxin Production of Staphylococcus aureus Strains Isolated from Clinical and Food Samples

    PubMed Central

    Attien, Paul; Sina, Haziz; Moussaoui, Wardi; Zimmermann-Meisse, Gaëlle; Dadié, Thomas; Keller, Daniel; Riegel, Philippe; Edoh, Vincent; Kotchoni, Simeon O.; Djè, Marcellin; Prévost, Gilles

    2014-01-01

    The aim of our study was to investigate the microbial quality of meat products and on some clinical samples in Abidjan focused on Staphylococcus genus and the toxin production profile of Staphylococcus aureus (S. aureus) isolated. Bacteria were collected from 240 samples of three meat products sold in Abidjan and 180 samples issued from clinical infections. The strains were identified by both microbiological and MALDI-TOF-MS methods. The susceptibility to antibiotics was determined by the disc diffusion method. The production of Panton-Valentine Leukocidin, LukE/D, and epidermolysins was screened using radial gel immunodiffusion. The production of staphylococcal enterotoxins and TSST-1 was screened by a Bio-Plex Assay. We observed that 96/240 of meat samples and 32/180 of clinical samples were contaminated by Staphylococcus. Eleven species were isolated from meats and 4 from clinical samples. Forty-two S. aureus strains were isolated from ours samples. Variability of resistance was observed for most of the tested antibiotics but none of the strains displays a resistance to imipenem and quinolones. We observed that 89% of clinical S. aureus were resistant to methicillin against 58% for those issued from meat products. All S. aureus isolates issued from meat products produce epidermolysins whereas none of the clinical strains produced these toxins. The enterotoxins were variably produced by both clinical and meat product samples. PMID:24987686

  3. Delta hepatitis agent: structural and antigenic properties of the delta-associated particle.

    PubMed Central

    Bonino, F; Hoyer, B; Shih, J W; Rizzetto, M; Purcell, R H; Gerin, J L

    1984-01-01

    Delta agent (delta) was serially passaged to a second and third hepatitis B surface antigen (HBsAg) carrier chimpanzee, using as inoculum the peak delta antigen (delta Ag) serum of an animal previously infected with human serum. The characteristics of serially transmitted delta Ag were similar to those described in first-passage animals. It was consistently detected before the development of anti-delta, in association with a 35- to 37-nm subpopulation of HBsAg particles and a unique low-molecular-weight (5.5 X 10(5)) RNA. RNase susceptibility of the delta-associated RNA and release of delta Ag activity upon treatment of delta-associated particles with detergent revealed that this particle is organized into a virion-like form with the RNA and delta Ag as internal components within a coat of HBsAg. Surface determinants of the delta-associated particle other than HBsAg were not detected by radioimmunoprecipitation experiments, using sera of humans and chimpanzees convalescent from delta hepatitis. The HBsAg-associated particle is the "candidate agent" of delta hepatitis. Images PMID:6698598

  4. Correlation between hepatitis B virus surface antigen level and alpha-fetoprotein in patients free of hepatocellular carcinoma or severe hepatitis.

    PubMed

    Akuta, Norio; Suzuki, Fumitaka; Kobayashi, Mariko; Hara, Tasuku; Sezaki, Hitomi; Suzuki, Yoshiyuki; Hosaka, Tetsuya; Kobayashi, Masahiro; Saitoh, Satoshi; Ikeda, Kenji; Kumada, Hiromitsu

    2014-01-01

    Alfa-fetoprotein (AFP) is used as a marker of early hepatocarcinogenesis. However, the impact of hepatitis B virus surface antigen (HBsAg) on this relationship in patients with HBV infection is not clear. The present study evaluated the relation between HBsAg and AFP levels at the initial visit in 1,610 untreated HBV patients, free of hepatocellular carcinoma (HCC) or severe hepatitis. The cumulative rate of HCC was significantly lower in patients with a low AFP level (≤10 µg/L; below the upper limit of normal) than in those with a high AFP level (≥11 µg/L) at the initial visit. In patients with HBsAg levels more than 500 IU/ml, HBsAg levels correlated significantly and negatively with AFP levels, and significantly with platelet count. Multivariate analysis of data of patients with HBsAg more than 500 IU/ml identified HBsAg (<7,000 IU/ml), albumin (<3.9 g/dl), platelet count (<20.0 × 10(4) /mm(3)), gamma-glutamyl transpeptidase (≥50 IU/L), aspartate aminotransferase (≥34 IU/L), HBeAg (positive), and HBV core-related antigen (≥3.0 log U/ml) as determinants of a high AFP. Especially, in patients with HBsAg more than 500 IU/ml and low transaminase levels (below the upper limit of normal), HBsAg was identified as significant determinant of a high AFP. On the other hand, in patients with HBsAg less than 500 IU/ml, multivariate analysis identified albumin, gamma-glutamyl transpeptidase, and HBV core-related antigen as determinants of a high AFP. The results indicated that HBsAg level seems to affect, at least in part, the AFP levels, and that it can be used as a surrogate marker of early hepatocarcinogenesis.

  5. Frequency analysis of HBsAg-specific B lymphocytes in high-responder individuals to recombinant hepatitis B vaccine: comparison of LDA and ELISPOT assays.

    PubMed

    Shokrgozar, M A; Sam, M R; Amirkhani, A; Shokri, F

    2006-11-01

    The determination of the frequency of antigen-specific lymphocytes may provide invaluable information for the evaluation of the immune response to different antigens and pathogens. Different methods are employed to determine the frequency of specific B lymphocytes in peripheral blood. In this study, the frequency of hepatitis B surface antigen (HBsAg)-specific B lymphocytes was determined by a limiting dilution assay (LDA) and an enzyme-linked immunospot assay (ELISPOT) in seven healthy adult high responders to recombinant HBsAg. Peripheral blood mononuclear cells isolated at different time intervals (1, 2, 4, 8 and 16 weeks) following administration of a booster dose were either transformed with Epstein-Barr virus (LDA) or stimulated with Pokeweed mitogen (ELISPOT). In an LDA, anti-HBs positive wells were screened by a sandwich ELISA and the frequency of specific B lymphocytes was estimated based on the Poisson statistical analysis. In an ELISPOT, coloured spots representing specific B lymphocytes were finally enumerated by stereomicroscope. Our results showed a significant increase in the number of specific B lymphocytes in the first week by an ELISPOT compared with an LDA (1:190 versus 1:13,462) (P < 0.001). No significant differences were observed at other time intervals. A significant correlation was observed between the serum titer of anti-HBs antibody and frequency of HBsAg-specific B cells obtained by LDA and ELISPOT methods at different time intervals. The highest correlation was found at fourth week in LDA (r = 0.83, P < 0.01) and ELISPOT (r = 0.85, P < 0.01) assays. Furthermore, a significant correlation was observed between an LDA and ELISPOT at different time intervals (highest correlation in second week, r = 0.88, P < 0.008). These findings suggest that in addition to technical advantages, such as speed and simplicity, an ELISPOT is a more sensitive assay, compared with an LDA.

  6. Dominance of hepatitis C virus (HCV) is associated with lower quantitative hepatitis B surface antigen and higher serum interferon-γ-induced protein 10 levels in HBV/HCV-coinfected patients.

    PubMed

    Wiegand, S B; Jaroszewicz, J; Potthoff, A; Höner Zu Siederdissen, C; Maasoumy, B; Deterding, K; Manns, M P; Wedemeyer, H; Cornberg, M

    2015-07-01

    Different viral dominance patterns have been documented in coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) based on HBV DNA and HCV RNA quantification. In most cases, HCV is dominant and suppresses HBV replication. In vitro studies revealed that there is most probably no direct interference between HBV and HCV replication. We hypothesized that indirect mechanisms mediated by host immune responses might be responsible for the different dominance patterns. In this study we analysed quantitative hepatitis B surface antigen (HBsAg) as a marker for immune control of HBV and interferon γ-induced protein 10 (IP-10) as host marker for the endogenous interferon in 85 patients with HBV/HCV coinfection. Levels of HBsAg were closely associated with viral dominance patterns in 85 HBV/HCV-coinfected patients. HBsAg levels were lowest in patients with HCV dominance, even lower compared with HBV-monoinfected patients undergoing treatment with nucleos(t)ide analogues (NA) but comparable to low replicative HBsAg carriers. An increase in HCV RNA during follow up was associated with HBsAg decline. Patients with HCV dominance had significantly higher serum IP-10 levels compared with HBV-dominant patients or HBV-monoinfected patients treated with NA. Lower HBsAg and higher IP-10 levels in HCV-dominant HBV/HCV-coinfected patients suggest that HCV suppresses HBV DNA replication and also HBsAg production by immune mechanisms.

  7. Antigens and allergic asthma

    SciTech Connect

    Reed, C.E.; Swanson, M.C.

    1987-06-01

    There are few reliable epidemiologic data on the overall frequency and importance of allergy. We describe a practical method for quantifying the concentration of both amorphous and morphologically defined antigens in the air. A high volume air sampler is used to collect airborne particles and has a facility to separate samples into different particle sizes. Samples are tested for allergenic activity by radioallergosorbent test inhibition assay. Preliminary findings from studies of community wide, amorphous and common household allergens are reported.

  8. [Comparison of antibody responses to hepatitis B surface antigen among four recipient groups of hepatitis B vaccines that have been approved in Japan: evaluation using passive hemagglutination assay and chemiluminescent immunoassay].

    PubMed

    Ogata, Norio

    2009-10-01

    In hepatitis B virus (HBV) infection-preventing programs, serum or plasma levels of antibody to hepatitis B surface antigen (anti-HBs) are important to determine whether individuals are protective or not. We compared anti-HBs responses using passive hemagglutination assay (Mycell) and chemiluminescent immunoassay (Architect) among four recipient groups of HB vaccines, Meinyu, HBY, Bimmugen and Heptavax II, that have been approved in Japan. Overall, in a total of 1875 vaccinees Mycell results showed recipient groups of Meinyu and HBY acquired higher anti-HBs levels than those of Bimmugen and Heptavax II. Comparison of anti-HBs responses by both Mycell and Architect in recipient groups of Meinyu (n=150), HBY (n=218), Bimmugen (n=260), and Heptavax II (n=47) demonstrated the order of vaccinees' responses, such as geometric mean titers, ratios of acquiring high antibody levels (Mycell titers over 1024, Architect measurements over 1000 mIU/mL), and ratios of having unsuccessful antibody responses (Mycell titers under 8, Architect measurements under 10 mIU/mL), were somewhat different between the two assays. Comparison of Architect measurements at given Mycell titers revealed Bimmugen-recipients showed significantly lower values than HBY- or Heptavax II-recipients. Around critical protective levels, 5 of 22 Bimmugen-recipients with Mycell titers 16 or 32 showed Architect measurements under 10 mIU/mL, while 8 of 11 Heptavax II-recipients with Mycell titers below 8 demonstrated Architect measurements over 10 mIU/mL. Thus, discrepancies in anti-HBs evaluation between Mycell and Architect seemed to partly depend on administered vaccines. These results indicate anti-HBs concentration should be evaluated carefully so that we could completely prevent HBV infection.

  9. Effects of 1,2,4,6-tetra-O-galloyl-β-D-glucose from P. emblica on HBsAg and HBeAg secretion in HepG2.2.15 cell culture.

    PubMed

    Xiang, Yang-Fei; Ju, Huai-Qiang; Li, Shen; Zhang, Ying-Jun; Yang, Chong-Ren; Wang, Yi-Fei

    2010-10-01

    A polyphenolic compound, 1,2,4,6-tetra-O-galloyl-β-D-glucose (1246TGG), was isolated from the traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae) and assayed for its potential as an anti-hepatitis B virus (HBV) agent. The cytotoxicity of 1246TGG on HepG2.2.15 as well as HepG2 cells was determined by observing cytopathic effects, and the effects of 1246TGG on secretion of HBsAg and HBeAg in HepG2.2.15 cells were assayed by enzyme immunoassay. Results indicates that treatment with 1246TGG (6.25 μg/mL, 3.13 μg/mL), reduced both HBsAg and HBeAg levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. This study provides a basis for further investigation of the anti-HBV activity and possible mechanism of action of 1246TGG.

  10. A frameshift mutation in the pre-S region of the human hepatitis B virus genome allows production of surface antigen particles but eliminates binding to polymerized albumin.

    PubMed Central

    Persing, D H; Varmus, H E; Ganem, D

    1985-01-01

    The coding region for the major polypeptide (p24S) of hepatitis B surface antigen (HBsAg) is preceded by an in-phase open reading frame termed pre-S. The coding potential of the pre-S region was examined in mouse L cells transformed with cloned hepatitis B virus DNA. Such cells produce three HBsAg-related polypeptides of Mr 24,000, 27,000, and 35,000 organized into complex particles of 22 nm diameter. These HBsAg particles bind to polymerized human albumin, but not to polyalbumins of several other species. In contrast, cells transformed with hepatitis B virus DNA bearing a frameshift mutation near the 3' end of the pre-S region secrete immunoreactive HBsAg particles containing only the 24,000 and 27,000 Mr species. These mutant particles, which lack the 35,000 Mr species, are unable to bind polymerized human albumin. These studies indicate that the pre-S region encodes the 35,000 Mr species, that this product accounts for the known polyalbumin-binding activity of HBsAg but is not required for assembly and secretion of HBsAg 22-nm particles, and that the major polypeptide of HBsAg is not derived primarily by cleavage of larger precursors encoded by the pre-S region. Images PMID:3858831

  11. Hepatitis B Surface Antigen Loss and Hepatocellular Carcinoma Development in Patients With Dual Hepatitis B and C Infection.

    PubMed

    Yang, Wan-Ting; Wu, Li-Wei; Tseng, Tai-Chung; Chen, Chi-Ling; Yang, Hung-Chih; Su, Tung-Hung; Wang, Chia-Chi; Kuo, Stephanie Fang-Tzu; Liu, Chen-Hua; Chen, Pei-Jer; Chen, Ding-Shinn; Liu, Chun-Jen; Kao, Jia-Horng

    2016-03-01

    Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are 2 major causes of chronic viral hepatitis. It is still unclear how HCV coinfection affects HBV replication and clinical outcomes in HBV/HCV coinfected patients.We conducted a longitudinal study, which enrolled 111 patients with HBV/HCV coinfection and 111 propensity score-matched controls with HBV monoinfection. Both groups had comparable baseline age, sex, fibrosis stage, levels of HBV DNA, and HBV surface antigen (HBsAg). The HCV coinfection and other host/viral factors were correlated with various outcomes, including HBsAg loss and cirrhosis/hepatocellular carcinoma (HCC) development.After a 10-year follow-up, we found that HCV coinfection itself was not associated with HBsAg loss. However, coinfected patients with alanine aminotransferase (ALT) level >80 U/L had a higher chance of HBsAg loss than those with ALT level ≤80 U/L [hazard ratio (95% confidence interval): 4.41 (1.75-11.15)] or matched controls with HBV monoinfection [hazard ratio (95% confidence interval): 3.40 (1.54-7.50)]. Besides, both HCV coinfection and higher ALT levels were associated with higher HCC risks and the HCC risks remained even after HBsAg loss in HBV/HCV con-infected patient.HCV coinfection is not associated with HBsAg loss. A higher ALT level is a major determinant of HBsAg loss in patients with HBV/HCV coinfection. Both HCV coinfection and a higher ALT level were independent risk factors of HCC.

  12. Hepatitis B surface antigen prevalence among 12 393 rural women of childbearing age in Hainan Province, China: a cross-sectional study

    PubMed Central

    2013-01-01

    Background Hepatitis B virus (HBV) infection is highly endemic in China and it threats human health seriously. The hepatitis B surface antigen (HBsAg) prevalence among women of childbearing age plays an important role in mother to child transmission of HBV, as 30% ~50% of chronic carriers can be attributed to maternal-infantile transmission. However, there are few studies which have reported on the prevalence of HBsAg among women of childbearing age in China. This study aimed to determine the prevalence of HBsAg and its associated risk factors among rural women of childbearing age in Hainan, which is the highest hepatitis B virus endemic province in China. Methods A cross-sectional, population-based study, which included 12393 rural women aged 15 ~ 49 years, enrolled by a multistage stratified cluster sampling, was carried out in Hainan province, China, from November 2007 to December 2008. Blood samples were obtained from each study participant, and screened for HBsAg. Results The overall HBsAg prevalence of childbearing age women was 9.51%. Risk factors for HBsAg positivity among rural women were: lower education level (OR=1.206), lower family monthly income (OR=1.233), having an HBsAg-positive family member (OR=1.300), without an immunization history (OR=1.243), tattooing (OR=1.190), body piercing (OR=1.293), vaginoscopy history (OR=1.103) and history of induced abortion (OR=1.142). Conclusions There is a high HBsAg seroprevalence rate among rural women of childbearing age in Hainan province. Hence, it is necessary to take preventive measures to reduce the seroprevalence of HBsAg and to control its associated risk factors. PMID:23332007

  13. [Asymptomatic carriers of HBsAg: is a follow-up necessary?].

    PubMed

    Vignote, M L; Gómez-Camacho, F; Poyato, A; González, R; Palomo, D; Hervás, A; Peña, J; Miño, G

    1995-10-01

    We evaluated the clinical and epidemiological data of 142 HBsAg carriers. This prospective trial is part of a program of study and follow-up in HVB patients. The median age was 34.58 years old, males 56.3%. The average follow-up was 32.4 months. Complete clinical history, routine analysis, liver function tests, alfa-fetoprotein, serology for HVB, HCV and HDV and abdominal ecography were done in all patients. DNA-HVB was done only in special cases. Patients with less than 6 months of follow-up were excluded. The 118 remaining carriers were classified into two groups, depending on ALT values. Group 1 (normal ALT): included 98 carriers, 3 of them developed an active chronic hepatitis that was treated with interferon. A small CHC was diagnosed in another patient and it was resected. Group 2 (elevated ALT): included 20 carriers, only 9 of them agreed to biopsy and we found severe hepatic lesions in 4 of them. No group presented coinfection with HCV or HDV. No patient died. We conclude that the study and follow-up of asymptomatic HBsAg carriers permits an early diagnosis and treatment of the complications of this pathology (chronic hepatitis, CHC, etc); in our study, three patients developed chronic hepatitis, successfully treated with interferon, and one small size CHC was diagnosed in another patient. The study of relatives permits also detect subclinic liver disease and facilitates vaccination to prevention transmission of this infection. PMID:8519537

  14. Morphological and physiological characteristics of transgenic cherry tomato mutant with HBsAg gene.

    PubMed

    Guan, Z J; Guo, B; Huo, Y L; Hao, H Y; Wei, Y H

    2011-08-01

    HBsAg gene was previously introduced into cherry tomato (Lycopersicum esculentum var. cerasiforme) by Agrobacterium-mediated transformation. To investigate the side effect of HBsAg gene in cherry tomato, we analyzed morphological and physiological characteristics of the transgenic mutant N244. The process was performed under field conditions. The results suggested that the mutant N244 exhibited morphological, cytological and physiological variation. First of all, compared with the wild plants NK, N244 had fleshy and dark green leaves, the fewer notches of leaf edge, more adventitious roots and barren seeds. Moreover, the chromosome of N244 were found to be triploid (n = 36) by flow cytometric analysis. Furthermore, N244 has obvious physiological alterations, as compared to NK. It was speculated that transformation of the genes probably led to ploidy variation, and further caused phenotype and physiological changes of plants. Our study will reveal side effects of the mutants, and promote cultivation of transgenic plants in the field. PMID:21954613

  15. Maternal ABO and rhesus blood group phenotypes and hepatitis B surface antigen carriage.

    PubMed

    Lao, T T; Sahota, D S; Chung, M-K; Cheung, T K W; Cheng, Y K Y; Leung, T Y

    2014-11-01

    In view of a persistently high prevalence of hepatitis B surface antigen (HBsAg) carriage in our obstetric population, we examined the association between HBsAg carriage with maternal ABO and rhesus (Rh) blood group phenotypes determined at routine antenatal screening. In a retrospective study, the antenatal screening results of women booked for confinement between 1998 and 2011 in our hospital were examined for the relationship between HBsAg carriage with the ABO and rhesus blood groups, taking into account also the effects of advanced maternal age (≥ 35 years) and parity status (nulliparous or multiparous), and year of birth before or following the availability of the hepatitis B vaccine (1984). HBsAg carriage was found in 9.9%, 9.6%, 9.1% and 10.2% (P = 0.037) for group-A (n = 20 581 or 26.1%), -B (n = 20 744 or 26.4%), -AB (n = 5138 or 6.5%) and -O (n = 32 242 or 41.0%) among the 78705 women in the study cohort. Rhesus negativity was found in 0.6%, and HBsAg carriage was 12.3% and 9.8%, respectively, for the Rh-negative and Rh-positive women (P = 0.071). Carriage rate between group-O and non-O was influenced by nulliparity, age ≥ 35 years and Rh-positive status. Regression analysis indicated that group-B (P = 0.044, aOR = 1.062, 95% CI 1.002-1.127) and group-AB (P = 0.016, aOR = 1.134, 95% CI 1.024-1.256) were associated with HBsAg carriage. Blood groups-B and -AB are associated with increased hepatitis B virus (HBV) infection in our population, and further studies are warranted to elucidate the implications of this on the sequelae of HBV infection.

  16. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

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

  17. Complexes of hepatitis B surface antigen and immunoglobulin M in the sera of patients with hepatitis B virus infection.

    PubMed Central

    Palla, M; Rizzi, R; Toti, M; Almi, P; Rizzetto, M; Bonino, F; Purcell, R

    1983-01-01

    Hepatitis B surface antigen (HBsAg) bound to immunoglobulin M (IgM) was detected in sera of HBsAg carriers by a radioimmunoassay based on selective absorption of the immunoglobulin on a solid phase coated with antiserum to human IgM. Isopycnic banding and rate-zonal sedimentation have shown that the reaction is related to particulate forms of the HBsAg complexed with IgM. The binding of IgM possibly occurred because of a selective affinity of these molecules to the surface of HBsAg particles. HBsAg/IgM was found transiently in 24 of 25 (96%) patients with acute self-limited hepatitis B and persistently in 6 of 25 patients whose acute hepatitis B progressed to chronicity. It was also found in 20 of 39 (51%) chronic HBsAg carriers with inactive and asymptomatic infection. The HBsAg/IgM phenomenon is not dependent on replication of hepatitis B virions; its persistence in patients with acute hepatitis B may provide complementary evidence of transition of the infection to chronicity. PMID:6309673

  18. Hepatitis B surface antigen titer is a good indicator of durable viral response after entecavir off-treatment for chronic hepatitis B

    PubMed Central

    Lee, Han Ah; Seo, Yeon Seok; Park, Seung Woon; Park, Sang Jung; Kim, Tae Hyung; Suh, Sang Jun; Jung, Young Kul; Kim, Ji Hoon; An, Hyunggin; Yim, Hyung Joon; Yeon, Jong Eun; Byun, Kwan Soo; Um, Soon Ho

    2016-01-01

    Background/Aims Clear indicators for stopping antiviral therapy in chronic hepatitis B (CHB) patients are not yet available. Since the level of hepatitis B surface antigen (HBsAg) is correlated with covalently closed circular DNA, the HBsAg titer might be a good indicator of the off-treatment response. This study aimed to determine the relationship between the HBsAg titer and the entecavir (ETV) off-treatment response. Methods This study analyzed 44 consecutive CHB patients (age, 44.6±11.4 years, mean±SD; men, 63.6%; positive hepatitis B envelope antigen (HBeAg) at baseline, 56.8%; HBV DNA level, 6.8±1.3 log10 IU/mL) treated with ETV for a sufficient duration and in whom treatment was discontinued after HBsAg levels were measured. A virological relapse was defined as an increase in serum HBV DNA level of >2000 IU/mL, and a clinical relapse was defined as a virological relapse with a biochemical flare, defined as an increase in the serum alanine aminotransferase level of >2 × upper limit of normal. Results After stopping ETV, virological relapse and clinical relapse were observed in 32 and 24 patients, respectively, during 20.8±19.9 months of follow-up. The cumulative incidence rates of virological relapse were 36.2% and 66.2%, respectively, at 6 and 12 months, and those of clinical relapse were 14.3% and 42.3%. The off-treatment HBsAg level was an independent factor associated with clinical relapse (hazard ratio, 2.251; 95% confidence interval, 1.076–4.706; P=0.031). When patients were grouped according to off-treatment HBsAg levels, clinical relapse did not occur in patients with an off-treatment HBsAg level of ≤2 log10 IU/mL (n=5), while the incidence rates of clinical relapse at 12 months after off-treatment were 28.4% and 55.7% in patients with off-treatment HBsAg levels of >2 and ≤3 log10 IU/mL (n=11) and >3 log10 IU/mL (n=28), respectively. Conclusion The off-treatment HBsAg level is closely related to clinical relapse after treatment cessation. A

  19. Concepts and applications for influenza antigenic cartography

    PubMed Central

    Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2011-01-01

    Influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. A robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. However, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incompleteness, high noises, and low reactors. To overcome these challenges, we developed a computational method, temporal Matrix Completion-Multidimensional Scaling (MC-MDS), by adapting the low rank MC concept from the movie recommendation system in Netflix and the MDS method from geographic cartography construction. The application on H3N2 and 2009 pandemic H1N1 influenza A viruses demonstrates that temporal MC-MDS is effective and efficient in constructing influenza antigenic cartography. The web sever is available at http://sysbio.cvm.msstate.edu/AntigenMap. PMID:21761589

  20. Preparation and testing of a Haemophilus influenzae Type b/Hepatitis B surface antigen conjugate vaccine.

    PubMed

    An, So Jung; Woo, Joo Sung; Chae, Myung Hwa; Kothari, Sudeep; Carbis, Rodney

    2015-03-24

    The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRPAH-DT conjugate induced strong anti-PRP and anti-DT responses, the Vi-AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRPAH-HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRPAH-HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine. PMID:25659268

  1. Preparation and testing of a Haemophilus influenzae Type b/Hepatitis B surface antigen conjugate vaccine.

    PubMed

    An, So Jung; Woo, Joo Sung; Chae, Myung Hwa; Kothari, Sudeep; Carbis, Rodney

    2015-03-24

    The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRPAH-DT conjugate induced strong anti-PRP and anti-DT responses, the Vi-AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRPAH-HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRPAH-HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine.

  2. Seroprevalence of Hepatitis B Surface Antigen and Occupational Risk Factors Among Health Care Workers in Ekiti State, Nigeria

    PubMed Central

    Alese, Oluwole Ojo; Ohunakin, Afolabi; Oluyide, Peter Olumuyiwa

    2016-01-01

    Introduction Hepatitis B virus (HBV) infection is contracted from blood and other body fluid making healthcare workers (HCW) prone to the infection especially in the developing world. Though it is a vaccine preventable disease, the level of awareness and universal precaution among HCW is low in sub-Saharan African and Asia. Aim The study was aimed at determining the seroprevalence of hepatitis B surface antigen and occupational risk factors among health care workers at Ekiti State University Teaching Hospital, Ado Ekiti. Materials and Methods One hundred and eighty-seven (187) blood samples were collected from volunteer subjects who comprised of medical doctors, nurses, health attendants, and porters who are in regular contact with blood, body fluids and patients after informed consent. Well detailed and structured questionnaires were used to obtain demographic and other relevant data from the subjects. Blood samples were tested by Enzyme Linked Immunosorbent assay (ELISA) for hepatitis B surface antigen. Results Out of the 187 HCWs there were 91 males (48.7%) and 96 (51.3%) females. Only 2 participants tested positive to hepatitis B surface antigen with a prevalence of 1.1%. Also, only 30 (16.0%) of the participants had been fully vaccinated against the infection while the remaining 157(84.0%) had no adult vaccination. Conclusion It is obvious that the awareness of the infection is low among the HCWs studied thus the need to incorporate screening for HbsAg and vaccination against HBV into the periodic/pre-employment health intervention programmes by employers to help in the protection of HCWs and control the spread of the virus. PMID:27042489

  3. HBsAg seroconversion after pegylated interferon alfa 2a rescue in a lamivudine-resistant patient with HBeAg-negative chronic hepatitis B and favourable IL28-B genotype.

    PubMed

    Stanzione, Maria; Stornaiuolo, Gianfranca; Rizzo, Viviana; Pontarelli, Agostina; Gaeta, Giovanni Battista

    2016-06-01

    Hepatitis B virus (HBV) surface antigen (HBsAg) seroconversion to anti-HBs antibody is the best final objective for all available chronic hepatitis B (CHB) treatments. Unfortunately, this goal is rarely achieved with the currently applied therapeutic approaches. Here we describe the case of an anti-HBe-positive CHB patient who was successfully treated with a particular therapeutic schedule. The patient was initially treated with lamivudine (LAM) for nine years. Breakthrough was observed after eight years of LAM therapy. HBV-DNA was 3x10E4 IU/mL and LAM resistance mutations were present. Subcutaneous pegylated interferon (PEG-IFN) alfa 2a, 180 mcg/week, was added to LAM and after 4 weeks LAM was discontinued and PEG-IFN alone was continued up to week 52. HBV-DNA became undetectable at week 4 of therapy; serum HBsAg started to decline from week 4 and became undetectable at week 36, with the subsequent appearance of anti-HBs antibodies. IL28-B was genotyped at the polymorphic site rs12979860 and the CC allele was detected. Rescue therapy with Peg-IFN may be an option for selected patients with resistance to nucleos(t)ide analogues. PMID:27367326

  4. Determination by enzyme-linked immunosorbent assay of immunoglobulin G (IgG), IgM, and IgA to Brucella melitensis major outer membrane proteins and whole-cell heat-killed antigens in sera of patients with brucellosis.

    PubMed Central

    Araj, G F; Kaufmann, A F

    1989-01-01

    An enzyme-linked immunosorbent assay was used to compare Brucella melitensis major outer membrane proteins (MOMP) and whole-cell heat-killed antigens (HK) in measuring antibrucella immunoglobulin G (IgG), IgM, and IgA in sera of brucellosis patients and controls. Antibodies to MOMP were generally similar to those against HK, and the correlation coefficients between the two antigens and IgG, IgM, and IgA in patients varied between 0.73 and 0.94. Both antigens are comparably suitable in detecting antibrucella immunoglobulin isotypes for the serologic diagnosis of patients with brucellosis, with high (greater than or equal to 95%) sensitivity and specificity. PMID:2768476

  5. Correlation between serum hepatitis B virus core-related antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients.

    PubMed

    Suzuki, Fumitaka; Miyakoshi, Hideo; Kobayashi, Mariko; Kumada, Hiromitsu

    2009-01-01

    Nucleos(t)ide analogues are utilized for the treatment of chronic HBV infection, and HBe seroconversion and HBV DNA levels are commonly used as markers of viral status and as primary treatment endpoints. Recently, a new assay was prepared for the detection of serum HBV core-related antigen (HBcrAg), consisting of HBcAg, HBeAg, and p22cr, which is a precore protein from amino acid -28 to at least amino acid 150, by coding the precore/core region. In this study, we examined the correlation between serum HBcrAg concentration and viral status by the analysis of serum HBeAg, HBsAg, peripheral HBV DNA, and intrahepatic covalently closed circular DNA (cccDNA) in 57 chronic hepatitis B patients. Intrahepatic cccDNA was detected in all 57 patients, 42 patients were HBcrAg-positive, and serum HBcrAg concentration level was closely correlated with cccDNA. Additionally, positive HBcrAg concentration level results were observed in 6 out of 13 HBsAg seroclearance patients and 20 out of 31 HBV DNA-negative patients. Moreover, the correlation between HBcrAg and cccDNA in these 31 HBV DNA-negative patients was statistically significant (r = 0.482, P = 0.006). These data suggest that serum HBcrAg concentration is well correlated with intrahepatic cccDNA level, and that the measurement of serum HBcrAg may be clinically useful for monitoring intrahepatic HBV viral status, especially in patients under treatment with nucleos(t)ide analogues.

  6. Combination of reverse transcription real-time polymerase chain reaction and antigen capture enzyme-linked immunosorbent assay for the detection of animals persistently infected with Bovine viral diarrhea virus.

    PubMed

    Yan, Lifang; Zhang, Shuping; Pace, Lanny; Wilson, Floyd; Wan, Henry; Zhang, Michael

    2011-01-01

    Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle. A successful control program requires early detection and removal of persistently infected (PI) animals. The objective of the current study was to develop, validate, and apply a cost-effective testing scheme for the detection of BVDV PI animals in exposed herds. Pooled samples were screened by using a real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and individual positives were identified with an antigen capture enzyme-linked immunosorbent assay (ACE). The detection limits of the optimized real-time RT-PCR were 10 and 100 RNA copies per reaction for BVDV-1 and BVDV-2, respectively. The semiquantitative results of real-time RT-PCR and ACE or real-time RT-PCR and immunohistochemistry were moderately correlated. The threshold cycle of real-time RT-PCR performed on pooled samples was significantly correlated with the pool size (R(2)  =  0.993). The least-cost pool sizes were 50 at a prevalence of 0.25-0.5% and 25 at a prevalence of 0.75-2.0%. By using the combined real-time RT-PCR and ACE procedure, 111 of 27,932 samples (0.4%) tested positive for BVDV. At this prevalence, cost reduction associated with the application of real-time RT-PCR and ACE ranged from 61% to 94%, compared with testing individual samples by ACE, immunohistochemistry, or real-time RT-PCR. Real-time RT-PCR screening also indicated that 92.94% of PI animals were infected with BVDV-1, 3.53% with BVDV-2, and 3.53% with both BVDV-1 and BVDV-2. Analysis of the 5'-untranslated region of 22 isolates revealed the predominance of BVDV-1b followed by BVDV-2a.

  7. Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae.

    PubMed

    Okada, Munenori; Asai, Tetsuo; Futo, Satoshi; Mori, Yasuyuki; Mukai, Tetsuya; Yazawa, Shigeto; Uto, Takehiko; Shibata, Isao; Sato, Shizuo

    2005-02-25

    To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP. PMID:15708823

  8. The Murine Humoral Immune Response to Hepatitis B Surface Antigen: Idiotype Network Pathways.

    NASA Astrophysics Data System (ADS)

    Schick, Michael Roy

    Recognition of a wide spectrum in disease outcomes following Hepatitis B Virus (HBV) infection has led to the suggestion that individual differences may be due to characteristics of the immune response. HBV, a hepatotropic virus, is not directly cytopathic to the host hepatocytes but the cellular damage which does not occur may be due to the host's own immune response. It is this variety in immune response capabilities following natural infection or vaccination which led to the present study in which the murine humoral immune response to hepatitis B surface antigen (HBsAg) was examined. Following immunization with purified HBsAg an anti-HBs response could be detected in 19 inbred strains of mice. The response, which varied among the strains, was linked to the major histocompatibility complex (MHC). Among high responders to HBsAg were two strains in which a poor response to a single epitope could be detected. Although quantitatively serum from these strains resembled serum from other high responders, there was a major difference in the qualitative aspects. Included within this study was the role of idotype networks within the murine anti-HBs response. By directly targeting HBsAg-specific B cells within the framework of an idiotype network by an Ab-2, it was possible to circumvent T cell-dependent regulation of an immune response. In each of five inbred strains of mice immunized with a polyclonal rabbit Ab-2 an Ab-3 population with HBsAg-specificity (Ab -1^') was induced. These mice were also immunized with HBsAg resulting in a higher anti-HBs response as compared to HBsAg immunization alone in all of the strains tested except for one. The response in this strain, normally a low responder to HBsAg, indicated that the mechanisms for genetic restriction of the anti -HBs response was still active, although it was not apparent during anti-Id immunization. The effects of an anti-Id on the murine antibody response to HBsAg may lead to insights on the presence of idiotype

  9. Dose-response association between hepatitis B surface antigen levels and liver cancer risk in Chinese men and women

    PubMed Central

    Yang, Yang; Gao, Jing; Li, Hong-Lan; Zheng, Wei; Yang, Gong; Zhang, Wei; Ma, Xiao; Tan, Yu-Ting; Rothman, Nat; Gao, Yu-Tang; Chow, Wong-Ho; Shu, Xiao-Ou; Xiang, Yong-Bing

    2016-01-01

    We aimed at evaluating the risk of liver cancer in different levels of HBsAg among Chinese men and women. We carried out a nested case-control study including 363 cases and 3,511 controls in two population-based cohorts in Shanghai. Plasma samples collected at enrollment were quantified for HBsAg levels using the Architect QT assay. Conditional logistic regression was performed to estimate the odds ratios (ORs) and 95% confidence intervals (95%CIs) for liver cancer, with adjustment for potential confounders. HBsAg was detected in 6.29% of control subjects overall (7.02% in men and 4.98% in women). HBsAg levels were positively associated with liver cancer risk in a dose-response manner (Ptrend<0.001). Such association showed a significant gender disparity. With increasing levels of HBsAg, liver cancer risks rose more steeply in men than in women. In men, the adjusted ORs increased from 7.27 (95%CI: 3.49–15.15) at the lowest detectable level of HBsAg (5–9 IU/ml) to 7.16 (95%CI: 3.21–15.96), 34.30 (95%CI: 16.94–69.44), and 47.33 (95%CI: 23.50–95.34) at the highest level of HBsAg (≥1,000 IU/ml) compared to those negative for HBsAg. The corresponding ORs were much lower for women, from 1.37 (95%CI: 0.25–7.47) to 3.81 (95%CI: 1.09–13.28), 7.36 (95%CI: 2.41–22.46), and 16.86 (95%CI: 7.24–39.27), respectively. HBsAg quantification has potential to distinguish individuals at different risks of liver cancer. Men with the lowest detectable level of HBsAg should still pay attention to their liver cancer risks, but those with a higher level may be given a higher priority in future liver cancer surveillance program. PMID:26990915

  10. DNA sequence and analysis of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 and serogroup-specific PCR assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined. There were 9 to 12 open reading frames (ORFs) identified, encoding genes required for O-antigen sugar biosynthesis, transfer, and processing. Primers based on the wzx...

  11. Enteric trimethyl chitosan nanoparticles containing hepatitis B surface antigen for oral delivery

    PubMed Central

    Farhadian, Asma; Dounighi, Naser Mohammadpour; Avadi, Mohammadreza

    2015-01-01

    Oral vaccination is the preferred route of immunization. However, the degradative condition of the gastrointestinal tract and the higher molecular size of peptides pose major challenges in developing an effective oral vaccination system. One of the most excellent methods used in the development of oral vaccine delivery system relies on the entrapment of the antigen in polymeric nanoparticles. In this work, trimethyl chitosan (TMC) nanoparticles were fabricated using ionic gelation teqnique by interaction hydroxypropyl methylcellulose phthalate (HPMCP), a pH-sensitive polymer, with TMC and the utility of the particles in the oral delivery of hepatitis B surface antigen (HBsAg) was evaluated employing solutions that simulated gastric and intestinal conditions. The particle size, morphology, zeta potential, loading capacity, loading efficiency, in vitro release behavior, structure, and morphology of nanoparticles were evaluated, and the activity of the loaded antigen was assessed. Size of the optimized TMC/HPMCP nanoparticles and that of the antigen-loaded nanoparticles were 85 nm and 158 nm, respectively. Optimum loading capacity (76.75%) and loading efficiency (86.29%) were achieved at 300 µg/mL concentration of the antigen. SEM images revealed a spherical shape as well as a smooth and near-homogenous surface of nanoparticles. Results of the in vitro release studies showed that formulation with HPMCP improved the acid stability of the TMC nanoparticles as well as their capability to preserve the loaded HBsAg from gastric destruction. The antigen showed good activity both before and after loading. The results suggest that TMC/HPMCP nanoparticles could be used in the oral delivery of HBsAg vaccine. PMID:26158754

  12. Serospecific antigens of Legionella pneumophila.

    PubMed Central

    Otten, S; Iyer, S; Johnson, W; Montgomery, R

    1986-01-01

    Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria. Images PMID:3017918

  13. High titre of antibody to hepatitis B core antigen detected by immune adherence haemagglutination in HBsAg-positive acute hepatitis.

    PubMed

    Ikegami, F; Takasu, S; Jo, K; Miyakawa, Y; Tsuda, F; Mayumi, M

    1982-08-01

    Nine patients with HBsAg-positive acute hepatitis were tested for antibody to hepatitis B core antigen (anti-HBc) by the immune adherence haemagglutination method. A high anti-HBc titre (2(15) or more) was found in three, while anti-HBc was not detectable in the remaining six. All of them recovered from hepatitis with the return of hepatic function tests to normal, but HBsAg persisted in the three patients whose acute-phase serum had revealed high anti-HBc titres. On the basis of these observations, the three patients were thought to be persistent HBsAg carriers who had contracted opportunistic acute hepatitis of non-B aetiology. Titration of anti-HBc may be indicated in patients with HBsAg-positive acute hepatitis, because it helps distinguish persistent HBsAg carriers with non-B hepatitis from patients with hepatitis B at the outset, during the episode of acute hepatitis.

  14. Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.

    PubMed

    Gurramkonda, Chandrasekhar; Zahid, Maria; Nemani, Satish Kumar; Adnan, Ahmad; Gudi, Satheesh Kumar; Khanna, Navin; Ebensen, Thomas; Lünsdorf, Heinrich; Guzmán, Carlos A; Rinas, Ursula

    2013-12-01

    Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.

  15. Total Hepatitis B Core Antigen Antibody, a Quantitative Non-Invasive Marker of Hepatitis B Virus Induced Liver Disease.

    PubMed

    Yuan, Quan; Song, Liu-Wei; Cavallone, Daniela; Moriconi, Francesco; Cherubini, Beatrice; Colombatto, Piero; Oliveri, Filippo; Coco, Barbara Agata; Ricco, Gabriele; Bonino, Ferruccio; Shih, James Wai Kuo; Xia, Ning-Shao; Brunetto, Maurizia Rossana

    2015-01-01

    Non invasive immunologic markers of virus-induced liver disease are unmet needs. We tested the clinical significance of quantitative total and IgM-anti-HBc in well characterized chronic-HBsAg-carriers. Sera (212) were obtained from 111 HBsAg-carriers followed-up for 52 months (28-216) during different phases of chronic-HBV-genotype-D-infection: 10 HBeAg-positive, 25 inactive-carriers (HBV-DNA≤2000IU/ml, ALT<30U/L), 66 HBeAg-negative-CHB-patients and 10 with HDV-super-infection. In 35 patients treated with Peg-IFN±nucleos(t)ide-analogues (NUCs) sera were obtained at baseline, end-of-therapy and week-24-off-therapy and in 22 treated with NUCs (for 60 months, 42-134m) at baseline and end-of-follow-up. HBsAg and IgM-anti-HBc were measured by Architect-assays (Abbott, USA); total-anti-HBc by double-antigen-sandwich-immune-assay (Wantai, China); HBV-DNA by COBAS-TaqMan (Roche, Germany). Total-anti-HBc were detectable in all sera with lower levels in HBsAg-carriers without CHB (immune-tolerant, inactive and HDV-superinfected, median 3.26, range 2.26-4.49 Log10 IU/ml) versus untreated-CHB (median 4.68, range 2.76-5.54 Log10 IU/ml), p<0.0001. IgM-anti-HBc positive using the chronic-hepatitis-cut-off" (0.130-S/CO) were positive in 102 of 212 sera (48.1%). Overall total-anti-HBc and IgM-anti-HBc correlated significantly (p<0.001, r=0.417). Total-anti-HBc declined significantly in CHB patients with response to Peg-IFN (p<0.001) and in NUC-treated patients (p<0.001); the lowest levels (median 2.68, range 2.12-3.08 Log10 IU/ml) were found in long-term responders who cleared HBsAg subsequently. During spontaneous and therapy-induced fluctuations of CHB (remissions and reactivations) total- and IgM-anti-HBc correlated with ALT (p<0.001, r=0.351 and p=0.008, r=0.185 respectively). Total-anti-HBc qualifies as a useful marker of HBV-induced-liver-disease that might help to discriminate major phases of chronic HBV infection and to predict sustained response to antivirals.

  16. Total Hepatitis B Core Antigen Antibody, a Quantitative Non-Invasive Marker of Hepatitis B Virus Induced Liver Disease

    PubMed Central

    Cavallone, Daniela; Moriconi, Francesco; Cherubini, Beatrice; Colombatto, Piero; Oliveri, Filippo; Coco, Barbara Agata; Ricco, Gabriele; Bonino, Ferruccio; Shih, James Wai Kuo; Xia, Ning-Shao; Brunetto, Maurizia Rossana

    2015-01-01

    Non invasive immunologic markers of virus-induced liver disease are unmet needs. We tested the clinical significance of quantitative total and IgM-anti-HBc in well characterized chronic-HBsAg-carriers. Sera (212) were obtained from 111 HBsAg-carriers followed-up for 52 months (28-216) during different phases of chronic-HBV-genotype-D-infection: 10 HBeAg-positive, 25 inactive-carriers (HBV-DNA≤2000IU/ml, ALT<30U/L), 66 HBeAg-negative-CHB-patients and 10 with HDV-super-infection. In 35 patients treated with Peg-IFN±nucleos(t)ide-analogues (NUCs) sera were obtained at baseline, end-of-therapy and week-24-off-therapy and in 22 treated with NUCs (for 60 months, 42-134m) at baseline and end-of-follow-up. HBsAg and IgM-anti-HBc were measured by Architect-assays (Abbott, USA); total-anti-HBc by double-antigen-sandwich-immune-assay (Wantai, China); HBV-DNA by COBAS-TaqMan (Roche, Germany). Total-anti-HBc were detectable in all sera with lower levels in HBsAg-carriers without CHB (immune-tolerant, inactive and HDV-superinfected, median 3.26, range 2.26-4.49 Log10 IU/ml) versus untreated-CHB (median 4.68, range 2.76-5.54 Log10 IU/ml), p<0.0001. IgM-anti-HBc positive using the chronic-hepatitis-cut-off" (0.130-S/CO) were positive in 102 of 212 sera (48.1%). Overall total-anti-HBc and IgM-anti-HBc correlated significantly (p<0.001, r=0.417). Total-anti-HBc declined significantly in CHB patients with response to Peg-IFN (p<0.001) and in NUC-treated patients (p<0.001); the lowest levels (median 2.68, range 2.12-3.08 Log10 IU/ml) were found in long-term responders who cleared HBsAg subsequently. During spontaneous and therapy-induced fluctuations of CHB (remissions and reactivations) total- and IgM-anti-HBc correlated with ALT (p<0.001, r=0.351 and p=0.008, r=0.185 respectively). Total-anti-HBc qualifies as a useful marker of HBV-induced-liver-disease that might help to discriminate major phases of chronic HBV infection and to predict sustained response to antivirals. PMID

  17. Induction of endoplasmic reticulum-derived oxidative stress by an occult infection related S surface antigen variant

    PubMed Central

    Lee, In-Kyung; Lee, Seoung-Ae; Kim, Hong; Won, You-Sub; Kim, Bum-Joon

    2015-01-01

    AIM: To investigate the mechanism of endoplasmic reticulum (ER) stress induction by an occult infection related hepatitis B virus S surface antigen (HBsAg) variant. METHODS: We used an HBsAg variant with lower secretion capacity, which was a KD variant from a Korean subject who was occultly infected with the genotype C. We compared the expression profiles of ER stress-related proteins between HuH-7 cells transfected with HBsAg plasmids of a wild-type and a KD variant using Western blot. RESULTS: Confocal microscopy indicated that the KD variant had higher levels of co-localization with ER than the wild-type HBsAg. The KD variant up-regulated ER stress-related proteins and induced reactive oxygen species (ROS) compared to the wild-type via an increase in calcium. The KD variant also down-regulated anti-oxidant proteins (HO-1, catalase and SOD) compared to the wild-type, which indicates positive amplification loops of the ER-ROS axis. The KD variant also induced apoptotic cell death via the up-regulation of caspase proteins (caspase 6, 9 and 12). Furthermore, the KD variant induced a higher level of nitric oxide than wild-type HBsAg via the up-regulation of the iNOS protein. CONCLUSION: Our data indicate that occult infection related HBsAg variants can lead to ER-derived oxidative stress and liver cell death in HuH-7 cells. PMID:26078563

  18. Quantitative Levels of Hepatitis B Virus DNA and Surface Antigen and the Risk of Hepatocellular Carcinoma in Patients with Hepatitis B Receiving Long-Term Nucleos(t)ide Analogue Therapy

    PubMed Central

    Kawanaka, Miwa; Nishino, Ken; Nakamura, Jun; Oka, Takahito; Urata, Noriyo; Goto, Daisuke; Suehiro, Mitsuhiko; Kawamoto, Hirofumi; Kudo, Masatoshi; Yamada, Gotaro

    2014-01-01

    Background Serum levels of hepatitis B virus (HBV) DNA are an important predictor of the risk of hepatocellular carcinoma (HCC) in patients with chronic HBV infection. However, little is known about whether high levels of hepatitis B surface antigen (HBsAg) increase the risk for HCC. Methods We investigated 167 patients who were treated with nucleos(t)ide analogues (NA) for at least 2 years (median: 5.8 years, range: 2-13.1 years). Relationships between reduced levels of HBsAg and various factors were evaluated. In addition, we evaluated the usefulness of quantitative serum levels of HBV DNA and HBsAg as predictors of HCC development in patients receiving long-term NA therapy. Results HCC developed in 9 of the 167 NA-treated patients. In the 9 patients with HCC, HBV DNA was undetectable (<2.1 log copies/mL), but HBsAg levels were ≥2000 C.O.I. in 7 patients. No maternal transmission, long NA treatment period, HBV DNA levels <3.0 log copies/mL, and reduced hepatitis B e antigen levels during the first 24 weeks of treatment were a significant factor of HBsAg levels <2000 C.O.I.. Conclusions Hepatocarcinogenesis was observed in patients with high HBsAg levels, despite the negative conversion of HBV DNA as a result of long-term NA therapy. Therefore, to suppress hepatocarcinogenesis, it is important to control not only HBV DNA levels but also HBsAg levels. PMID:24804176

  19. Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids

    SciTech Connect

    Simonsen, C.C.; Levinson, A.D.

    1983-12-01

    The authors examined the transcription of the hepatitis B virus surface antigen (HBsAg) gene in COs cells transfected with simian virus 40-based recombinant plasmids. When positioned behind the simian virus 40 late promoter, three transcripts were identified which hybridized to the HBsAg gene: a 2,000-nucleotide transcript colinear with a gene, a 1,100-nucleotide transcript representing a spliced molecule in which a major portion of the sequences encoding HBsAg were deleted, and an 800-nucleotide transcript derived primarily from sequences 3' to the HBsAg gene. The splice acceptor site utilized by the 1,100-nucleotide transcript is located immediately upstream of an open reading frame of unknown function contained within the 3' nontranslated region of the HBsAg gene. The HBsAg-specific mRNA species terminate 12 to 19 base pairs 3' of the sequence UAUAAA, similar to the concensus hexanucleotide which is thought to promote polyadenylation (AAUAAA). They constructed a series of plasmids with progressive deletions from the region surrounding where these transcripts terminate. Analysis of mRNA produced by cells transfected with these plasmids indicated that the signal hexanucleotide is in itself unable to promote the efficient processing of mRNA in the absence of downstream hepatitis B virus sequences. Processing proceeds properly, however, from plasmids containing an additional 30 nucleotides 3' of this signal.

  20. Asymptomatic rectal mucosal lesions and hepatitis B surface antigen at sites of sexual contact in homosexual men with persistent hepatitis B virus infection.

    PubMed

    Reiner, N E; Judson, F N; Bond, W W; Francis, D P; Petersen, N J

    1982-02-01

    To ascertain why active and passive oral-anal and genital anal intercourse correlate strongly with hepatitis B virus (HBV) infection in homosexual men, we studied 22 men with HBV infection for the presence of hepatitis B surface antigen (HBsAg) in gingival and anorectal mucosa, feces, and semen. Thirteen of 22 (59%) patients had asymptomatic rectal mucosal lesions consisting of punctate bleeding points. Seventy-seven percent of swabbed specimens from lesions, 62% from feces, 59% from rectal mucosa, and 50% from anal canal mucosa contained HBsAg. Sera diluted serially and tested for HBsAg by radioimmunoassay showed that men with serum titers of 105 or greater were more likely to have HBsAg in specimens from lesions (p = 0.034), feces (p = 0.032), and normal mucosa (p = 0.001) than men with titers under 10 5. Asymptomatic rectal bleeding is frequent in homosexual men with persistent HBV infection. Rectal mucosa, feces, and anal canal mucosa become contaminated with HBsAg, creating a setting for de facto parenteral transmission when there is contact with oral or urethral mucosa. PMID:7059064

  1. Hepatitis B virus genome replication triggers toll-like receptor 3-dependent interferon responses in the absence of hepatitis B surface antigen.

    PubMed

    Real, Catherine Isabell; Lu, Mengji; Liu, Jia; Huang, Xuan; Trippler, Martin; Hossbach, Markus; Deckert, Jochen; Jahn-Hofmann, Kerstin; Ickenstein, Ludger Markus; John, Matthias Johannes; Gibbert, Kathrin; Dittmer, Ulf; Vornlocher, Hans-Peter; Schirmbeck, Reinhold; Gerken, Guido; Schlaak, Joerg Friedrich; Broering, Ruth

    2016-01-01

    The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV. PMID:27121087

  2. Hepatitis B virus genome replication triggers toll-like receptor 3-dependent interferon responses in the absence of hepatitis B surface antigen

    PubMed Central

    Real, Catherine Isabell; Lu, Mengji; Liu, Jia; Huang, Xuan; Trippler, Martin; Hossbach, Markus; Deckert, Jochen; Jahn-Hofmann, Kerstin; Ickenstein, Ludger Markus; John, Matthias Johannes; Gibbert, Kathrin; Dittmer, Ulf; Vornlocher, Hans-Peter; Schirmbeck, Reinhold; Gerken, Guido; Schlaak, Joerg Friedrich; Broering, Ruth

    2016-01-01

    The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV. PMID:27121087

  3. Stable solid-phase Rh antigen.

    PubMed

    Yared, M A; Moise, K J; Rodkey, L S

    1997-12-01

    Numerous investigators have attempted to isolate the Rh antigens in a stable, immunologically reactive form since the discovery of the Rh system over 56 years ago. We report here a successful and reproducible approach to solubilizing and adsorbing the human Rh antigen(s) to a solid-phase matrix in an antigenically active form. Similar results were obtained with rabbit A/D/F red blood cell antigens. The antigen preparation was made by dissolution of the red blood cell membrane lipid followed by fragmentation of the residual cytoskeleton in an EDTA solution at low ionic strength. The antigenic activity of the soluble preparations was labile in standard buffers but was stable in zwitterionic buffers for extended periods of time. Further studies showed that the antigenic activity of these preparations was enhanced, as was their affinity for plastic surfaces, in the presence of acidic zwitterionic buffers. Adherence to plastic surfaces at low pH maintained antigenic reactivity and specificity for antibody was retained. The data show that this approach yields a stable form of antigenically active human Rh D antigen that could be used in a red blood cell-free assay for quantitative analysis of Rh D antibody and for Rh D antibody immunoadsorption and purification.

  4. Prevalence of Hepatitis B Surface Antigen in US-Born and Foreign-Born Asian/Pacific Islander College Students

    ERIC Educational Resources Information Center

    Quang, Yen N.; Vu, Joanne; Yuk, Jihey; Li, Chin-Shang; Chen, Moon; Bowlus, Christopher L.

    2010-01-01

    The prevalence of chronic hepatitis B (HBV) among college-age US-born Asian and Pacific Islanders (A/PI) is not well known. Objectives: To compare the prevalence of hepatitis B surface antigen (HBsAg) seropositivity in US-born to A/PI-born students at a public university. Participants: Undergraduate who self-identified themselves as A/PI. Results:…

  5. The underlying mechanisms for the "simultaneous HBsAg and anti-HBs serological profile".

    PubMed

    Pondé, R A A

    2011-11-01

    The course of HBV infection is determined by the interplay between viral replication via HBV protein production and the host's immune response. Therefore, the diagnosis of infection in clinical practice is established by the serological detection of HBV protein products as well as antibodies produced by the host. Although the serological findings for assessing the clinical course of infection are already well established, the expression of viral proteins and the dynamics of antibody production may vary during the natural course of infection. This causes the HBV infection to be occasionally associated with the presence of unusual serologic profiles, which can lead to doubts in the interpretation of results and mistaken serological diagnosis. The simultaneous detection of HBsAg and anti-HBs in the blood stream comprises an atypical serological profile, somewhat incoherent, whose significance can be complicated to establish. Outlined in this article are some immunological and molecular mechanisms which could justify the existence of this profile in which there is a great laboratorial and clinical interest.

  6. Demonstration and partial characterization of 22-nm HBsAg and Dane particles of subtype HBsAg/ady.

    PubMed

    Hess, G; Shih, J W; Arnold, W; Gerin, J L; zum Büschenfelde, K H

    1979-09-01

    The present paper describes the demonstration of d, y, w, and r HBsAg determinants in one serum. It was shown that there are two populations of HBsAg particles: HBsAg/ad and HBsAg/ady. All complete Dane particles were of subtype HBsAg/ady. Further characterization of HBsAg/ady particles did not reveal morphologic differences when they were compared with HBsAg/ad and HBsAg/ay particles. An HBsAg/ady phenotype may be the result of a double infection with hepatitis B viruses or exchanges of DNA sequences that determine HBsAg/ay and HBsAg/ad to form a new genotype. PMID:89163

  7. Demonstration and partial characterization of 22-nm HBsAg and Dane particles of subtype HBsAg/ady.

    PubMed

    Hess, G; Shih, J W; Arnold, W; Gerin, J L; zum Büschenfelde, K H

    1979-09-01

    The present paper describes the demonstration of d, y, w, and r HBsAg determinants in one serum. It was shown that there are two populations of HBsAg particles: HBsAg/ad and HBsAg/ady. All complete Dane particles were of subtype HBsAg/ady. Further characterization of HBsAg/ady particles did not reveal morphologic differences when they were compared with HBsAg/ad and HBsAg/ay particles. An HBsAg/ady phenotype may be the result of a double infection with hepatitis B viruses or exchanges of DNA sequences that determine HBsAg/ay and HBsAg/ad to form a new genotype.

  8. Production of highly concentrated, heat stable hepatitis B surface antigen in maize

    PubMed Central

    Hayden, Celine A.; Egelkrout, Erin M.; Moscoso, Alessa M.; Enrique, Cristina; Keener, Todd K.; Jimenez-Flores, Rafael; Wong, Jeffrey C.; Howard, John A.

    2012-01-01

    Summary Plant-based oral vaccines are a promising emergent technology that could help alleviate disease burden worldwide by providing a low-cost, heat stable, oral alternative to parenterally administered commercial vaccines. Here we describe high-level accumulation of the hepatitis B surface antigen (HBsAg) at a mean concentration of 0.51%TSP in maize T1 seeds using an improved version of the globulin1 promoter. This concentration is more than four-fold higher than any previously reported lines. HBsAg expressed in maize seeds was extremely heat stable, tolerating temperatures up to 55°C for one month without degradation. Optimal heat stability was achieved after oil extraction of ground maize material, either by supercritical fluid extraction or hexane treatment. The contributions of this material towards the development of a practical oral vaccine delivery system are discussed. PMID:22816734

  9. A PILOT EXTERNAL QUALITY ASSURANCE STUDY OF TRANSFUSION SCREENING FOR HIV, HCV AND HBSAG IN TWELVE AFRICAN COUNTRIES

    PubMed Central

    Bloch, Evan M; Shah, Avani; Kaidarova, Zhanna; Laperche, Syria; Lefrere, Jean-Jacques; van Hasselt, James; Zacharias, Peter; Murphy, Edward L

    2014-01-01

    Background and Objectives Serologic screening for the major transfusion transmissible viruses (TTV) is critical to blood safety and has been widely implemented. However, actual performance as measured by proficiency testing has not been well studied in Sub-Saharan Africa. Therefore, we conducted an external quality assessment of laboratories engaged in transfusion screening in the region. Materials and Methods Blinded test panels, each comprising 25 serum samples that were pedigreed for HIV, HBsAg, HCV and negative status, were sent to participating laboratories. The panels were tested using the laboratories’ routine donor screening methods and conditions. Sensitivity and specificity were calculated and multivariable analysis was used to compare performance against mode of testing, country and infrastructure. Results A total of 12 African countries and 44 laboratories participated in the study. The mean (range) sensitivities for HIV, HBsAg and HCV were 91.9% (14.3-100), 86.7% (42.9-100) and 90.1% (50-100), respectively. Mean specificities for HIV, HBsAg and HCV were 97.7%, 97% and 99.5% respectively. After adjusting for country and infrastructure, rapid tests had significantly lower sensitivity than enzyme immunoassays (EIA) for both HBsAg (p<0.0001) and HCV (p<0.05). Sensitivity also varied by country and selected infrastructure variables. Conclusion While specificity was high, sensitivity was more variable and deficient in a substantial number of testing laboratories. These findings underscore the importance of proficiency testing and quality control, particularly in Africa where TTV prevalence is high. PMID:25052195

  10. Optimization of in vitro HBV replication and HBsAg production in HuH7 cell line.

    PubMed

    Cavallone, Daniela; Moriconi, Francesco; Colombatto, Piero; Oliveri, Filippo; Bonino, Ferruccio; Brunetto, Maurizia Rossana

    2013-04-01

    The Gunther's vector-free method (GM), using PCR-amplified full length HBV-DNA (fl-HBV-DNA), is currently the best in vitro HBV replication system despite the low intracellular HBV-DNA production. The replication efficiency and HBsAg secretion of 12 isolates from HBsAg/HBeAg positive sera by GM, Monomer-Linear-Sticky-Ends-DNA (MLSE) and Monomer-Circular-Closed (MCC) were compared in HuH7 cells. Eight of twelve genomes (67%) were replication competent by GM; however direct sequencing (DS) showed that more than 80% of input DNA was undigested in spite of SapI treatment. Replication Intermediates (RI) were detected earlier (24 vs. 48h) and in higher amounts (2.51±0.32 and 6.43±0.43 fold) by MCC than GM or MLSE. By MCC 10 of 12 genomes (83%) were replication competent and 7 produced high RI levels. RI and HBsAg kinetics correlated positively in MCC (R=0.696, p=0.017 overall; R=0.928, p=0.008), but not in GM (R=-0.437, p=0.179 overall; R=-0.395, p=0.439) in genotype D isolates. In conclusion, HBV-DNA circularization prior transfection improves in vitro viral replication and replication competent HBsAg production, mimicking better the in vivo conditions.

  11. New Enzyme Immunoassay for Detection of Hepatitis B Virus Core Antigen (HBcAg) and Relation between Levels of HBcAg and HBV DNA

    PubMed Central

    Kimura, Tatsuji; Rokuhara, Akinori; Matsumoto, Akihiro; Yagi, Shintaro; Tanaka, Eiji; Kiyosawa, Kendo; Maki, Noboru

    2003-01-01

    A new enzyme immunoassay specific for hepatitis B virus (HBV) core antigen (HBcAg) was developed. In order to detect HBcAg, specimens were pretreated with detergents to release HBcAg from the HBV virion and disassemble it to dimers, and simultaneously, the treatment inactivated anti-HBc antibodies. HBcAg detected by the assay peaked with HBV DNA in density gradient fractions of HBV-positive sera. The assay showed a wide detection range from 2 to 100,000 pg/ml. We observed no interference from anti-HBc antibody or blood components, but the assay was inhibited by very high concentrations (>1 μg/ml; corresponding to 80 signal/cutoff) of HBeAg. When the cutoff value was tentatively set at 4 pg/ml, all healthy control (HBsAg and HBV DNA negative, n = 160) and anti-hepatitis C virus-positive (n = 55) sera were identified as negative. HBcAg concentrations correlated very closely with HBV DNA (r = 0.946, n = 145) in 216 samples from 72 hepatitis B patients. In seroconversion panels, HBcAg concentrations changed in parallel with HBV DNA levels. The assay, therefore, offers a simple method for monitoring hepatitis B patients. With a series of sera during lamivudine therapy, HBV DNA levels fell sharply and the HBcAg concentration also decreased, but the change in HBcAg was smaller and more gradual. The supposed mechanism of these changes and their clinical significance are discussed. PMID:12734224

  12. Epidemiological characteristics of the carriers with coexistence of HBsAg and anti-HBs based on a community cohort study.

    PubMed

    Pu, Z; Li, D; Wang, A; Su, H; Shao, Z; Zhang, J; Ji, Z; Gao, J; Choi, B C K; Yan, Y

    2016-04-01

    The coexistence of HBsAg and anti-HBs is an atypical serological pattern in HBV infection. There is no epidemiological characteristics of this serological pattern in the community and there is controversy over the molecular mechanisms underlying this pattern. We investigated the epidemiological characteristics of the carriers with HBsAg and anti-HBs in a longitudinal community cohort study. The prevalence of this atypical serological pattern was 2.93% (122/4169) in HBsAg-positive populations. The prevalence progressively increased with age from 40 to 70 years old. The rate of HBeAg positive and detectable HBV DNA were both significantly higher in carriers with this pattern than in carriers who were HBsAg positive but anti-HBs negative (26/122 verse 598/4047, P = 0.046; 86/122 verse 275/529,P < 0.001). After 1 year of follow-up, 85.19% of the carriers still had coexistence HBsAg and anti-HBs, 14.81% of the carriers lost their anti-HBs. Viral sequencing showed that carriers with coexistence of HBsAg and anti-HBs had higher numbers of residue changes within the S gene than carriers who were HBsAg positive but anti-HBs negative (2.42 verse 1.33 changes per 100 residues, P < 0.05). Hence, the coexistence of HBsAg and anti-HBs is a unique serological pattern which may be associated with an increased risk of adverse clinical outcome and may be related to HBsAg immune variants which have genotypic heterogeneity.

  13. [Comparison of adsorbent with varying arm length and ligand density for the purification of recombinant hepatitis B virus surface antigen].

    PubMed

    Li, Rui-Hong; Li, Yan; Bi, Jing-Xiu; Zhao, Lan; Zhou, Wei-Bin; Huang, Yong-Dong; Zhang, Yan; Sun, Li-Jing; Wang, Hua-Jun; Su, Zhi-Guo

    2007-07-01

    Novel hydrophobic absorbents were synthesized by immobilizing butyl derivative onto the highly cross-linked agarose beads manufatured in China, which are used as matrix. The effect of the spacer arm length (3C, 8C and 10C) and ligand density (from 13 to 45 micromol/mL) on the hydrophobicity were investigated using purified Hepatitis B surface antigen (HBsAg) expressed by CHO cell lines. Also considering the effects of salt concentration and pH on HBsAg recovery and purification factor, orthogonal experiment design method was used to evaluated the absorbents. The results showed the butyl-S absorbent with the spacer arm length of C8, the ligand density of 22 micromol/mL gel showed the best performance for the separation of HBsAg. Approximately 100% HBsAg recovery and 60 as purification fold were achieved by this media under the operating condition of pH 7.0 and 9% of salt concentrateion.

  14. Study of the immunogenicity of hepatitis B surface antigen synthesized in transgenic potato plants with increased biosafety.

    PubMed

    Rukavtsova, Elena B; Rudenko, Natalya V; Puchko, Elena N; Zakharchenko, Natalya S; Buryanov, Yaroslav I

    2015-06-10

    Oral immunogenicity of the hepatitis B surface antigen (HBsAg) synthesized in the tubers of marker-free potato plants has been demonstrated. Experiments were performed in the two groups of outbred NMRI mice. At the beginning of investigations, the mice of experimental group were fed the tubers of transgenic potato synthesizing the HBsAg three times. The mice of control group were fed nontransgenic potato. Intraperitoneal injection of the commercial vaccine against hepatitis B (0.5μg/mouse) was made on day 71 of the experiment. Enzyme-linked immunoassay (ELISA) of the serum of immunized animals showed an increase in the level of HBsAg antibodies significantly above the protective value, which was maintained for 1 year after the immunization. In 1 year, the experimental group of mice underwent additional oral immunization with HBsAg-containing potato tubers. As a result, the level of antibodies against the HBsAg increased and remained at a high protective level for several months. The findings show the possibility of using transgenic plants as a substance for obtaining a safe edible vaccine against hepatitis B. PMID:25840367

  15. Prevalence, Risk Factors, and Impact of Isolated Antibody to Hepatitis B Core Antigen and Occult Hepatitis B Virus Infection in HIV-1–Infected Pregnant Women

    PubMed Central

    Khamduang, Woottichai; Ngo-Giang-Huong, Nicole; Gaudy-Graffin, Catherine; Jourdain, Gonzague; Suwankornsakul, Weerapong; Jarupanich, Tapnarong; Chalermpolprapa, Veeradate; Nanta, Sirisak; Puarattana-aroonkorn, Noossara; Tonmat, Sakchai; Lallemant, Marc; Goudeau, Alain; Sirirungsi, Wasna

    2013-01-01

    Background. Prevalence and risk factors for isolated antibody to hepatitis B core antigen (anti-HBc) and occult hepatitis B virus (HBV) infection are not well known in human immunodeficiency virus type 1 (HIV-1)–infected pregnant women. It is unclear if women with occult infections are at risk of transmitting HBV to their infants. Methods. HIV-1–infected and HBV surface antigen (HBsAg)–negative pregnant women were tested for antibody to HBsAg (anti-HBs) and anti-HBc using enzyme immunoassay. Women with isolated anti-HBc were assessed for occult HBV infection, defined as HBV DNA levels >15 IU/mL, using the Abbott RealTime HBV DNA assay. Infants born to women with isolated anti-HBc and detectable HBV DNA were tested at 4 months of age for HBV DNA. Logistic regression analysis was used to identify factors associated with isolated anti-HBc and occult HBV infection. Results. Among 1812 HIV-infected pregnant women, 1682 were HBsAg negative. Fourteen percent (95% confidence interval [CI], 12%–15%) of HBsAg-negative women had an isolated anti-HBc that was independently associated with low CD4 count, age >35 years, birth in northern Thailand, and positive anti–hepatitis C virus serology. Occult HBV infection was identified in 24% (95% CI, 18%–30%) of women with isolated anti-HBc, representing 2.6% (95% CI, 1.9%–3.5%) of HIV-1–infected pregnant women, and was inversely associated with HIV RNA levels. None of the women with isolated anti-HBc and occult HBV infection transmitted HBV to their infants. Conclusions. HIV-1–infected pregnant women with isolated anti-HBc and occult HBV infection have very low HBV DNA levels and are thus at very low risk to transmit HBV to their infants. PMID:23487379

  16. The use of sucrose-acetone-extracted Rift Valley fever virus antigen derived from cell culture in an indirect enzyme-linked immunosorbent assay and haemagglutination-inhibition test.

    PubMed

    Paweska, J T; Barnard, B J; Williams, R

    1995-12-01

    A sucrose-acetone-extracted, Madin-Darby-bovine-kidney (MDBK)-derived Rift Valley fever virus (RVFV) antigen was tested both in an indirect ELISA and a haemagglutination-inhibition test for its ability to detect serum antibodies to RVFV. Optimal conditions for antigen concentration, serum and conjugate dilutions for the ELISA were established by checkerboard titration. The specificity and sensitivity of ELISA were determined by the use of paired pre- and post-vaccination sheep-serum samples. Compared with the virus neutralization test, the overall ELISA specificity and sensitivity were 97.4 and 97.3%, respectively. There was a 100% correlation between the results obtained in haemagglutination-inhibition tests with a RVFV sucrose-acetone-extracted antigen derived from hamster liver, and from MDBK cells. A total of 10 582 field-serum samples (84 cattle, 3,659 sheep, 6,839 goats) collected in 1994-1995 from animals of unknown vaccination status in different regions of South Africa were tested with ELISA for antibodies against RVFV. There were no seropositive cattle, 0.16% seropositive sheep and 0.12% seropositive goats. This study demonstrates the potential diagnostic application of cell-culture-derived, sucrose-acetone-extracted RVFV antigen in an indirect ELISA and HI test.

  17. Experimental studies on the inhibition effects of 1000 Chinese medicinal herbs on the surface antigen of hepatitis B virus.

    PubMed

    Zheng, M; Zheng, Y

    1992-09-01

    The reverse passive hemagglutination inhibition test was used in the screening of 1000 Chinese materia medica for inhibitors of hepatitis B virus surface antigen. The herbal drugs were commercially available and the surface antigen was from sera of hepatitis B patients. 127 effective drugs were obtained from the survey of which 28 were highly inhibitory (8:1), 35 were moderately effective (4:1), and 64 mildly effective (2:1). Further experiments with varying dosages of the drug, dosages of HBsAg and duration of contact showed 10 drugs to be of optimal effect. PMID:1453758

  18. A computational framework for influenza antigenic cartography.

    PubMed

    Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2010-10-07

    Influenza viruses have been responsible for large losses of lives around the world and continue to present a great public health challenge. Antigenic characterization based on hemagglutination inhibition (HI) assay is one of the routine procedures for influenza vaccine strain selection. However, HI assay is only a crude experiment reflecting the antigenic correlations among testing antigens (viruses) and reference antisera (antibodies). Moreover, antigenic characterization is usually based on more than one HI dataset. The combination of multiple datasets results in an incomplete HI matrix with many unobserved entries. This paper proposes a new computational framework for constructing an influenza antigenic cartography from this incomplete matrix, which we refer to as Matrix Completion-Multidimensional Scaling (MC-MDS). In this approach, we first reconstruct the HI matrices with viruses and antibodies using low-rank matrix completion, and then generate the two-dimensional antigenic cartography using multidimensional scaling. Moreover, for influenza HI tables with herd immunity effect (such as those from Human influenza viruses), we propose a temporal model to reduce the inherent temporal bias of HI tables caused by herd immunity. By applying our method in HI datasets containing H3N2 influenza A viruses isolated from 1968 to 2003, we identified eleven clusters of antigenic variants, representing all major antigenic drift events in these 36 years. Our results showed that both the completed HI matrix and the antigenic cartography obtained via MC-MDS are useful in identifying influenza antigenic variants and thus can be used to facilitate influenza vaccine strain selection. The webserver is available at http://sysbio.cvm.msstate.edu/AntigenMap.

  19. Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus.

    PubMed Central

    Salfeld, J; Pfaff, E; Noah, M; Schaller, H

    1989-01-01

    The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. The single conformational determinant responsible for HBc antigenicity in the assembled core (HBc) and a linear HBe-related determinant (HBe1) were both mapped to an overlapping hydrophilic sequence around amino acid 80; a second HBe determinant (HBe2) was assigned to a location in the vicinity of amino acid 138 but found to require for its antigenicity the intramolecular participation of the extended sequence between amino acids 10 and 140. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis B virus nucleocapsid. Images PMID:2463383

  20. Evaluation of a new fourth generation enzyme-linked immunosorbent assay, the LG HIV Ag-Ab Plus, with a combined HIV p24 antigen and anti-HIV-1/2/O screening test.

    PubMed

    Yeom, Joon-Sup; Jun, Gyo; Chang, Young; Sohn, Mi-Jin; Yoo, Seungbum; Kim, Eunkyung; Ryu, Seung-Ho; Kang, Hee-Jung; Kim, Young-A; Ahn, Sun-Young; Cha, Je-Eun; Youn, Sung-Tae; Park, Jae-Won

    2006-11-01

    The LG HIV Ag-Ab Plus, a new fourth generation diagnostic assay for HIV infection, was evaluated in comparison to the Enzygnost HIV Integral, an established fourth generation HIV assay. The LG assay showed 100% sensitivity with 109 samples with anti-HIV-1, anti-HIV-2 or anti-HIV-1 group O reactivity. It also detected correctly all 51 positives on three BBI performance panels, slightly outperforming the Enzygnost HIV Integral, which detected 50. The specificity of the LG HIV Ag-Ab Plus was 99.9% with 999 sera from healthy blood donors, which was slightly inferior to the performance of the Enzygnost HIV Integral, which had 100% specificity. The LG assay showed 100% specificity with 81 specimens with underlying diseases including hepatitis B, demonstrating a low risk of cross-reactivity with other infections. The reduction of the diagnostic window by the LG HIV Ag-Ab Plus, compared to a third generation HIV assay, was 6.3 days. The LG assay also showed sufficiently high intra-person and inter-person reproducibility. The overall performance of this new fourth generation HIV assay was adequate for screening and diagnosis of HIV infection.

  1. Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus

    SciTech Connect

    Salfeld, J.; Pfaff, E.; Noah, M.; Schaller, H.

    1989-02-01

    The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen (HBcAg)) and a secreted processed protein (p17e, serologically defined as HBe antigen (HBeAg)). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis virus nucleocapsid.

  2. Integrating influenza antigenic dynamics with molecular evolution

    PubMed Central

    Bedford, Trevor; Suchard, Marc A; Lemey, Philippe; Dudas, Gytis; Gregory, Victoria; Hay, Alan J; McCauley, John W; Russell, Colin A; Smith, Derek J; Rambaut, Andrew

    2014-01-01

    Influenza viruses undergo continual antigenic evolution allowing mutant viruses to evade host immunity acquired to previous virus strains. Antigenic phenotype is often assessed through pairwise measurement of cross-reactivity between influenza strains using the hemagglutination inhibition (HI) assay. Here, we extend previous approaches to antigenic cartography, and simultaneously characterize antigenic and genetic evolution by modeling the diffusion of antigenic phenotype over a shared virus phylogeny. Using HI data from influenza lineages A/H3N2, A/H1N1, B/Victoria and B/Yamagata, we determine patterns of antigenic drift across viral lineages, showing that A/H3N2 evolves faster and in a more punctuated fashion than other influenza lineages. We also show that year-to-year antigenic drift appears to drive incidence patterns within each influenza lineage. This work makes possible substantial future advances in investigating the dynamics of influenza and other antigenically-variable pathogens by providing a model that intimately combines molecular and antigenic evolution. DOI: http://dx.doi.org/10.7554/eLife.01914.001 PMID:24497547

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

    PubMed

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

    2016-08-30

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

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

    PubMed

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

    2016-08-30

    The monoclonal antibody-based circulating antigen detecting ELISA (B158/B60 Ag-ELISA) has been used elaborately in several studies for the diagnosis of human, bovine and porcine cysticercosis. Interpretation of test results requires a good knowledge of the test characteristics, including the repeatability and the effect of the borders of the ELISA plates. Repeatability was tested for 4 antigen-negative and 5 antigen-positive reference bovine serum samples by calculating the Percentage Coefficient of Variation (%CV) within and between plates, within and between runs, overall, for two batches of monoclonal antibodies and by 2 laboratory technicians. All CV values obtained were below 20% (except one: 24.45%), which indicates a good repeatability and a negligible technician error. The value of 24.45% for indicating the variability between batches of monoclonal antibodies for one positive sample is still acceptable for repeatability measures. Border effects were determined by calculating the %CV values between the inner and outer wells of one plate for 2 positive serum samples. Variability is a little more present in the outer wells but this effect is very small and no significant border effect was found. PMID:27523940

  5. Immunogenicity and safety of Advax™, a novel polysaccharide adjuvant based on delta inulin, when formulated with hepatitis B surface antigen: a randomized controlled Phase 1 study.

    PubMed

    Gordon, David; Kelley, Peter; Heinzel, Susanne; Cooper, Peter; Petrovsky, Nikolai

    2014-11-12

    There is a need for additional safe and effective human vaccine adjuvants. Advax™ is a novel adjuvant produced from semi-crystalline particles of delta inulin. In animal studies Advax enhanced humoral and cellular immunity to hepatitis B surface antigen (HBsAg) without inducing local or systemic reactogenicity. This first-in-man Phase 1 clinical trial tested the safety and tolerability of three intramuscular doses of HBsAg formulated with Advax in a group of healthy adult subjects. Advax was well tolerated with injection site pain scores not significantly different to subjects receiving HBsAg alone and no adverse events were reported in subjects that received Advax. Seroprotection and HBsAb geometric mean titers (GMT) after three immunizations were higher in the Advax 5mg (seroprotection 5/6, 83.3%, GMT 40.7, 95% CI 11.9-139.1) and 10mg (seroprotection 4/5, 80%, GMT 51.6, 95% CI 10.0-266.2) groups versus HBsAg alone (seroprotection 1/5, 20%, GMT 4.1, 95% CI 1.3-12.8). Similarly the proportion of subjects with positive CD4 T-cell responses to HBsAg was higher in the Advax 5mg (4/6, 67%) and Advax 10mg (4/5, 80%) groups versus HBsAg alone (1/5, 20%). These results confirm the safety, tolerability and immunogenicity of Advax adjuvant observed in preclinical studies. Advax may represent a suitable replacement for alum adjuvants in prophylactic human vaccines subject to confirmation of current results in larger studies. Australia and New Zealand Clinical Trial Registry: ACTRN12607000598482.

  6. Improvement of arbovirus HA antigens by treatment with a colloidal silica gel and sonication.

    PubMed

    Traavik, T

    1977-01-01

    A remarkable increase in HA titers for weakly haemagglutinating Norwegian arbovirus strains, Uukuniemi and Runde viruses, was achieved by including treatment with the colloidal silica gel Aerosil in the antigen preparation scheme. By combining this procedure with sonication, the titers of sucrose-aceton extracted, infected suckling mouse brains could be increased several hundred times. Good antigens also were obtained from virus grown in BHK21/c 13 cell cultures and concentrated by polyethylene glycol 6000/NaCl. Rubella virus HA antigen and HBsAg were adsorbed to the gel, and excluded from a preparation by treatment with Aerosil. This indicates a limitation to the universal use of the method, presumably related to the particle size.

  7. Complement fixing hepatitis B core antigen immune complexes in the liver of patients with HBs antigen positive chronic disease.

    PubMed Central

    Rizzetto, M; Bonino, F; Crivelli, O; Canese, M G; Verme, G

    1976-01-01

    One hundred and fifty-two biopsies from serologically HBsAg positive and negative patients with liver disease were studied in immunofluorescence: for the presence of the surface (HBs) and the core (HBc) antigenic determinants foeterminants of the hepatitis B virus, of immunoglobulins and complement (C) deposits, and for the capacity to fix human C. Circumstantial evidence is presented suggesting that HBc immune-complexes are a relevant feature in the establishment and progression of chronic HBSAg liver disease. C fixation by liver cells was shown in all HBC positive patients with chronic hepatitis; an active form was present in every case, except two with a persistent hepatitis, an inverse ratio of HBc to C binding fluorescence being noted between active chronic hepatitis and cirrhotic patients. HBc without C fixation was observed in only three patients in the incubation phase of infectious hepatitis. IgG deposits were often found in HBc containing, C fixing nuclei. No C binding or IgG deposits were observed in acute self-limited type B hepatitis, in serologically positive patients with normal liver or minimal histological lesions, with and without HBs cytoplasmic fluorescence in their biopsy, or in serologically negative individuals. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:1001973

  8. Development of an in-house enzyme-linked immunosorbent assay based on surface whole cell antigen for diagnosis of Helicobacter pylori infection in patients with gastroduodenal ulcer disease.

    PubMed

    Aziz, Faisal; Sherwani, Sikander Khan; Akhtar, Syed Shakeel; Kazmi, Shahana Urooj

    2014-01-01

    Helicobacter pylori (H. pylori) is a causative agent of gastritis, gastroduodenal ulcers and gastric adenocarcinoma. More than 50% world population is colonized by H. pylori, which is closely related to the chronic gastritis and gastric ulcer infection. In this study, a total of 214 gastritis patient's serum samples were screened for anti-H. pylori IgG antibody. A 96-well plate coated with 20 μg/ml antigen and hundred-fold diluted patient's serum was allowed to react. After extensive washing with buffer, 1:2,500 diluted conjugated secondary antibody was added. Later substrate was added to observe positivity by measuring the intensity of color. Statistical analyses were performed, and p value of <0.01 was taken as significant; 84% male patients and 89% female patients, respectively, tested positive for H. pylori, while agewise distribution was 35-45 years males (40%) and 35-55 years females (52%) were found highest number of H. pylori infected patients. In-house ELISA based on surface whole cell antigen (wELISA) showed a sensitivity of 93%, specificity of 100%, accuracy 94% and κ value 0.86 with significant correlation R-0.77020; p < 0.0001. We conclude that H. pylori local isolates surface antigen was satisfactory for diagnosis as different parameters were adjusted according to the local H. pylori isolates. Fluctuations in serum antibody titer predict the variation in an individual's response of the host against H. pylori. In-house wELISA could provide a reliable and a clinically useful method for the diagnosis of H. pylori infection in patients of Karachi, Pakistan.

  9. Prevalence study of Bovine viral diarrhea virus by evaluation of antigen capture ELISA and RT-PCR assay in Bovine, Ovine, Caprine, Buffalo and Camel aborted fetuses in Iran

    PubMed Central

    2011-01-01

    Bovine viral diarrhea virus is a pestivirus in the family Flaviviridae that cause abortions and stillbirths in livestock and its traditional diagnosis is based on cell culture and virus neutralization test. In this study, for more sensitive, specific detection and determined the prevalence of virus in aborted Bovine, Ovine, Caprine, Buffalo and Camel fetuses the antigen capture ELISA and RT-PCR were recommended. From the total of 2173 aborted fetuses, 347 (15.96%) and 402 (18.49%) were positive for presence of Bovine viral diarrhea virus by antigen capture ELISA and RT-PCR respectively. Statistical analysis of data showed significant differences between ELISA and RT-PCR for detection of virus in aborted fetuses. These results indicate a high presence of this pathogen in Iran and that RT- PCR is considerably faster and more accurate than ELISA for identification of Bovine viral diarrhea virus. To our knowledge the Camels and Bovine are the most resistant and sensitive to Bovine viral diarrhea's abortions respectively and the prevalence of virus in Caprine is more than Ovine aborted fetuses. This study is the first prevalence report of Bovine viral diarrhea virus in aborted Bovine, Ovine, Caprine, Buffalo and Camel fetuses by evaluation of ELISA and RT-PCR in Iran. PMID:22018096

  10. Tumor-associated antigens in gynecologic cancer.

    PubMed

    DiSaia, P J

    1975-12-01

    If the study of tumor immunology is to have a profound impact on clinical medicine, certain hypotheses must be proven to be valid. First and foremost, it must be demonstrated that malignant tissue possesses antigenic substances (probably protein moieties) that are unique to that particular malignant process. In addition, these antigenic substances must be very similar in histologically similar tumors. Second, the host defense mechanisms must be capable of reacting to these tumor-associated antigens. The reaction is, of course, necessary in order to develop both diagnostic and therapeutic routes of application. The reaction of the immunologic system to these tumor-associated antigens could be monitored as an early serodiagnostic tool for subclinical cancer, and the cytotoxic reaction holds great promise as an immunotherapeutic tool. The essence of tumor immunologic research can thus be stated in the form of the following questions: 1. Do histologically similar cancers from identical primary sites share common tumor-associated antigens? 2. Does the immunologic system react to these antigens? 3. Can this reaction be assayed on one hand for serodiagnosis and augmented on the other for immunotherapy? Specific antigens have been found in animal tumors and have been divided into two classes: the viral induced tumors, which share common antigens when caused by the same viral agent, and carcinogen-induced tumors, which appear to have unique antigenic determinants for each tumor. In recent years a great many human tumors have been found to have tumor-associated antigens; these include colonic carcinoma, neuroblastoma, melanoma, soft tissue and osteogenic sarcoma, bladder carcinoma and Burkitt's lymphoma. This report includes evidence for the existence of such antigens in adenocarcinoma of the ovary and squamous cell carcinoma of the cervix. The laboratory evidence that has been presented would suggest that there are both a cell-mediated response and humoral response to the

  11. Antigenic diversity of lipooligosaccharides of nontypable Haemophilus influenzae.

    PubMed Central

    Campagnari, A A; Gupta, M R; Dudas, K C; Murphy, T F; Apicella, M A

    1987-01-01

    The lipooligosaccharides (LOS) of nontypable Haemophilus influenzae are an antigenically heterogeneous group of macromolecules. Immunodiffusion and enzyme-linked immunosorbent assay inhibition studies with phenol-water-extracted LOS and absorbed antisera specific for the oligosaccharide portion of the LOS identified six LOS strain-specific antigens. To facilitate screening large numbers of strains to search for LOS antigenic heterogeneity, a system utilizing proteinase K whole cell digests in Western blots was developed. Seventy-two nontypable H. influenzae LOS extracts were analyzed in this Western blot assay. Thirty-seven of these extracts could be segregated into 10 antigenically distinct LOS groups based on immunologic recognition by one or more of the rabbit antisera. Thirty-five of the strains did not contain these LOS antigens. These results demonstrate that antigenic differences exist among the LOS of nontypable H. influenzae strains, and this heterogeneity has the potential to be used to establish an LOS-based serogrouping system. Images PMID:3549563

  12. Synthesis of antibodies to hepatitis B virus by cultured lymphocytes from chronic hepatitis B surface antigen carriers

    SciTech Connect

    Dusheiko, G.M.; Hoofnagle, J.H.; Cooksley, W.G.; James, S.P.; Jones, E.A.

    1983-05-01

    It has been postulated that host immune defects are responsible for the development and persistence of the hepatitis B surface antigen (HBsAg) carrier state. The synthesis of both anti-HBs and antibody to hepatitis B core antigen (anti-HBc) in cultures containing peripheral blood mononuclear cells from chronic HBsAg carriers and from control (antibody-positive) patients was measured in the presence of pokeweed mitogen. Similar amounts of polyclonal IgG and IgM were synthesized by cultures containing lymphocytes from chronic carriers and controls. Anti-HBc was detectable in lymphocyte supernatants from 2 of 20 controls and from 21 of 29 carriers. The presence of anti-HBc synthesis in vitro correlated with high serum titers of anti-HBc. In contrast, anti-HBs was detected in lymphocyte supernatants from 6 of 20 controls (predominantly in those who had high serum titers of anti-HBs) but in none of the supernatants from 29 HBsAg carriers. Co-culture experiments were performed using T and B lymphocyte fractions that had been purified by affinity chromatography. B lymphocytes from carriers co-cultured with allogeneic irradiated (''helper'') T lymphocytes from controls synthesized normal amounts of IgG, IgM, and anti-HBc but still did not synthesize detectable amounts of anti-HBs. In the converse experiments, B lymphocytes from controls were co-cultured with irradiated T lymphocytes from carriers. The T lymphocytes from 16 of 24 carriers augmented anti-HBs production by control B cells normally, the remaining eight did not. Finally, mixtures of control B cells and control irradiated T lymphocytes were co-cultured with T lymphocytes from chronic HBsAg carriers. 5 of 12 carriers demonstrated active suppression of anti-HBs production, and in three this suppression was specific, as IgG and IgM production remained normal.

  13. Rate of hepatitis B virus infection in pregnant women determined by a monoclonal hepatitis B surface antigen immunoassay.

    PubMed

    Gotstein, Melissa G; Aide, Paula M; Coleman, Paul F; Sanborn, Mark R

    2002-09-01

    The rate of HBsAg in 6,976 B-human chorionic gonadotropin (B-hCG)-positive specimens, as determined by the Auszyme Monoclonal assay (Abbott Laboratories, Abbott Park, Ill.), was 0.56% (39 of 6,986 repeatedly reactive [RR] and confirmed-positive specimens). All RR and confirmed specimens were hepatitis B virus positive by at least one additional test, yielding an assay specificity of 99.96%. The findings argue against unique attributes in the pregnant population that might produce inaccurate assay results. PMID:12202601

  14. Frequency and significance of antibodies against hepatitis B core (anti-HBc) antigen as the only serological marker for hepatitis B infection in Lebanese blood donors.

    PubMed Central

    Ramia, S.; Ramlawi, F.; Kanaan, M.; Klayme, S.; Naman, R.

    2005-01-01

    During a 2-year period, blood samples from 2505 Lebanese blood donors were chosen at random, at various periods of time at one blood donation centre (Hotel Dieu de France, Beirut, Lebanon) and were screened for markers of HBV infection (HBsAg, anti-HBc and anti-HBs). The study showed HBsAg positivity of 0.6% and an overall exposure rate to HBV of 10.0%. Out of the 2505 blood donors screened, 56 (22%) were found to be 'anti-HBc alone' positive which is almost four times the HBsAg positivity. The 56 'anti-HBc alone' samples were retested by another ELISA kit commercially available and 54 samples were 'anti-HBc alone' positive by both assays. The 54 samples had no serological markers as evidence of infection with human immunodeficiency virus (HIV) or hepatitis C virus (HCV). Only seven (13%) out of the 54 samples were HBV DNA positive by PCR and all were HBV genotype D. All seven HBV DNA-positive samples had HBV DNA levels below 400 copies/ml. Although any circulating HBV DNA among our 'anti-HBc alone' blood donors was below the detection limit of our Amplicor Monitor assay, some of these samples had circulating virus. A national study, where a larger number of blood donors from different blood donation centres across the country will perhaps determine whether screening for anti-HBc in addition to HBsAg detection is needed in Lebanese blood donors. PMID:16050516

  15. Mutations of pre-core and basal core promoter before and after hepatitis B e antigen seroconversion

    PubMed Central

    Kamijo, Nozomi; Matsumoto, Akihiro; Umemura, Takeji; Shibata, Soichiro; Ichikawa, Yuki; Kimura, Takefumi; Komatsu, Michiharu; Tanaka, Eiji

    2015-01-01

    AIM: To investigate the role of pre-core and basal core promoter (BCP) mutations before and after hepatitis B e antigen (HBeAg) seroconversion. METHODS: The proportion of pre-core (G1896A) and basal core promoter (A1762T and G1764A) mutant viruses and serum levels of hepatitis B virus (HBV) DNA, hepatitis B surface antigen (HBsAg), and HB core-related antigen were analyzed in chronic hepatitis B patients before and after HBeAg seroconversion (n = 25), in those who were persistently HBeAg positive (n = 18), and in those who were persistently anti-HBe positive (n = 43). All patients were infected with HBV genotype C and were followed for a median of 9 years. RESULTS: Although the pre-core mutant became predominant (24% to 65%, P = 0.022) in the HBeAg seroconversion group during follow-up, the proportion of the basal core promoter mutation did not change. Median HBV viral markers were significantly higher in patients without the mutations in an HBeAg positive status (HBV DNA: P = 0.003; HBsAg: P < 0.001; HB core-related antigen: P = 0.001). In contrast, HBV DNA (P = 0.012) and HBsAg (P = 0.041) levels were significantly higher in patients with the pre-core mutation in an anti-HBe positive status. CONCLUSION: There is an opposite association of the pre-core mutation with viral load before and after HBeAg seroconversion in patients with HBV infection. PMID:25593470

  16. Single-assay combination of Epstein-Barr Virus (EBV) EBNA1- and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening.

    PubMed

    Fachiroh, J; Paramita, D K; Hariwiyanto, B; Harijadi, A; Dahlia, H L; Indrasari, S R; Kusumo, H; Zeng, Y S; Schouten, T; Mubarika, S; Middeldorp, J M

    2006-04-01

    Assessment of immunoglobulin A (IgA) antibody responses to various Epstein-Barr virus (EBV) antigen complexes, usually involving multiple serological assays, is important for the early diagnosis of nasopharyngeal carcinoma (NPC). Through combination of two synthetic peptides representing immunodominant epitopes of EBNA1 and viral capsid antigen (VCA)-p18 we developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of EBV reactive IgG and IgA antibodies in NPC patients (EBV IgG/IgA ELISA). Sera were obtained from healthy donors (n = 367), non-NPC head and neck cancer patients (n = 43), and biopsy-proven NPC patients (n = 296) of Indonesian and Chinese origin. Higher values of optical density at 450 nm for EBV IgG were observed in NPC patients compared to the healthy EBV carriers, but the large overlap limits its use for NPC diagnosis. Using either EBNA1 or VCA-p18 peptides alone IgA ELISA correctly identified 88.5% and 79.8% of Indonesian NPC patients, with specificities of 80.1% and 70.9%, whereas combined single-well coating with both peptides yielded sensitivity and specificity values of 90.1 and 85.4%, respectively. The positive and negative predictive values (PPV and NPV, respectively) for the combined EBNA1 plus VCA EBV IgA ELISA were 78.7% and 93.9%, respectively. In the Indonesia panel, the level of EBV IgA reactivity was not associated with NPC tumor size, lymph node involvement, and metastasis stage, sex, and age group. In the China panel the sensitivity/specificity values were 86.2/92.0% (EBNA1 IgA) and 84.1/90.3% (VCA-p18 IgA) for single-peptide assays and 95.1/90.6% for the combined VCA plus EBNA1 IgA ELISA, with a PPV and an NPV for the combined EBV IgA ELISA of 95.6 and 89.3%, respectively. Virtually all NPC patients had abnormal anti-EBV IgG diversity patterns as determined by immunoblot analysis. On the other hand, healthy EBV carriers with positive EBV IgA ELISA result showed normal IgG diversity patterns

  17. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies.

  18. Large hepatitis delta antigen in packaging and replication inhibition: role of the carboxyl-terminal 19 amino acids and amino-terminal sequences.

    PubMed

    Lee, C Z; Chen, P J; Chen, D S

    1995-09-01

    Hepatitis delta virus (HDV) encodes two proteins, the small delta antigen (SHDAg) and large delta antigen (LHDAg). The latter is identical to the former except for the presence of additional 19 amino acids at the C terminus. While SHDAg is required for HDV replication, LHDAg inhibits replication and, together with hepatitis B surface antigen (HBsAg), is required for the assembly of HDV. The last 19 C-terminal amino acids of LHDAg are essential for HDV assembly. Most of LHDAg (amino acids 19 to 146 and 163 to 195) had been shown to be dispensable for packaging with HBsAg. To discern whether the last 19 C-terminal amino acids solely constitute the signal for packaging with HBsAg, we constructed two LHDAg deletion mutants and tested their abilities to be packaged with HBsAg in cotransfection experiments. We found that deletion of amino acids 2 to 21 and 142 to 165 did not affect LHDAg packaging. This result suggested that only the last 19 C-terminal amino acids of LHDAg are required for packaging. We further constructed two plasmids which expressed c-H-ras with or without additional 19 C-terminal amino acids identical to those in LHDAg. Only c-H-ras with additional 19 amino acids could be cosecreted with HBsAg in the cotransfection experiment. This result confirmed that the C-terminal 19 amino acids are the packaging signal for HBsAg. We also tested the trans activation activity and trans-dominant inhibitory activity of the deletion mutants of SHDAg and LHDAg, respectively. In contrast to deletion of amino acids 142 to 165, deletion of amino acids 2 to 21 impaired the trans-dominant inhibitory activity of LHDAg. Deletion of amino acids 2 to 21 and 142 to 165 did not affect the trans activation activity of SHDAg. This result suggested that a functional domain which is important for the trans-dominant inhibitory activity of LHDAg exists in the amino terminus of HDAg. PMID:7636976

  19. Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

    PubMed

    Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

    2013-11-01

    In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

  20. Serodiagnosis of Lyme disease by kinetic enzyme-linked immunosorbent assay using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with 2-tiered testing using whole-cell lysates.

    PubMed

    Bacon, Rendi Murphree; Biggerstaff, Brad J; Schriefer, Martin E; Gilmore, Robert D; Philipp, Mario T; Steere, Allen C; Wormser, Gary P; Marques, Adriana R; Johnson, Barbara J B

    2003-04-15

    In a study of US patients with Lyme disease, immunoglobulin (Ig) G and IgM antibody responses to recombinant Borrelia burgdorferi antigen VlsE1 (rVlsE1), IgG responses to a synthetic peptide homologous to a conserved internal sequence of VlsE (C6), and IgM responses to a synthetic peptide comprising the C-terminal 10 amino acid residues of a B. burgdorferi outer-surface protein C (pepC10) were evaluated by kinetic enzyme-linked immunoassay. At 99% specificity, the overall sensitivities for detecting IgG antibody to rVlsE1 or C6 in samples from patients with diverse manifestations of Lyme disease were equivalent to that of 2-tiered testing. When data were considered in parallel, 2 combinations (IgG responses to either rVlsE1 or C6 in parallel with IgM responses to pepC10) maintained high specificity (98%) and were significantly more sensitive than 2-tiered analysis in detecting antibodies to B. burgdorferi in patients with acute erythema migrans. In later stages of Lyme disease, the sensitivities of the in parallel tests and 2-tiered testing were high and statistically equivalent.

  1. Cellulase Assays

    NASA Astrophysics Data System (ADS)

    Zhang, Y. H. Percival; Hong, Jiong; Ye, Xinhao

    Cellulose is a heterogeneous polysaccharide, and its enzymatic hydrolysis requires endoglucanase, exoglucanase (cellobiohydrolase), and β-glucosidase to work together. We summarize the most commonly used assays for individual enzymes and cellulase mixture.

  2. Phylogenetic analysis of HDV isolates from HBsAg positive patients in Karachi, Pakistan

    PubMed Central

    2012-01-01

    Background In spite of a high occurrence of Hepatitis Delta in the province of Sindh in Pakistan, no genetic study of Hepatitis Delta virus (HDV) isolates from this region was carried out. The aim of this study is to analyze the genetic proximity within local HDV strains, and relationship with other clades of HDV, using phylogenetic analysis. Results Phylogenetic analysis of nucleotide sequences of the Hepatitis Delta Antigen (HDAg) R0 region obtained in this study, showed considerable diversity among the local strains with a potential subgroup formation within clade I. The multiple sequence alignment of predicted amino acids within clade I showed many uncommon amino acid substitutions within some conserved regions that are crucial for replication and assembly of HDV. Conclusions The studied strains showed a range of genetic diversity within HDV clade I. There is clustering of sequences into more than one group, along with formation of potential subgroup within clade I. Clustering shows the genetic closeness of strains and indicates a common origin of spread of HDV infection. Further phylogeny-based studies may provide more information about subgroup formation within clade I and may be used as an effective tool in checking and/or preventing the spread of hepatitis D virus infection in this region. PMID:22894717

  3. Antibody secreting cell assay for influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ELISPOT assay to enumerate B-cells producing antibodies specific to a given antigen, also known as an antibody secreting cell (ASC) assay, was adapted to detect B-cells specific for influenza A virus (IAV). The assay is performed ex vivo and enumerates ASC at a single cell level. A simple ASC det...

  4. Functional study of hepatitis delta virus large antigen in packaging and replication inhibition: role of the amino-terminal leucine zipper.

    PubMed

    Chen, P J; Chang, F L; Wang, C J; Lin, C J; Sung, S Y; Chen, D S

    1992-05-01

    The large hepatitis delta antigen (HDAg) has been found to be essential for the assembly of the hepatitis delta virion. Furthermore, in a cotransfection experiment, the large HDAg itself, without the hepatitis delta virus (HDV) genome and small HDAg, could be packaged into hepatitis B surface antigen (HBsAg) particles. By deletion analysis, it was shown that the amino-terminal leucine zipper domain was dispensable for packaging. The large HDAg could also help in copackaging of the small HDAg into HBsAg particles without the need for HDV RNA. This process was probably mediated through direct interaction of the two HDAgs as a mutated large HDAg whose leucine zipper domain was deleted such that it could not help in copackaging of the small HDAg. This mutated large HDAg did not suppress HDV replication, suggesting that this effect is probably also via protein interaction. These results indicated that functional domains of the large HDAg responsible for packaging with HBsAg particles and for the trans-negative effect on HDV replication can be separated. PMID:1560529

  5. Flow cytometer measurement of binding assays

    DOEpatents

    Saunders, George C.

    1987-01-01

    A method of measuring the result of a binding assay that does not require separation of fluorescent smaller particles is disclosed. In a competitive binding assay the smaller fluorescent particles coated with antigen compete with antigen in the sample being analyzed for available binding sites on larger particles. In a sandwich assay, the smaller, fluorescent spheres coated with antibody attach themselves to molecules containing antigen that are attached to larger spheres coated with the same antibody. The separation of unattached, fluorescent smaller particles is made unnecessary by only counting the fluorescent events triggered by the laser of a flow cytometer when the event is caused by a particle with a light scatter measurement within a certain range corresponding to the presence of larger particles.

  6. Assessing stability and assembly of the hepatitis B surface antigen into virus-like particles during down-stream processing.

    PubMed

    Zahid, Maria; Lünsdorf, Heinrich; Rinas, Ursula

    2015-07-17

    The hepatitis B surface antigen (HBsAg) is a recombinant protein-based vaccine being able to form virus-like particles (VLPs). HBsAg is mainly produced using yeast-based expression systems, however, recent results strongly suggest that VLPs are not formed within the yeast cells during the cultivation but are formed in a gradual manner during the following down-stream procedures. VLPs are also not detectable during the first down-stream steps including mechanical and EDTA/detergent-assisted cell destruction. Moreover, VLPs are not detectable in the cell lysate treated with polyethylene glycol and colloidal silica. The first VLP resembling structures appear after elution of HBsAg from colloidal silica to which it binds through hydrophobic interaction. These first VLP resembling structures are non-symmetrical as well as heterodisperse and exhibit a high tendency toward cluster formation presumably because of surface exposed hydrophobic patches. More symmetrical and monodisperse VLPs appear after the following ion-exchange and size-exclusion chromatography most likely as the result of buffer changes during these purification steps (toward more neutral pH and less salt). Final treatment of the VLPs with the denaturant KSCN at moderate concentrations with following KSCN removal by dialysis does not cause unfolding and VLP disassembly but results in a re- and fine-structuring of the VLP surface topology.

  7. Hepatitis B core-related antigen levels are associated with response to entecavir and peginterferon add-on therapy in hepatitis B e antigen-positive chronic hepatitis B patients.

    PubMed

    van Campenhout, M J H; Brouwer, W P; van Oord, G W; Xie, Q; Zhang, Q; Zhang, N; Guo, S; Tabak, F; Streinu-Cercel, A; Wang, J; Pas, S D; Sonneveld, M J; de Knegt, R J; Boonstra, A; Hansen, B E; Janssen, H L A

    2016-06-01

    Hepatitis B core-related antigen (HBcrAg), a new serum marker, may be useful in monitoring chronic hepatitis B infection. HBcrAg was measured in 175 hepatitis B e antigen-positive patients treated with entecavir (ETV) with or without peginterferon (PEG-IFN) add-on therapy. Decline in HBcrAg was stronger in patients with vs. without combined response (ETV: -3.22 vs. -1.71 log U/mL, p <0.001; PEG-IFN add-on: -3.16 vs. -1.83 IU/mL, p <0.001) and in patients with vs. without hepatitis B surface antigen (HBsAg) response (ETV: -2.60 vs. -1.74 log U/mL, p <0.001; PEG-IFN add-on: -2.38 vs. -2.15 log U/mL, p = 0.31). HBcrAg was associated with combined response (adjusted odds ratio 0.3, 95% confidence interval 0.2-0.5, p <0.001), but was not superior to quantitative HBsAg (qHBsAg).

  8. Risk of underdiagnosing amebic dysentery due to false-negative Entamoeba histolytica antigen detection.

    PubMed

    Prim, Núria; Escamilla, Pilar; Solé, Roser; Llovet, Teresa; Soriano, Germán; Muñoz, Carmen

    2012-08-01

    Entamoeba histolytica antigen assays on stool are widely used to diagnose amebiasis. We report a case of confirmed amebic colitis with a false-negative antigen detection that became positive after treatment. Our results indicate that these assays may underdiagnose acute amebic infection when used alone and should be used cautiously.

  9. Identification of an immunodominant epitope in glycoproteins B and G of herpes simplex viruses (HSVs) using synthetic peptides as antigens in assay of antibodies to HSV in herpes simplex encephalitis patients.

    PubMed

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

    2014-01-01

    Herpes simplex encephalitis (HSE) is a severe viral infection of the central nervous system (CNS). Assay of antibody response is widely used in diagnostics of HSE. The aim of this study was to identify an immunodominant epitope determining the antibody response to herpes simplex viruses (HSVs) in cerebrospinal fluid (CSF) of HSE patients. The synthetic peptides that resembled type-common as well as type-specific domains of glycoproteins B (gB) and G (gG) of these viruses were evaluated for binding with IgM and IgG antibodies in CSF samples from HSE and non-HSE patients in ELISA. The QLHDLRF peptide, derived from gB of HSV was found to be an immunodominant epitope in the IgM and IgG antibody response. The patients with confirmed and suspected HSE showed in ELISA against this peptide 26% and 23% positivities for IgM, 43% and 37% positivities for IgG and 17% and 15% for both IgM and IgG antibodies, respectively. The total positivities of 86% and 75% for both IgM and IgG antibodies were obtained in the patients with confirmed and suspected HSE, respectively. These results demonstrate that a synthetic peptide-based diagnostics of HSE can be an efficient and easily accessible alternative. This is the first report describing the use of synthetic peptides derived from HSVs in diagnostics of HSE using patientsʹ CSF samples.

  10. Inhibitor-neutralisation assay and electro-immuno assay of human factor IX (Christmas factor).

    PubMed

    Bertina, R M; van der Linden, I K

    1977-06-15

    A rabbit antibody specifically precipitating human factor IX has been used in the assay of factor IX antigen. The results obtained with two different methods (inhibitor-neutralisation assay and electro-immunoassay) have been compared in a group of healthy individuals and in a group of hemophilia B patients and carriers. In general, identical results are obtained with both methods, except in some hemophilia B+ carriers and patients, where the electroimmuno assay gives 1.5-2.0 times higher levels. Results obtained by electroimmuno assay are more accurate and reproducible than those obtained by inhibitor-neutralisation assay, which is of importance for its potential use in carrier detection.

  11. Efficacy of Post-Exposure Prophylaxis in Infants Born to HBsAg Positive Mothers in Iran; Is It Authentic?

    PubMed Central

    Ahmadinejad, Zahra; Abdi Liae, Zahra; Salehizadeh, Saideh; Mansori, Sedighe; Alijani, Neda

    2016-01-01

    Background Hepatitis B infection is a universal concern. This infection can lead to chronic liver disease and hepatocellular carcinoma. Neonates born to HBsAg-positive mothers are at high risk of chronic hepatitis B virus (HBV) infection, especially for HBeAg-positive mothers or neonates who have not received hepatitis B immunoglobulin (HBIg) and HBV vaccines. Objectives The aim of this study was to evaluate the efficacy of post-exposure prophylaxis in these infants to prevent infection. Patients and Methods Thirty-eight infants born to HBsAg-positive mothers between September 2006 and September 2013 were followed. The investigation evaluated whether the standard prevention protocol of neonatal HBV transmission including HBIg at birth and receiving three doses of vaccine at birth and 2 and 6 months of age was performed, followed by post-vaccination tests (evaluation of HBsAg and HBsAb titer at 9 to 18 months of age) to determine subsequent infection. HBsAb titer ≥ 10 was considered as criterion for effectiveness of the prophylaxis procedure. The acquired data were analyzed using SPSS software (Version 18). The results are reported in descriptive tabulations. Results Ninety seven percent (97%) of infants received HBIg at birth in the hospital. Generally, all of them received the first, second and third doses of vaccine at birth, 2 months, and 6 months after birth, respectively. Information for 35 mothers infected with HBV and 38 infants was available. The mean age of the mothers was 30.3 years. The results indicated that 20% of mothers were HBeAg positive. HBsAg was positive in one (2.6%) infant born to an HBeAg-positive mother. Around 94% of infants’ HBsAb titers were ≥ 10, and 5.8% were reported as non-responders. Conclusions The vertical transmission prevention program used in the study population in Tehran, which had an appropriate sample size, is effective. Additional doses of the vaccine can be useful in raising the effectiveness of immunoprophylaxis for

  12. Prevalence of Serologic Hepatitis B Markers in Blood Donors From Puebla, Mexico: The Association of Relatively High Levels of Anti-Core Antibodies With the Detection of Surface Antigen and Genomic DNA

    PubMed Central

    Sosa-Jurado, Francisca; Hilda Rosas-Murrieta, Nora; Guzman-Flores, Belinda; Perez Zempoaltecalt, Cintia; Patricia Sanchez Torres, Ana; Ramirez Rosete, Leticia; Bernal-Soto, Maribel; Marquez-Dominguez, Luis; Melendez-Mena, Daniel; Angel Mendoza Torres, Miguel; Teresa Lopez Delgado, Maria; Reyes-Leyva, Julio; Vallejo-Ruiz, Veronica; Santos-Lopez, Gerardo

    2016-01-01

    Background The hepatitis B virus (HBV) causes chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. Surface antigen (HBsAg) detection is a definitive test that can confirm HBV infection, while the presence of antibodies against the core protein (anti-HBc) suggests either a previous or ongoing infection or occult hepatitis B infection (OBI). Objectives The aim of the present study was to determine the prevalence of anti-HBc and HBsAg in blood donors. Further, the study aimed to estimate the anti-HBc level at which HBV DNA is detected in putative OBI cases, as well as to search for mutations in the “a” determinant associated with the non-detection of HBsAg in serum. Patients and Methods We conducted a cross-sectional study from 2003–2009. The study included 120,552 blood donors from the state of Puebla, Mexico. Different commercial systems based on microparticles (enzymatic (MEIA) or chemiluminescent (CMIA)) were used to determine the HBsAg and anti-HBc levels. For the detection of HBV DNA, a nested polymerase chain reaction (nested PCR) was used and the genotypes were determined using Sanger sequencing. Results Of the 120,552 blood donors, 1437 (1.19%, 95% CI: 1.12 - 1.26) were reactive to anti-HBc, while 82 (0.066%, 95% CI: 0.053 - 0.079) were reactive to HBsAg. Some 156 plasma samples collected in 2009 from anti-HBc-positive/HBsAg-negative blood donors were submitted for HBV DNA detection in a search for probable OBI. Viral DNA was detected in 27/156 (17.3%, 95% CI: 11.5 - 23.1). Our results show an association between HBV DNA or HBsAg and anti-HBc S/CO levels ≥ 4.0. All DNA samples were identified as genotype H and some “a” determinant mutations were identified, although none corresponded to mutations previously reported to hinder the detection of HBsAg by commercial immunoassays. Conclusions We observed that as the anti-HBc levels increase, there is a higher prevalence of the viral protein HBsAg in blood donors. Samples testing positive

  13. Prevalence of Serologic Hepatitis B Markers in Blood Donors From Puebla, Mexico: The Association of Relatively High Levels of Anti-Core Antibodies With the Detection of Surface Antigen and Genomic DNA

    PubMed Central

    Sosa-Jurado, Francisca; Hilda Rosas-Murrieta, Nora; Guzman-Flores, Belinda; Perez Zempoaltecalt, Cintia; Patricia Sanchez Torres, Ana; Ramirez Rosete, Leticia; Bernal-Soto, Maribel; Marquez-Dominguez, Luis; Melendez-Mena, Daniel; Angel Mendoza Torres, Miguel; Teresa Lopez Delgado, Maria; Reyes-Leyva, Julio; Vallejo-Ruiz, Veronica; Santos-Lopez, Gerardo

    2016-01-01

    Background The hepatitis B virus (HBV) causes chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. Surface antigen (HBsAg) detection is a definitive test that can confirm HBV infection, while the presence of antibodies against the core protein (anti-HBc) suggests either a previous or ongoing infection or occult hepatitis B infection (OBI). Objectives The aim of the present study was to determine the prevalence of anti-HBc and HBsAg in blood donors. Further, the study aimed to estimate the anti-HBc level at which HBV DNA is detected in putative OBI cases, as well as to search for mutations in the “a” determinant associated with the non-detection of HBsAg in serum. Patients and Methods We conducted a cross-sectional study from 2003–2009. The study included 120,552 blood donors from the state of Puebla, Mexico. Different commercial systems based on microparticles (enzymatic (MEIA) or chemiluminescent (CMIA)) were used to determine the HBsAg and anti-HBc levels. For the detection of HBV DNA, a nested polymerase chain reaction (nested PCR) was used and the genotypes were determined using Sanger sequencing. Results Of the 120,552 blood donors, 1437 (1.19%, 95% CI: 1.12 - 1.26) were reactive to anti-HBc, while 82 (0.066%, 95% CI: 0.053 - 0.079) were reactive to HBsAg. Some 156 plasma samples collected in 2009 from anti-HBc-positive/HBsAg-negative blood donors were submitted for HBV DNA detection in a search for probable OBI. Viral DNA was detected in 27/156 (17.3%, 95% CI: 11.5 - 23.1). Our results show an association between HBV DNA or HBsAg and anti-HBc S/CO levels ≥ 4.0. All DNA samples were identified as genotype H and some “a” determinant mutations were identified, although none corresponded to mutations previously reported to hinder the detection of HBsAg by commercial immunoassays. Conclusions We observed that as the anti-HBc levels increase, there is a higher prevalence of the viral protein HBsAg in blood donors. Samples testing positive

  14. Assays for B lymphocyte function.

    PubMed

    Bondada, Subbarao; Robertson, Darrell A

    2003-11-01

    This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation. PMID:18432909

  15. Neuraminidase assays.

    PubMed

    Aymard, M; Ferraris, O; Gerentes, L; Jolly, J; Kessler, N

    2003-01-01

    Anti-neuraminidase (NA) antibodies (Ab) play a role in protection against influenza and in combination with anti-HA Ab they increase the protection in mice. To control the NA content of vaccines, which should improve vaccine standardisation and may benefit vaccine efficacy, a series of questions must be addressed: 1) The antigenic characterization of NA in vaccine strains and seed lots is based on the measurement of the enzymatic (E) activity using fetuin as substrate. The antigenic profile is established by inhibiting the E activity with post infectious ferret antisera. Overnight incubation ensures sensitivity, and fetuin substrate gives specificity by detection of variant specific antibodies. Several difficulties have to be overcome, such as the low level of E activity in MDCK grown viruses, and the lability of N1. 2) The NA protein content of the vaccines (in bulk or final product) can be measured by an ELISA capture test but the lability of the NA proteins at 4 degrees C must be checked. 3) The anti NA Ab response can be measured using a neuraminidase inhibition test. --The steric hindrance by HI antibodies does not exceed a titre of 20 in human sera. --Triton treatment of viruses reduces the steric hindrance in polyclonal sera and monoclonal antibodies but unmasks epitopes. 4) The correlations between neuraminidase inhibition, neutralization and protection, has been established in the mouse model, but remains to be shown in humans. 5) The use of a small fluorescent (MUN) or chemiluminescent (NA-STAR) substrate can be used for the rapid differentiation of N1 from N2 and NB, but not for the titration of protective NI antibodies.

  16. Plaque assay for murine norovirus.

    PubMed

    Gonzalez-Hernandez, Mariam B; Bragazzi Cunha, Juliana; Wobus, Christiane E

    2012-01-01

    Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques. Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50). This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration. However, its limit of detection is higher compared to a plaque assay. In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of

  17. Breaking B and T cell tolerance using cationic lipid--DNA complexes (CLDC) as a vaccine adjuvant with hepatitis B virus (HBV) surface antigen in transgenic mice expressing HBV.

    PubMed

    Morrey, John D; Motter, Neil E; Chang, Stella; Fairman, Jeffery

    2011-06-01

    Cationic lipid DNA complexes (CLDC), referred to here as JVRS-100, were evaluated as an adjuvant for hepatitis B surface antigen (HBsAg) for eliciting B and T cell responses in transgenic mice expressing hepatitis B virus (HBV). To confirm the immunogenicity of HBsAg+JVRS-1000, a study was conducted in C57BL/6 mice, the genetic background of the HBV transgenic mice used in the study. HBsAg+JVRS-100 elicited a T cell response and B cell response as evidenced by interferon-gamma (IFN-γ) secretion by re-stimulated splenocytes and anti-HBsAg IgG induction, respectively, whereas, HBsAg only elicited a B cell response. In HBV transgenic mice, HBsAg did not elicit either T or B cell responses, unlike the HBsAg+JVRS-100 that elicited both. Energix-B vaccine did perform better than the HBsAg by eliciting a B cell response in the transgenic mice, but it did not perform as HBsAg+JVRS-100 since it did not elicit a T cell response. The response by HBsAg+JVRS-100 was not sufficient to cause destruction of infected liver cells, but it did suppress HBV DNA non-cytolytically. From these results, JVRS-100 might be considered for further development as an adjuvant for HBV therapeutic vaccines. PMID:21545812

  18. Multiplex assays to diagnose celiac disease.

    PubMed

    Lochman, Ivo; Martis, Peter; Burlingame, Rufus W; Lochmanová, Alexandra

    2007-08-01

    Patients with celiac disease are sensitive to the gluten fractions of wheat. Symptoms include gastrointestinal problems and a failure to thrive in children, but may range from headaches to general malaise in adults. Thus, it is difficult to diagnose celiac disease by symptoms alone. The standard diagnostic criteria include the presence of the characteristic anti-gliadin or anti-tissue transglutaminase antibodies (anti-tTG) in serum, flattened mucosa on intestinal biopsy, and improved symptoms on a gluten-free diet. Because of the ease of use of the tTG enzyme-linked immunosorbent assay (ELISA) compared to endomysial by indirect immunofluorescence assay, there has been much more screening for celiac disease in recent years. This increased screening showed that celiac disease was more prevalent than previously believed. We compared a new multiplex assay that includes a novel form of deamidated gliadin and recombinant human tTG as the antigens to other assays using standard antigens. In addition, the new assay detects the presence of selective IgA deficiency, which shows a 10-fold increase in prevalence in patients with celiac disease compared to the general population. The combination of sensitivity and specificity of the new multiplex assay was equal or better than those for standard assays. Thus the performance, ease of use, and ability to measure three clinically important parameters in a single test make the new multiplex assay a viable alternative to standard assays in a clinical lab.

  19. New diagnostic antigens for early trichinellosis: the excretory-secretory antigens of Trichinella spiralis intestinal infective larvae.

    PubMed

    Sun, Ge Ge; Liu, Ruo Dan; Wang, Zhong Quan; Jiang, Peng; Wang, Li; Liu, Xiao Lin; Liu, Chun Yin; Zhang, Xi; Cui, Jing

    2015-12-01

    The excretory-secretory (ES) antigens from Trichinella spiralis muscle larvae (ML) are the most commonly used diagnostic antigens for trichinellosis, but anti-Trichinella IgG antibodies cannot be detected until 2-3 weeks after infection; there is an obvious window period between Trichinella infection and antibody positivity. Intestinal infective larvae (IIL) are the first invasive stage during Trichinella infection, and their ES antigens are firstly exposed to the immune system and might be the early diagnostic markers of trichinellosis. The aim of this study was to evaluate the early diagnostic values of IIL ES antigens for trichinellosis. The IIL were collected from intestines of infected mice at 6 h postinfection (hpi), and IIL ES antigens were prepared by incubation for 18 h. Anti-Trichinella IgG antibodies in mice infected with 100 ML were detectable by ELISA with IIL ES antigens as soon as 10 days postinfection (dpi), but ELISA with ML ES antigens did not permit detection of infected mice before 12 dpi. When the sera of patients with trichinellosis at 19 dpi were assayed, the sensitivity (100 %) of ELISA with IIL ES antigens was evidently higher than 75 % of ELISA with ML ES antigens (P < 0.05) The specificity (96.86 %) of ELISA with IIL ES antigens was also higher than 89.31 % of ELISA with ML ES antigens (P < 0.05). The IIL ES antigens provided a new source of diagnostic antigens and could be considered as a potential early diagnostic antigen for trichinellosis.

  20. Expression of Plasmodium falciparum surface antigens in Escherichia coli.

    PubMed Central

    Ardeshir, F; Flint, J E; Reese, R T

    1985-01-01

    The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity. Images PMID:3887406

  1. Transcutaneous antigen delivery system

    PubMed Central

    Lee, Mi-Young; Shin, Meong-Cheol; Yang, Victor C.

    2013-01-01

    Transcutaneous immunization refers to the topical application of antigens onto the epidermis. Transcutaneous immunization targeting the Langerhans cells of the skin has received much attention due to its safe, needle-free, and noninvasive antigen delivery. The skin has important immunological functions with unique roles for antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells. In recent years, novel vaccine delivery strategies have continually been developed; however, transcutaneous immunization has not yet been fully exploited due to the penetration barrier represented by the stratum corneum, which inhibits the transport of antigens and adjuvants. Herein we review recent achievements in transcutaneous immunization, focusing on the various strategies for the enhancement of antigen delivery and vaccination efficacy. [BMB Reports 2013; 46(1): 17-24] PMID:23351379

  2. MicroRNA-122 as a predictor of HBsAg seroclearance in hepatitis B and C dual infected patients treated with interferon and ribavirin

    PubMed Central

    Yen, Yi-Hao; Huang, Chao-Min; Wei, Kuo-Liang; Wang, Jing-Houng; Lu, Sheng-Nan; Lee, Chuan-Mo; Hung, Chao-Hung; Chen, Chien-Hung; Tseng, Po-Lin; Chang, Kuo-Chin; Tsai, Ming-Chao; Lin, Ming-Tsung; Wu, Cheng-Kun; Yang, Cheng-Hong; Moi, Sin-Hua; Cho, Chung-Lung; Hu, Tsung-Hui

    2016-01-01

    It has been demonstrated that microRNA-122 (miR-122) plays key roles in the modulation of hepatitis B virus (HBV) replication. This study examined the role of miR-122 in patients with hepatitis C virus (HCV)-HBV dual infection with active hepatitis C who received pegylated interferon-α and ribavirin dual therapy. We enrolled 121 patients with HCV-HBV dual infection after dual therapy. Stored serum was collected before treatment. RT-PCR was used to analyze miR-122. HBsAg seroclearance was noted in 37 (30.1%) cases during a median follow-up period of 5.4 years. miR-122 was significantly lower in HBsAg seroclearance patients than in non-HBsAg seroclearance patients (P < 0.014). Multivariate analysis showed that miR-122 was an independent factor of HBsAg seroclearance (OR: 0.30, 95% CI: 0.09–0.98, P = 0.046). miR-122 was significantly higher in patients who were qHBsAg > 100 IU/mL versus ≤100 IU/mL (P < 0.001). We concluded that in patients with HBV-HCV dual infection with active hepatitis C, miR-122 was associated with HBsAg seroclearance after therapy and qHBsAg level before therapy, indicating that miR-122 plays key roles in modulating HBV replication. PMID:27665934

  3. Dot-immunobinding assay (Dot-Iba).

    PubMed

    Surendran, Sumi; Mathai, Annamma; Radhakrishnan, Vishnampet Venkataraman

    2015-01-01

    Dot-immunobinding assay (Dot-Iba) is a simple and highly reproducible immunodiagnostic method. Antibody or antigen is dotted directly onto nitrocellulose membrane (NCM) discs. The diagnostic material to be checked can be incubated on this disc. Presence of antigen-antibody complex in NCM discs can be directly demonstrated with enzyme-conjugated antiglobulins and substrate. Development of a purple-pink colored, insoluble substrate product in the nitrocellulose membrane will be considered a positive result in the assay. This assay allows the processing of multiple specimens at a time and the entire operational procedures required only 4-6 h. Dot-IBA is rapid and the technical steps involved in the assay are much simpler than the other immunoassays such as enzyme-linked immunosorbent assay in detecting circulating antigen and antibody in clinical samples. The Dot-Iba showed an overall sensitivity of 60 % for tuberculous meningitis diagnosis and no false positive results were encountered. Hence this assay is highly specific for the diagnosis of paucibacillary diseases like extrapulmonary tuberculosis. Dot-Iba is best suited to laboratories in developing world where there are constraints in laboratory resources.

  4. Use of antigenic cartography in vaccine seed strain selection.

    PubMed

    Fouchier, Ron A M; Smith, Derek J

    2010-03-01

    Human influenza A viruses are classic examples of antigenically variable pathogens that have a seemingly endless capacity to evade the host's immune response. The viral hemagglutinin (HA) and neuraminidase (NA) proteins are the main targets of our antibody response to combat infections. HA and NA continuously change to escape from humoral immunity, a process known as antigenic drift. As a result of antigenic drift, the human influenza vaccine is updated frequently. The World Health Organization (WHO) coordinates a global influenza surveillance network that, by the hemagglutination inhibition (HI) assay, routinely characterizes the antigenic properties of circulating strains in order to select new seed viruses for such vaccine updates. To facilitate a quantitative interpretation and easy visualization of HI data, a new computational technique called "antigenic cartography" was developed. Since its development, antigenic cartography has been applied routinely to assist the WHO with influenza surveillance activities. Until recently, antigenic variation was not considered a serious issue with influenza vaccines for poultry. However, because of the diversification of the Asian H5N1 lineage since 1996 into multiple genetic clades and subclades, and because of the long-term use of poultry vaccines against H5 in some parts of the world, this issue needs to be re-addressed. The antigenic properties of panels of avian H5N1 viruses were characterized by HI assay, using mammalian or avian antisera, and analyzed using antigenic cartography methods. These analyses revealed antigenic differences between circulating H5N1 viruses and the H5 viruses used in poultry vaccines. Considerable antigenic variation was also observed within and between H5N1 clades. These observations have important implications for the efficacy and long-term use of poultry vaccines.

  5. Human humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-molecular-mass antigens.

    PubMed Central

    Laal, S; Samanich, K M; Sonnenberg, M G; Zolla-Pazner, S; Phadtare, J M; Belisle, J T

    1997-01-01

    The selection of antigens of Mycobacterium tuberculosis for most studies of humoral responses in tuberculosis patients has been restricted to molecules that were either immunodominant in immunized animals or amenable to biochemical purification rather than those that were reactive with the human immune system. Delineation of antigens that elicit humoral responses during the natural course of disease progression in humans has been hindered by the presence of cross-reactive antibodies to conserved regions on ubiquitous prokaryotic antigens in sera from healthy individuals and tuberculosis patients. The levels of cross-reactive antibodies in the sera were reduced by preadsorption with Escherichia coli lysates, prior to studying their reactivity against a large panel of M. tuberculosis antigens to which the human immune system may be exposed during natural infection and disease. Thus, reactivity against pools of secreted, cellular, and cell wall-associated antigens of M. tuberculosis was assessed by an enzyme-linked immunosorbent assay (ELISA). Initial results suggested that the secreted protein preparation contained antigens most frequently recognized by the humoral responses of pulmonary tuberculosis patients. The culture filtrate proteins were subsequently size fractionated by preparative polyacrylamide gel electrophoresis, characterized by reaction with murine monoclonal antibodies to known antigens of M. tuberculosis by an ELISA, and assessed for reactivity with tuberculous and nontuberculous sera. Results show that a secreted antigen of 88 kDa elicits a strong antibody response in a high percentage of patients with pulmonary tuberculosis. This and other antigens identified on the basis of their reactivity with patient sera may prove useful for developing serodiagnosis for tuberculosis. PMID:9008280

  6. Genetic and antigenic changes in porcine rubulavirus

    PubMed Central

    Sánchez-Betancourt, José I.; Trujillo, María E.; Mendoza, Susana E.; Reyes-Leyva, Julio; Alonso, Rogelio A.

    2012-01-01

    Blue eye disease, caused by a porcine rubulavirus (PoRV), is an emergent viral swine disease that has been endemic in Mexico since 1980. Atypical outbreaks were detected in 1990 and 2003. Growing and adult pigs presented neurological signs, mild neurological signs were observed in piglets, and severe reproductive problems were observed in adults. Amino acid sequence comparisons and phylogenetic analysis of the hemagglutinin-neuraminidase (HN) protein revealed genetically different lineages. We used cross-neutralization assays, with homologous and heterologous antisera, to determine the antigenic relatedness values for the PoRV isolates. We found antigenic changes among several strains and identified a highly divergent one, making up a new serogroup. It seems that genetically and antigenically different PoRV strains are circulating simultaneously in the swine population in the geographical region studied. The cross neutralization studies suggest that the HN is not the only antigenic determinant participating in the antigenic changes among the different PoRV strains. PMID:22754092

  7. Immunological evaluation of colonic delivered Hepatitis B surface antigen loaded TLR-4 agonist modified solid fat nanoparticles.

    PubMed

    Sahu, Kantrol Kumar; Pandey, Ravi Shankar

    2016-10-01

    Hepatitis B is one of the leading liver diseases and remains a major global health problem. Currently available vaccines provide protection but often results in weaker/minimal mucosal immunity. Thus the present study is devoted to the development and in-vivo exploration of the colonically delivered biomimetic nanoparticles which capably enhance humoral as well as cellular immune response. In present work, Hepatitis B surface antigen (HBsAg) entrapped nanoparticles containing Monophosphoryl lipid A (MPLA) (HB+L-NP) were prepared by solvent evaporation method and characterized for particle size (~210nm), shape, zeta potential (-24mV±0.68), entrapment efficiency (58.45±1.68%), in-vitro release and antigen integrity. Dose escalation study was done to confirm prophylactic immune response following defined doses of prepared nanoparticulate formulations with or without MPLA. Intramuscular administered alum based marketed HBsAg (Genevac B) was used as standard (10μg) and were able to induce significant systemic (IgG) but remarkably low mucosal immune (IgA) response. Notably, HB+L-NP (0.5ml-10μg) induced strong systemic and robust mucosal immunity (510 and 470 mIU/ml respectively, p<0.001) from which mucosal was more significant due to the involvement of Common Mucosal Immune System (CMIS). Likewise, significant cellular immune response was elicited by HB+L-NP through T-cell activation (mixed Th1 and Th2) as confirmed by significantly increased cytokines level (IL-2 and Interferon-γ) in spleen homogenates. This study supports that delivery of HBsAg to the colon may open new vista in designing oral vaccines later being one of most accepted route for potential vaccines in future. PMID:27526270

  8. Immunological evaluation of colonic delivered Hepatitis B surface antigen loaded TLR-4 agonist modified solid fat nanoparticles.

    PubMed

    Sahu, Kantrol Kumar; Pandey, Ravi Shankar

    2016-10-01

    Hepatitis B is one of the leading liver diseases and remains a major global health problem. Currently available vaccines provide protection but often results in weaker/minimal mucosal immunity. Thus the present study is devoted to the development and in-vivo exploration of the colonically delivered biomimetic nanoparticles which capably enhance humoral as well as cellular immune response. In present work, Hepatitis B surface antigen (HBsAg) entrapped nanoparticles containing Monophosphoryl lipid A (MPLA) (HB+L-NP) were prepared by solvent evaporation method and characterized for particle size (~210nm), shape, zeta potential (-24mV±0.68), entrapment efficiency (58.45±1.68%), in-vitro release and antigen integrity. Dose escalation study was done to confirm prophylactic immune response following defined doses of prepared nanoparticulate formulations with or without MPLA. Intramuscular administered alum based marketed HBsAg (Genevac B) was used as standard (10μg) and were able to induce significant systemic (IgG) but remarkably low mucosal immune (IgA) response. Notably, HB+L-NP (0.5ml-10μg) induced strong systemic and robust mucosal immunity (510 and 470 mIU/ml respectively, p<0.001) from which mucosal was more significant due to the involvement of Common Mucosal Immune System (CMIS). Likewise, significant cellular immune response was elicited by HB+L-NP through T-cell activation (mixed Th1 and Th2) as confirmed by significantly increased cytokines level (IL-2 and Interferon-γ) in spleen homogenates. This study supports that delivery of HBsAg to the colon may open new vista in designing oral vaccines later being one of most accepted route for potential vaccines in future.

  9. An enzyme-linked immunosorbent assay for the detection of marine caliciviruses.

    PubMed

    Ferris, N P; Oxtoby, J M

    1994-11-01

    An indirect, sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of antigens of vesicular exanthema, San Miguel sea lion viruses and other marine caliciviruses is described. The assay which uses rabbit and guinea-pig antisera to purified antigens of each calicivirus serotype has high sensitivity and is almost totally type-specific. PMID:7886934

  10. Comparison of peptide cocktails and purified protein derivatives for use in the Bovigam assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Currently, the Bovigam™ assay is used as an official complementary test within the bovine tuberculosis eradication program. This assay measures Interferon-gamma (IFN-') produced by lymphocytes in response to specific antigens. The objectives of the present study were to compare in vitro antigen prep...

  11. Monoclonal antibodies to hepatitis B surface antigen: production and characterization.

    PubMed

    Hlozánek, I; Dostálová, V; Korec, E; Zelený, V; König, J; Nĕmecek, V

    1986-01-01

    Hybridomas secreting anti-HBsAg antibodies were produced by fusion of the mouse myeloma cell line SP2/0 with lymphocytes from mice immunized with purified HBsAg. All clones produced antibodies of the IgG1 idiotype that react with the subtype a determinant of HBsAg. An enzyme immunoassay for detection of HBsAg in human sera using monoclonal antibodies was developed and compared with commercial Sevatest ELISA HBsAg/micro I kit for detection of HBsAg in clinical serum samples. PMID:3527770

  12. [The frequency of the e-antigen in acute and chronic hepatitis (author's transl)].

    PubMed

    Fay, O H; Tanno, H; Roncoroni, M; Findor, J; Igartua, E B; Domecq, R

    1975-08-01

    Five groups of patients were studied: (a) 88 HBsAg-positive cases of acute viral hepatitis (AVH) who completely cured within 90 days; (b) 75 cases of acute HBsAg-negative hepatitis with early resolution; (c) 15 AVH patients who became chronic; (d) 94 HBsAg-positive cases of chronic hepatitis (CH) and liver cirrhosis; (e) HBsAg-negative cases with CH. The e-antigen was investigated in every patient and was found in 9 of the AVH cases with favorable evolution, and in 8 of the AVH patients evolving to chronicity. Amont the CH and cirrhosis group, 48 of the HBsAg carriers were positive while e-antigen could not be detected in any of the HBsAg-negative ones. The high incidence of e-antigen carriers among patients with chronic hepatitis and liver cirrhosis as well as among those with AVH who later entered the chronic stage of the disease, deserves especial consideration. A possible role of e-antigen in the evolution to chronicity and a prognostic significance is suggested.

  13. An ultrastructural and immunohistochemical study on the delta antigen associated with the hepatitis B virus.

    PubMed

    Canese, M G; Rizzetto, M; Arico, S; Crivelli, O; Zanetti, A R; Macchiorlatti, E; Ponzetto, A; Leone, L; Mollo, F; Verme, G

    1979-08-01

    Thirteen liver biopsies in which the delta antigen was detected by immunofluorescence were studied by electron microscopy and immune electron microscopy with peroxidase labelled IgG and F(ab1)2 fraction obtained from a human antiserum containing high-titre anti-delta antibodies. The findings were compared with those obtained in 11 HBcAg positive and in two HBsAg negative controls. Neither unique particulate morphology nor any HB virus ultrastructural component were visualised in the delta positive specimens; 20-23 nm naked core particles were observed in 10 of 11 biopsies displaying the HBcAg in immunofluorescence. Delta positive nuclei frequently contained dense round structures of diameter varying between 20 and 30 nm with a soft indistinct edge. These granules did not exhibit characteristic ultrastructural features which enabled them to be distinguished from other granular material observed occasionally in nuclei of normal and diseased livers. However, their association with the delta antigen has been proved by the deposition on identical structures of peroxidase labelled anti-delta antibody. These results suggest that the delta antigen is unrelated to the Dane particle, the putative HB virus. The granules observed in the delta positive nuclei are composed of an amorphous matrix, possibly insoluble aggregates of the delta antigen.

  14. The differences in short- and long-term varicella-zoster virus (VZV) immunoglobulin G levels following varicella vaccination of healthcare workers measured by VZV fluorescent-antibody-to-membrane-antigen assay (FAMA), VZV time-resolved fluorescence immunoassay and a VZV purified glycoprotein enzyme immunoassay.

    PubMed

    Maple, P A C; Haedicke, J; Quinlivan, M; Steinberg, S P; Gershon, A A; Brown, K E; Breuer, J

    2016-08-01

    Healthcare workers (HCWs) reporting no history of varicella frequently receive varicella vaccination (vOka) if they test varicella-zoster virus (VZV) immunoglobulin G (IgG) negative. In this study, the utilities of VZV-IgG time-resolved fluorescence immunoassay (VZV-TRFIA) and a commercial VZV-IgG purified glycoprotein enzyme immunoassay (gpEIA) currently used in England for confirming VZV immunity have been compared to the fluorescent-antibody-to-membrane-antigen assay (FAMA). A total of 110 HCWs received two doses of vOka vaccine spaced 6 weeks apart and sera collected pre-vaccination (n = 100), at 6 weeks post-completion of vaccination (n = 86) and at 12-18 months follow-up (n = 73) were analysed. Pre-vaccination, by FAMA, 61·0% sera were VZV IgG negative, and compared to FAMA the sensitivities of VZV-TRFIA and gpEIA were 74·4% [95% confidence interval (CI) 57·9-87·0] and 46·2% (95% CI 30·1-62·8), respectively. Post-completion of vaccination the seroconversion rate by FAMA was 93·7% compared to rates of 95·8% and 70·8% determined by VZV-TRFIA and gpEIA, respectively. At 12-18 months follow-up seropositivity rates by FAMA, VZV-TRFIA and gpEIA were 78·1%, 74·0% and 47·9%, respectively. Compared to FAMA the sensitivities of VZV-TRFIA and gpEIA for measuring VZV IgG following vaccination were 96·4% (95% CI 91·7-98·8) and 74·6% (95% CI 66·5-81·6), respectively. Using both FAMA and VZV-TRFIA to identify healthy adult VZV susceptibles and measure seroconversion showed that vOka vaccination of HCWs is highly immunogenic.

  15. Modulation of antigenicity of mycelial antigens during developmental cycle of Karnal bunt (Tilletia indica) of wheat.

    PubMed

    Rai, G; Kumar, A; Singh, A; Garg, G K

    2000-05-01

    Indirect enzyme linked immunosorbent assays (ELISA) were developed using polyclonal antibodies against soluble cytoplasmic (SCA) and insoluble cell wall antigens (ICWA) for monitoring modulation of mycelial antigens during growth cycle of T. indica. With SCA, continuous decrease in ELISA reactivity was observed in maturing fungus cultures, suggesting that SCA were expressed predominantly during early vegetative phase and their decreasing role was apparent as the fungus matures possibly towards sporogenous mycelium. In case of ICWA, the reaction profile showed an increase up to exponential phase of growth probably due to increase in the cell division and branching of mycelium. But later, ICWA antibody reactivity was decreased which may be due to conversion of mycelial phase to sporogenous phase, a quiescent stage of growth. Characterization of changes in antigenic configuration during developmental cycle of Tilletia indica by these antibodies could prove to be useful in identification of developmentally related and virulence marker(s).

  16. Size-exclusion HPLC provides a simple, rapid, and versatile alternative method for quality control of vaccines by characterizing the assembly of antigens.

    PubMed

    Yang, Yanli; Li, Hao; Li, Zhengjun; Zhang, Yan; Zhang, Songping; Chen, Yi; Yu, Mengran; Ma, Guanghui; Su, Zhiguo

    2015-02-25

    The assembly of antigen structure is often crucial to the potency of vaccines. Currently adopted methods like animal testing and ultracentrifugation take long time and are difficult to automate for multiple samples. Here we develop a size-exclusion high-performance liquid chromatography (SE-HPLC) method to characterize the assembly of antigen structure during both manufacturing process and storage. Three important vaccine antigens including inactivated foot and mouth disease virus (FMDV), which is a virus vaccine; and two virus-like particles (VLPs) vaccines involving hepatitis B core antigen (HBcAg) VLPs, and hepatitis B surface antigen (HBsAg) VLPs, were successfully analyzed using commercially available TSK gel columns with pore size above 45nm. Combined with other analytical methods including SDS-PAGE, dynamic light scattering, wavelength scan, and multi-angle laser light scattering, the SE-HPLC method was proven to be a simple, rapid, and reliable tool for antigen particles assembly analysis. Specifically, for FMDV whole virus particle, SE-HPLC was used to analyze 146S content in vaccine preparations and the thermal dissociation of the 146S. For HBcAg-VLPs that are expressed in recombinant Escherichia coli, its expression level during cell culture process was quantitatively monitored by SE-HPLC. The SE-HPLC also showed applicability for quality check of HBsAg vaccine preparations by monitoring the product consistency of different lot number and the product stability during storage. Results shown in this work clearly demonstrated that SE-HPLC method has potential as a versatile alternative technology for control of the final product by both manufacturers and the regulatory agencies.

  17. Heat stability of protective antigen of Leptospira interrogans serovar lai.

    PubMed Central

    Masuzawa, T; Nakamura, R; Shimizu, T; Yanagihara, Y

    1990-01-01

    Protective antigen (PAg; glycolipid antigen; molecular size, 23 to 30 kilodaltons), the serogroup-specific antigen partially purified from leptospiral cells, is one of the most important protective antigens. The heat stability of PAg was compared with that of whole-cell (WC) antigen by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, protective activity, opsonin-inducing activity, agglutinating antibody-inducing activity, and an inhibition test in an enzyme-linked immunosorbent assay. A band of 23 to 30 kilodaltons of PAg, which was seen in untreated PAg and WC, shifted to a position with a molecular size of ca. 20 kilodaltons after heat treatment of PAg at 80 degrees C for 30 min and WC at 100 degrees C for 30 min. In the enzyme-linked immunosorbent assay inhibition test with monoclonal antibody LW2 and a sonicated antigen of WC, the inhibition rate of PAg and WC to sonicated WC was reduced by heat treatment at 80 degrees C for 30 min and at 100 degrees C for 30 min, respectively. Agglutinating antibody-inducing activities and opsonin-inducing activities of PAg and WC in mice were reduced by heat treatment under the same conditions; these activities were assayed by a microscopic agglutination test and by chemical luminescence response in serum from immunized mice, respectively. Protective activity of heated PAg and heated WC in cyclophosphamide-pretreated mice agreed with the results of immunogenicity in mice. These results indicate that the Leptospira PAg is one of the important protective antigens and is altered by heat treatment at 80 degrees C. Furthermore, the immunogenicity and antigenicity of the PAg present in WC are more stable than that of the extracted PAg, and the coexistence of other cellular components with PAg might protect and stabilize PAg from the heat treatment. Images PMID:2332463

  18. Hepatitis D Virus Infection Among Hepatitis B Surface Antigen Carriers and in “Isolated anti-HBc” Antibodies Profile in Central Tunisia

    PubMed Central

    Mhalla, Salma; Kadri, Yosr; Alibi, Sana; Letaief, Amel; Boukadida, Jalel; Hannachi, Naila

    2016-01-01

    Background: Hepatitis D Virus (HDV) causes accelerated liver diseases in patients with Hepatitis B Virus (HBV) infection. There is lack of data about its prevalence, related risk factors and interaction with HBV carriers in our country. Objectives: The aim of this study was to estimate the prevalence of hepatitis delta and associated risk factors among Hepatitis B surface antigen (HBsAg) and “isolated anti-HBc” profile carriers in central Tunisia. Patients and Methods: In this cross-sectional study, 540 patients with positive HBsAg and 109 “isolated anti-HBc” profile receiving care in a teaching hospital were tested for the presence of HDV serum-markers using commercially available enzyme immunoassay kit. HBV-DNA was detected by nested PCR in “isolated anti-HBc” profile group. Results: Prevalence of HDV was 8.1% in HBsAg carriers group, but it was significantly higher in active than inactive hepatitis (30.2% and 4.5%, respectively, OR = 9, 95% CI: [4.48-18.58]). There was no significant association between studied risk factors and HDV infection. In the “isolated anti-HBc” profile group, prevalence of HDV was 4.6% and HBV-DNA had negative result in all patients with positive results for HDV. Conclusions: Although HDV had low prevalence in our area, it is vital to plan preventive strategies for HDV spread as well as HBV prevention. It is particularly important to suspect HDV infection in active HBV carriers to manage a particularly severe dual infection. HDV infection should be suspected even in negative HBsAg patients having “isolated anti-HBc” profile. PMID:27110257

  19. Discriminating antigen and non-antigen using proteome dissimilarity: bacterial antigens

    PubMed Central

    Ramakrishnan, Kamna; Flower, Darren R

    2010-01-01

    It has been postulated that immunogenicity results from the overall dissimilarity of pathogenic proteins versus the host proteome. We have sought to use this concept to discriminate between antigens and non-antigens of bacterial origin. Sets of 100 known antigenic and nonantigenic peptide sequences from bacteria were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we were unable to determine a threshold able to separate meaningfully antigen from non-antigen. Thus, antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to the human genome. PMID:20975907

  20. Presentation of hepatocellular antigens

    PubMed Central

    Grakoui, Arash; Crispe, Ian Nicholas

    2016-01-01

    The liver is an organ in which antigen-specific T-cell responses manifest a bias toward immune tolerance. This is clearly seen in the rejection of allogeneic liver transplants, and multiple other phenomena suggest that this effect is more general. These include tolerance toward antigens introduced via the portal vein, immune failure to several hepatotropic viruses, the lack of natural liver-stage immunity to malaria parasites, and the frequent metastasis of cancers to the liver. Here we review the mechanisms by which T cells engage with hepatocellular antigens, the context in which such encounters occur, and the mechanisms that act to suppress a full T-cell response. While many mechanisms play a role, we will argue that two important processes are the constraints on the cross-presentation of hepatocellular antigens, and the induction of negative feedback inhibition driven by interferons. The constant exposure of the liver to microbial products from the intestine may drive innate immunity, rendering the local environment unfavorable for specific T-cell responses through this mechanism. Nevertheless, tolerance toward hepatocellular antigens is not monolithic and under specific circumstances allows both effective immunity and immunopathology. PMID:26924525

  1. Hepatitis B virus and hepatitis D virus in blood donors from Argentina: circulation of HBsAg and reverse transcriptase mutants.

    PubMed

    Delfino, Cecilia María; Gentile, Emiliano Alberto; Castillo, Amalia Inés; Cuestas, María Luján; Pataccini, Gabriela; Cánepa, Camila; Malan, Richard; Blejer, Jorgelina; Berini, Carolina; Eirin, María Emilia; Pedrozo, Williams; Oubiña, José Raúl; Biglione, Mirna Marcela; Mathet, Verónica Lidia

    2014-05-01

    In Argentina, current procedures to ensure the safety of the blood supply for transfusion include the serologic detection of specific blood-borne infections. The aim of this study was to evaluate the prevalence and the genetic diversity of hepatitis B virus (HBV) and hepatitis D virus (HDV) in blood donor populations from two distantly located Argentine regions. Data from 56,983 blood donations from the Favaloro Foundation, in the city of Buenos Aires (Central Region), and the Central Blood Bank of Misiones Province (Northeast Region) were analyzed. Samples that were reactive for HBsAg were analyzed for HBV-DNA characterization and HDV serological and molecular analysis. The HBV prevalence was 0.12 % for HBsAg and 1.68 % for anti-HBc antibodies in Buenos Aires, and 0.73 % and 8.55 %, respectively, in Misiones. Seventy-seven HBsAg-reactive samples were analyzed by polymerase chain reaction for HBV-DNA. Subgenotypes A2, B2, C2, F1b and F4 (Buenos Aires) and F1b and D3 (Misiones) were detected. Several mutations within the major hydrophilic region of HBsAg, the reverse transcriptase, the basal core promoter, and the precore/core were detected. HDV genotype 1 was identified in Buenos Aires. This study confirms the circulation of several HBV subgenotypes, as well as known and newly identified variants, and the presence of HDV1 in this population. A thorough investigation has to be carried out to evaluate the clinical importance of some of the documented mutations as well as those detected in the HDV1 case.

  2. Hepatitis B virus and hepatitis D virus in blood donors from Argentina: circulation of HBsAg and reverse transcriptase mutants.

    PubMed

    Delfino, Cecilia María; Gentile, Emiliano Alberto; Castillo, Amalia Inés; Cuestas, María Luján; Pataccini, Gabriela; Cánepa, Camila; Malan, Richard; Blejer, Jorgelina; Berini, Carolina; Eirin, María Emilia; Pedrozo, Williams; Oubiña, José Raúl; Biglione, Mirna Marcela; Mathet, Verónica Lidia

    2014-05-01

    In Argentina, current procedures to ensure the safety of the blood supply for transfusion include the serologic detection of specific blood-borne infections. The aim of this study was to evaluate the prevalence and the genetic diversity of hepatitis B virus (HBV) and hepatitis D virus (HDV) in blood donor populations from two distantly located Argentine regions. Data from 56,983 blood donations from the Favaloro Foundation, in the city of Buenos Aires (Central Region), and the Central Blood Bank of Misiones Province (Northeast Region) were analyzed. Samples that were reactive for HBsAg were analyzed for HBV-DNA characterization and HDV serological and molecular analysis. The HBV prevalence was 0.12 % for HBsAg and 1.68 % for anti-HBc antibodies in Buenos Aires, and 0.73 % and 8.55 %, respectively, in Misiones. Seventy-seven HBsAg-reactive samples were analyzed by polymerase chain reaction for HBV-DNA. Subgenotypes A2, B2, C2, F1b and F4 (Buenos Aires) and F1b and D3 (Misiones) were detected. Several mutations within the major hydrophilic region of HBsAg, the reverse transcriptase, the basal core promoter, and the precore/core were detected. HDV genotype 1 was identified in Buenos Aires. This study confirms the circulation of several HBV subgenotypes, as well as known and newly identified variants, and the presence of HDV1 in this population. A thorough investigation has to be carried out to evaluate the clinical importance of some of the documented mutations as well as those detected in the HDV1 case. PMID:24306325

  3. HBsAg and aflatoxins in sera of rural (Igbo-Ora) and urban (Ibadan) populations in Nigeria.

    PubMed

    Olubuyide, I O; Maxwell, S M; Akinyinka, O O; Hart, C A; Neal, G E; Hendrickse, R G

    1993-12-01

    The purpose of this study was to screen for the presence of hepatitis B surface antigen and aflatoxins in the sera of 100 non-hospitalized individuals from the rural population of Igbo-Ora and 89 non-hospitalized individuals from the urban population of Ibadan, Nigeria. Hitherto, such a study as this has not been undertaken in this environment. The proportions of hepatitis B surface antigen carriage and serum 'pathologic' levels of aflatoxins were high (47-49%, 8.2-9.0% respectively) but varied very little between the two different populations sampled. These findings indicate that determined efforts should be instituted to reduce or eliminate hepatitis B virus infection and aflatoxin contamination of high risk foodstuffs from this environment.

  4. Detection of antibodies to Epstein-Barr virus capsid antigen by immune adherence hemagglutination.

    PubMed

    Lennette, E T; Ward, E; Henle, G; Henle, W

    1982-01-01

    The immune adherence hemagglutination assay was found to be as sensitive and specific as the indirect immunofluorescence technique for titration of antibodies to Epstein-Barr virus capsid antigen. Satisfactory virus capsid antigen-specific and negative control antigens for the immune adherence hemagglutination assay were prepared from cell extracts of the Epstein-Barr virus producer P3HR-1 and the Epstein-Barr virus genome-negative BJAB lymphoblastoid cell lines, respectively. As the immune adherence hemagglutination assay can be used to titrate antibodies to both the heterophil antigen of the Paul-Bunnell type and to virus capsid antigen, it offers a promising alternative to the immunofluorescence methods in the serodiagnosis of Epstein-Barr virus infections which can be performed by most diagnostic laboratories.

  5. Radioimmunoassay for hepatitis B core antigen

    SciTech Connect

    Sagnelli, E.; Pereira, C.; Triolo, G.; Vernace, S.; Paronetto, F.

    1982-02-01

    Serum hepatitis B core antigen (HBcAg) is an important marker of hepatitis B virus replication. We describe an easy, sensitive radioimmunoassay for determination of HBcAg in detergent-treated serum pellets containing Dane particles. Components of a commercial kit for anticore determination are used, and HBcAG is measured by competitive inhibition of binding of /sub 125/I-labeled antibodies to HBcAg with HBcAg-coated beads. We assayed for HBcAG in the sera of 49 patients with hepatitis B surface antigen (HBsAg)-positive chronic hepatitis, 50 patients with HBsAg-negative chronic hepatitis, and 30 healthy volunteers. HBcAg was detected in 41% of patients with HBsAg-positive chronic hepatitis but not in patients with HBsAg-negative chronic hepatitis. Hepatitis Be antigen (an antigen closely associated with the core of Dane particles) determined in the same sera by radioimmunoassay, was not detected in 50% of HBcAg-positive sera.

  6. An assay for adjuvanticity

    PubMed Central

    Dresser, D. W.

    1968-01-01

    Adult mice injected with an adequate amount of a non-immunogenic antigen progress to a specific state of immunological paralysis, unless a substance with `extrinsic' adjuvanticity is injected before the induction of paralysis is completed. Consequently incipiently paralysed mice can be used to assay substances for adjuvanticity. Conventional adjuvants such as Freund's adjuvant and pertussis possess adjuvanticity; other substances with varying degrees of adjuvanticity are listed in the tables. It has been shown that the adjuvanticity effect of an injection of pertussis lasts for only a few days, although the effect of such an injection of pertussis on phagocytosis of carbon particles does not reach a maximum until 2 weeks after the injection. The dose-effectiveness of alum precipitated (highly phagocytosable) bovine γ-globulin was greatly increased by the intraperitoneal injection of pertussis. The evidence is considered to be incompatible with increased phagocytosis being either an essential factor in the role of pertussis as a conventional adjuvant, or in the adjuvanticity effect of pertussis. PMID:4179956

  7. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

  8. Anti-HCV immunoassays based on a multiepitope antigen and fluorescent lanthanide chelate reporters.

    PubMed

    Salminen, Teppo; Juntunen, Etvi; Khanna, Navin; Pettersson, Kim; Talha, Sheikh M

    2016-02-01

    There is a need for simple to produce immunoassays for hepatitis C virus (HCV) antibody capable of detecting all genotypes worldwide. Current commonly used third generation immunoassays use three to six separate recombinant proteins or synthetic peptides. We have developed and expressed in Escherichia coli a single recombinant antigen incorporating epitopes from different HCV proteins. This multiepitope protein (MEP) was used to develop two types of HCV antibody immunoassays: a traditional antibody immunoassay using a labeled secondary antibody (indirect assay) and a double-antigen assay with the same MEP used as capture binder and labeled binder. The secondary antibody assay was evaluated with 171 serum/plasma samples and double-antigen assay with 148 samples. These samples included an in-house patient sample panel, two panels of samples with different HCV genotypes and a seroconversion panel. The secondary antibody immunoassay showed 95.6% sensitivity and 100% specificity while the double-antigen assay showed 91.4% sensitivity and 100% specificity. Both assays detected samples from all six HCV genotypes. The results showed that combining a low-cost recombinant MEP binder antigen with a high sensitivity fluorescent lanthanide reporter can provide a sensitive and specific immunoassay for HCV serology. The results also showed that the sensitivity of HCV double-antigen assays may suffer from the low avidity immune response of acute infections.

  9. Rotor assembly and assay method

    DOEpatents

    Burtis, Carl A.; Johnson, Wayne F.; Walker, William A.

    1993-01-01

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor.

  10. Rotor assembly and assay method

    DOEpatents

    Burtis, C.A.; Johnson, W.F.; Walker, W.A.

    1993-09-07

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor. 34 figures.

  11. Radioimmune assay of human platelet prostaglandin synthetase

    SciTech Connect

    Roth, G.J.; Machuga, E.T.

    1982-02-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH/sub 2/ from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and (/sup 125/I)-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the (/sup 125/I)antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10/sup 9/ platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency.

  12. Reversal of B-cell hyperactivation and functional impairment is associated with HBsAg seroconversion in chronic hepatitis B patients

    PubMed Central

    Xu, Xiangsheng; Shang, Qinghua; Chen, Xinyue; Nie, Weimin; Zou, Zhengsheng; Huang, Ang; Meng, Ming; Jin, Lei; Xu, Ruonan; Zhang, Ji-Yuan; Fu, Junliang; Wang, Lifeng; Tang, Zirong; Xie, Yunbo; Yang, Xiaoming; Zhang, Zheng; Wang, Fu-Sheng

    2015-01-01

    B cells play an important role in the clearance of hepatitis B virus (HBV) and protection against reinfection. However, the functional characteristics of these cells that are associated with the outcome of chronic HBV infection remain unknown. We comprehensively investigated the frequency, phenotype, and function of peripheral B-cell subsets from CHB patients in different phases: immune tolerance (IT), immune activation (IA), immune clearance (IC), responders with HBsAg seroconversion (resolved patients, RP), and healthy controls (HC). IA patients displayed lower percentages of peripheral blood memory B cells compared with the other groups. Overall polyclonal activation of B cells, indicated by higher levels of activation markers and secretion of IgG and IgM, was observed in IA patients. This B-cell hyperactivation could be induced by increased IFN-α and soluble CD40 ligands in IA patients. Notably, the expression of the co-stimulator molecule CD80 and serum HBsAb and the frequency of HBsAg-specific B cells were significantly decreased in IT, IA, and IC patients compared with HC subjects. More importantly, the B-cell hyperactivation, co-stimulatory molecule downregulation and HBsAg-specific B-cell impairment were reversed in RP patients. The reversal of B-cell hyperactivation and functional impairment is associated with HBsAg seroconversion in chronic hepatitis B patients. PMID:25849120

  13. Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand

    PubMed Central

    Nawtaisong, Pruksa; Tanganuchitcharnchai, Ampai; Smith, Derek J.; Day, Nicholas P. J.; Paris, Daniel H.

    2016-01-01

    Background Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. Methodology/Principal Findings This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. Conclusions/Significance Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes. PMID:27248711

  14. Enzyme-linked fluorescence assay: Ultrasensitive solid-phase assay for detection of human rotavirus.

    PubMed Central

    Yolken, R H; Stopa, P J

    1979-01-01

    Enzyme-linked immunosorbent assay (ELISA) has proven to be a useful assay system for the direct detection of infectious agents. However, when the usual color-producing substrates are employed, relatively large amounts of substrate must be hydrolyzed by the bound enzyme before detection can be achieved. We attempted to improve the sensitivity of ELISA by utilizing a substrate that yields a fluorescent product on enzyme action. The enzyme-linked fluorescence assay (ELFA) based on this principle was approximately 100 times more sensitive than the corresponding ELISA or radioimmunoassay for the detection of human rotavirus in a standard stool suspension. In addition, the ELFA for human rotavirus was capable of detecting antigen in six specimens that were negative by ELISA. Five of these specimens were obtained late in the course of confirmed rotavirus infections. ELFA provides a simple, reliable, ultrasensitive method for the rapid detection of viral antigen. PMID:226564

  15. Immunonephelometric and immunoturbidimetric assays for proteins.

    PubMed

    Whicher, J T; Price, C P; Spencer, K

    1983-01-01

    Immunonephelometric and immunoturbidimetric techniques for the measurement of proteins have developed and expanded rapidly in recent years and are fast replacing the time-honoured gel precipitation techniques. The reasons for this are primarily an increased awareness of the value of specific protein measurements with the impetus of improved chemical reagents and advances in instrument technology. This review discusses the background to such free fluid phase immunochemical measurement systems and the developments which have occurred in this field. The following topics are reviewed. (1) The theoretical background of light scattering theory as applied to the measurement of antibody-antigen complexes. (2) The nature and kinetics of the antibody-antigen reaction in fluid media and the effect of enhancing polymers. (3) The sample and antibody requirements for nephelometric and turbidimetric assay. (4) Instrumental systems for nephelometry. (5) Instrumental systems for turbidimetry. (6) Methods of establishing, assessing and monitoring nephelometric assays for specific proteins in the laboratory.

  16. Electrophoretic and immunoblot analyses of Rhizopus arrhizus antigens.

    PubMed Central

    Wysong, D R; Waldorf, A R

    1987-01-01

    Four antigen preparations from Rhizopus arrhizus were made and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and column chromatography. Electrophoretic analyses of these antigens indicated that there are 18 to 28 component bands with a molecular mass range of approximately 10,500 to 83,000 daltons. Seven of these bands appear to be components common to three antigen preparations. Several of the bands identified by SDS-PAGE were composed of glycoproteins or carbohydrates as determined by their affinity for concanavalin A. Western blots, using sera from five patients with mucormycosis, consistently identified five different determinants in the R. arrhizus antigens separated by SDS-PAGE. This suggests that several of the Rhizopus antigens are present during mucormycosis. Four of the antigenic determinants recognized by patient sera reacted with the concanavalin A-peroxidase stain, indicating that they are composed of glycoproteins or carbohydrate. Enzyme-linked immunosorbent assays of sera from five patients with mucormycosis and with rabbit antisera resulted in antibody titers ranging from 1:64 to 1:32,000 for the R. arrhizus antigens. Images PMID:3546367

  17. Electrophoretic and immunoblot analyses of Rhizopus arrhizus antigens.

    PubMed

    Wysong, D R; Waldorf, A R

    1987-02-01

    Four antigen preparations from Rhizopus arrhizus were made and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and column chromatography. Electrophoretic analyses of these antigens indicated that there are 18 to 28 component bands with a molecular mass range of approximately 10,500 to 83,000 daltons. Seven of these bands appear to be components common to three antigen preparations. Several of the bands identified by SDS-PAGE were composed of glycoproteins or carbohydrates as determined by their affinity for concanavalin A. Western blots, using sera from five patients with mucormycosis, consistently identified five different determinants in the R. arrhizus antigens separated by SDS-PAGE. This suggests that several of the Rhizopus antigens are present during mucormycosis. Four of the antigenic determinants recognized by patient sera reacted with the concanavalin A-peroxidase stain, indicating that they are composed of glycoproteins or carbohydrate. Enzyme-linked immunosorbent assays of sera from five patients with mucormycosis and with rabbit antisera resulted in antibody titers ranging from 1:64 to 1:32,000 for the R. arrhizus antigens.

  18. Duality of β-glucan microparticles: antigen carrier and immunostimulants

    PubMed Central

    Baert, Kim; De Geest, Bruno G; De Greve, Henri; Cox, Eric; Devriendt, Bert

    2016-01-01

    Designing efficient recombinant mucosal vaccines against enteric diseases is still a major challenge. Mucosal delivery of recombinant vaccines requires encapsulation in potent immunostimulatory particles to induce an efficient immune response. This paper evaluates the capacity of β-glucan microparticles (GPs) as antigen vehicles and characterizes their immune-stimulatory effects. The relevant infectious antigen FedF was chosen to be loaded inside the microparticles. The incorporation of FedF inside the particles was highly efficient (roughly 85%) and occurred without antigen degradation. In addition, these GPs have immunostimulatory effects as well, demonstrated by the strong reactive oxygen species (ROS) production by porcine neutrophils upon their recognition. Although antigen-loaded GPs still induce ROS production, antigen loading decreases this production by neutrophils for reasons yet unknown. However, these antigen-loaded GPs are still able to bind their specific β-glucan receptor, demonstrated by blocking complement receptor 3, which is the major β-glucan receptor on porcine neutrophils. The dual character of these particles is confirmed by a T-cell proliferation assay. FedF-loaded particles induce a significantly higher FedF-specific T-cell proliferation than soluble FedF. Taken together, these results show that GPs are efficient antigen carriers with immune-stimulatory properties. PMID:27330289

  19. Intracellular targeting of antigens internalized by membrane immunoglobulin in B lymphocytes.

    PubMed

    Mitchell, R N; Barnes, K A; Grupp, S A; Sanchez, M; Misulovin, Z; Nussenzweig, M C; Abbas, A K

    1995-05-01

    An important function of membrane immunoglobulin (mIg), the B cell antigen receptor, is to endocytose limiting quantities of antigen for efficient presentation to class II-restricted T cells. We have used a panel of mIg mutants to analyze the mechanism of mIg-mediated antigen presentation, and specifically to explore the ability of mIg to target internalized antigen to intracellular processing compartments. Transfected mIgs carrying substitutions for the transmembrane Tyr587 residue fail to efficiently present specifically bound antigen. However, these mutants internalize antigen normally, and their defect cannot be attributed to a lack of mIg-associated Ig alpha/Ig beta molecules. A novel functional assay for detecting antigenic peptides in subcellular fractions shows that wild-type mIg transfectants generate class II-peptide complexes intracellularly, whereas only free antigenic peptides are detectable in the mutant mIg transfectants. Furthermore, an antigen competition assay reveals that antigen internalized by the mutant mIgs fails to enter the intracellular processing compartment accessed by wild-type mIg. Therefore, mIg specifically targets bound and endocytosed antigen to the intracellular compartment where processed peptides associate with class II molecules, and the transmembrane Tyr587 residue plays an obligatory role in this process. Targeting of internalized antigen may be mediated by receptor-associated chaperones, and may be a general mechanism for optimizing the presentation of specifically bound and endocytosed antigens in b lymphocytes and other antigen-presenting cells.

  20. Confocal detection of planar homogeneous and heterogeneous immunosorbent assays

    NASA Astrophysics Data System (ADS)

    Ghafari, Homanaz; Zhou, Yanzhou; Ali, Selman; Hanley, Quentin S.

    2009-11-01

    Optically sectioned detection of fluorescence immunoassays using a confocal microscope enables the creation of both homo- and heterogeneous planar format assays. We report a set assays requiring optically sectioned detection using a model system and analysis procedures for separating signals of a surface layer from an overlying solution. A model sandwich assay with human immunoglobulin G as the target antigen is created on a glass substrate. The prepared surfaces are exposed to antigen and a FITC-labeled secondary antibody. The resulting preparations are either read directly to provide a homogeneous assay or after wash steps, giving a heterogeneous assay. The simplicity of the object shapes arising from the planar format makes the decomposition of analyte signals from the thin film bound to the surface and overlayer straightforward. Measured response functions of the thin film and overlayer fit well to the Cauchy-Lorentz and cumulative Cauchy-Lorentz functions, respectively, enabling the film and overlayer to be separated. Under the conditions used, the detection limits for the homogeneous and heterogeneous forms of the assay are 2.2 and 5.5 ng/ml, respectively. Planar format, confocally read fluorescence assays enable wash-free detection of antigens and should be applicable to a wide range of assays involving surface-bound species.

  1. Confocal detection of planar homogeneous and heterogeneous immunosorbent assays.

    PubMed

    Ghafari, Homanaz; Zhou, Yanzhou; Ali, Selman; Hanley, Quentin S

    2009-01-01

    Optically sectioned detection of fluorescence immunoassays using a confocal microscope enables the creation of both homo- and heterogeneous planar format assays. We report a set assays requiring optically sectioned detection using a model system and analysis procedures for separating signals of a surface layer from an overlying solution. A model sandwich assay with human immunoglobulin G as the target antigen is created on a glass substrate. The prepared surfaces are exposed to antigen and a FITC-labeled secondary antibody. The resulting preparations are either read directly to provide a homogeneous assay or after wash steps, giving a heterogeneous assay. The simplicity of the object shapes arising from the planar format makes the decomposition of analyte signals from the thin film bound to the surface and overlayer straightforward. Measured response functions of the thin film and overlayer fit well to the Cauchy-Lorentz and cumulative Cauchy-Lorentz functions, respectively, enabling the film and overlayer to be separated. Under the conditions used, the detection limits for the homogeneous and heterogeneous forms of the assay are 2.2 and 5.5 ng/ml, respectively. Planar format, confocally read fluorescence assays enable wash-free detection of antigens and should be applicable to a wide range of assays involving surface-bound species.

  2. New broadly reactive neutralizing antibodies against hepatitis B virus surface antigen.

    PubMed

    Kucinskaite-Kodze, Indre; Pleckaityte, Milda; Bremer, Corinna M; Seiz, Pia L; Zilnyte, Milda; Bulavaite, Aiste; Mickiene, Gitana; Zvirblis, Gintautas; Sasnauskas, Kestutis; Glebe, Dieter; Zvirbliene, Aurelija

    2016-01-01

    Hepatitis B virus (HBV) surface antigen (HBsAg) is considered to be the most important target for the diagnosis and immune prophylaxis of HBV infection. HBsAg-specific monoclonal antibodies (MAbs) are extensively used for studying the complex structure of the HBsAg, mapping the neutralizing epitopes and development of HBV diagnostic tests. However, the efficiency of anti-HBV binding strongly depends on the epitope structure and MAb capability to recognize different HBV variants. In the current study, 9 MAbs against yeast-expressed HBsAg of ayw2 serotype were generated and 7 of them were shown to recognize a linear epitope comprising amino acid (aa) residues 119-GPCRTCT-125 within the main antigenic "a" determinant of HBsAg. One MAb of the highest affinity (clone HB1) was selected for detailed cross-reactivity studies, generation of recombinant single-chain antibody (scFv) and molecular modelling of antibody-epitope interaction. The importance of each aa residue within the identified MAb epitope was determined by alanine substitution study that revealed aa residues C(121), T(123), C(124) and T(125) as essential for binding. These aa residues are highly conserved among HBV variants. In contrast, alanine substitution of G119, P120 and R122 had no or minor influence on the reactivity with the MAb. Certain aa residues at position 122 (either R or K) define different HBV serotypes (either d or y), therefore, the affinity of the MAb HB1 for the epitope with R122K substitution was determined to evaluate its diagnostic potential. The MAb recognized both epitope variants with high affinity. Sequence alignment of the MAb epitope within different HBV strains demonstrated that the shortest peptide recognized by the MAb 121-CR(K)TCT-125 is identical among different human HBV genotypes (HBV A-F, H) and monkey HBV species (HBVCP, HBVGO, HBVGB, WMHBV). In line with these data, the MAb HB1 was cross-reactive in Western blot with a large panel of antigens derived from different HBV

  3. A phase I study of the safety and immunogenicity of recombinant hepatitis B surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant.

    PubMed

    Halperin, Scott A; Van Nest, Gary; Smith, Bruce; Abtahi, Simin; Whiley, Heather; Eiden, Joseph J

    2003-06-01

    Certain oligodeoxynuclotides with CpG motifs provide enhanced immune response to co-delivered antigens. We performed a phase I, observer-blinded, randomized study in healthy anti-hepatitis B surface antigen (anti-HBsAg) antibody negative adults to explore safety and immunogenicity of co-injection of recombinant HBsAg combined with an immunostimulatory DNA sequence (ISS) 1018 ISS. Four ISS dosage groups (N=12 per group) were used: 300, 650, 1000 or 3000 microg. For each group, two controls received 20 microg HBsAg alone, two controls received ISS alone, and eight subjects received ISS+20 microg HBsAg. Subjects received two doses 8 weeks apart. Injection site reactions (tenderness and pain on limb movement) were more frequent at higher ISS+HBsAg doses but were mainly mild and of short duration. Higher anti-HBsAg antibody levels were associated with higher ISS doses. Four weeks after the first dose, a seroprotective titer (>or=10 mIU/ml) was noted for 0, 25, 75, and 87.5% of subjects by increasing ISS dose group (P<0.05) for those who received ISS+HBsAg; 1 month after the second dose this increased to 62.5, 100, 100, and 100%, respectively. Geometric mean anti-HBsAg antibody levels by increasing ISS+HBsAg dose were 1.22, 5.78, 24.75, and 206.5 mIU/ml after the first dose and 65.37, 877.6, 1545, and 3045 mIU/ml after the second dose. We conclude that 1018 ISS+HBsAg was well tolerated and immunogenic in this phase I study in healthy adults and may offer the potential for enhancement of hepatitis B virus (HBV) immunization and protection after one or two doses or in individuals who fail to respond to the standard vaccine regimen. PMID:12744879

  4. Cooperative Immunoassays: Ultrasensitive Assays with Mixed Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ehrlich, Paul H.; Moyle, William R.

    1983-07-01

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

  5. Cancer vaccine--Antigenics.

    PubMed

    2002-01-01

    Antigenics is developing a therapeutic cancer vaccine based on heat-shock proteins (HSPs). The vaccine [HSPPC-96, Oncophage] is in a pivotal phase III clinical trial for renal cancer at 80 clinical sites worldwide. The trial is enrolling at least 500 patients who are randomised to receive surgical removal of the primary tumour followed by out-patient treatment with Oncophage((R)) or surgery only. This study was initiated on the basis of results from a pilot phase I/II study and preliminary results from a phase II study in patients with renal cell cancer. In October 2001, Oncophage was designated as a fast-track product by the Food and Drug Administration in the US for the treatment of renal cell carcinoma. Oncophage is in phase I/II trials in Italy for colorectal cancer (30 patients) and melanoma. The trials in Italy are being conducted at the Istituto dei Tumouri, Milan (in association with Sigma-Tau). Preliminary data from the phase II trial for melanoma was presented at the AACR-NCI-EORTC International Conference in Florida, USA, in October 2001. Oncophage is also in a phase I/II (42 patients) and a phase II trial (84 patients) in the US for renal cell cancer, a phase II trial in the US for non-Hodgkin's lymphoma (35 patients), a phase II trial in the US for sarcoma (20-35 patients), a phase I/II trial in the US for melanoma (36 patients), and phase I/II trials in Germany for gastric (30 patients) and pancreatic cancers. A pilot phase I trial in patien