Science.gov

Sample records for antigen hbsag assays

  1. Comparison between Elecsys HBsAg II and Architect HBsAg QT Assays for Quantification of Hepatitis B Surface Antigen among Patients Coinfected with HIV and Hepatitis B Virus

    PubMed Central

    Maylin, Sarah; Boyd, Anders; Delaugerre, Constance; Zoulim, Fabien; Lavocat, Fabien; Simon, François; Girard, Pierre-Marie

    2012-01-01

    Hepatitis B surface antigen (HBsAg) quantification has been steadily gaining interest as a clinical marker of therapeutic efficacy, for which two commercial assays are currently available: Architect HBsAg QT (Architect) and Elecsys HBsAg II (Elecsys). HBsAg quantification was evaluated using both assays in 126 human immunodeficiency virus (HIV) and hepatitis B virus (HBV)-coinfected patients initiating treatment with tenofovir dipivoxil fumarate. Linear regression and correlation were used to establish the relationship between the two methods. Bland-Altman analysis was performed to determine mean between-assay difference and limits of agreement (LOA) (±2 standard deviations [SD]) both overall and stratified on HBV (hepatitis B envelope antigen [HBeAg] status, replication, genotype, HBV mutants) or HIV (CD4+ cell count) cofactors. There was a significant correlation between Elecsys and Architect assays (correlation coefficient, r = 0.959; P < 0.001). HBsAg quantification using the Elecsys assay was on average 0.200 log10 IU/ml (LOA, −0.500, 0.800) higher than that using Architect, which was consistent across levels of CD4+ cell count, presence of precore and YMDD mutations, and HBeAg status. A slightly larger mean between-assay difference was observed with genotypes A and G (0.196 and 0.201, respectively) versus HBV genotypes D and E (0.036 and 0.030, respectively). Mutations on the S region at position s120/s145 were the only determinant in which the mean between-assay difference in HBsAg quantification was lower than the null value (−0.078). In conclusion, the Elecsys assay, with automatic on-board dilution, is capable of quantifying serum HBsAg levels in HIV-HBV-coinfected patients, with very high correlation with the Architect assay. PMID:22190396

  2. Comparison between Elecsys HBsAg II and architect HBsAg QT assays for quantification of hepatitis B surface antigen among patients coinfected with HIV and hepatitis B virus.

    PubMed

    Maylin, Sarah; Boyd, Anders; Delaugerre, Constance; Zoulim, Fabien; Lavocat, Fabien; Simon, François; Girard, Pierre-Marie; Lacombe, Karine

    2012-02-01

    Hepatitis B surface antigen (HBsAg) quantification has been steadily gaining interest as a clinical marker of therapeutic efficacy, for which two commercial assays are currently available: Architect HBsAg QT (Architect) and Elecsys HBsAg II (Elecsys). HBsAg quantification was evaluated using both assays in 126 human immunodeficiency virus (HIV) and hepatitis B virus (HBV)-coinfected patients initiating treatment with tenofovir dipivoxil fumarate. Linear regression and correlation were used to establish the relationship between the two methods. Bland-Altman analysis was performed to determine mean between-assay difference and limits of agreement (LOA) (±2 standard deviations [SD]) both overall and stratified on HBV (hepatitis B envelope antigen [HBeAg] status, replication, genotype, HBV mutants) or HIV (CD4(+) cell count) cofactors. There was a significant correlation between Elecsys and Architect assays (correlation coefficient, r = 0.959; P < 0.001). HBsAg quantification using the Elecsys assay was on average 0.200 log(10) IU/ml (LOA, -0.500, 0.800) higher than that using Architect, which was consistent across levels of CD4(+) cell count, presence of precore and YMDD mutations, and HBeAg status. A slightly larger mean between-assay difference was observed with genotypes A and G (0.196 and 0.201, respectively) versus HBV genotypes D and E (0.036 and 0.030, respectively). Mutations on the S region at position s120/s145 were the only determinant in which the mean between-assay difference in HBsAg quantification was lower than the null value (-0.078). In conclusion, the Elecsys assay, with automatic on-board dilution, is capable of quantifying serum HBsAg levels in HIV-HBV-coinfected patients, with very high correlation with the Architect assay. PMID:22190396

  3. Performance evaluation of 70 hepatitis B virus (HBV) surface antigen (HBsAg) assays from around the world by a geographically diverse panel with an array of HBV genotypes and HBsAg subtypes

    PubMed Central

    Scheiblauer, H; El-Nageh, M; Diaz, S; Nick, S; Zeichhardt, H; Grunert, H-P; Prince, A

    2010-01-01

    Background and Objectives This study was conducted by the International Consortium for Blood Safety (ICBS) to identify high-quality test kits for detection of hepatitis B virus (HBV) surface antigen (HBsAg) for the benefit of developing countries. Materials and Methods The 70 HBsAg test kits from around the world were evaluated comparatively for their clinical sensitivity, analytical sensitivity, sensitivity to HBV genotypes and HBsAg subtypes, and specificity using 394 (146 clinical, 48 analytical and 200 negative) ICBS Master Panel members of diverse geographical origin comprising the major HBV genotypes A-F and the HBsAg subtypes adw2,4, adr and ayw1-4. Results Seventeen HBsAg enzyme immunoassay (EIA) kits had high analytical sensitivity <0·13 IU/ml, showed 100% diagnostic sensitivity, and were even sensitive for the various HBV variants tested. An additional six test kits had high sensitivity (<0·13 IU/ml) but missed HBsAg mutants and/or showed reduced sensitivity to certain HBV genotypes. Twenty HBsAg EIA kits were in the sensitivity range of 0·13–1 IU/ml. The other eight EIAs and the 19 rapid assays had analytical sensitivities of 1 to >4 IU/ml. These assays were falsely negative for 1–4 clinical samples and 17 of these test kits showed genotype dependent sensitivity reduction. Analytical sensitivities for HBsAg of >1 IU/ml significantly reduce the length of the HBsAg positive period which renders them less reliable for detecting HBsAg in asymptomatic HBV infections. Reduced sensitivity for HBsAg with genetic diversity of HBV occurred with genotypes/subtypes D/ayw3, E/ayw4, F/adw4 and by S gene mutants. Specificity of the HBsAg assays was ≥99·5% in 57 test kits and 96·4–99·0% in the remaining test kits. Conclusion Diagnostic efficacy of the evaluated HBsAg test kits differed substantially. Laboratories should therefore be aware of the analytical sensitivity for HBsAg and check for the relevant HBV variants circulating in the relevant population

  4. Decreased antigenicity profiles of immune-escaped and drug-resistant hepatitis B surface antigen (HBsAg) double mutants

    PubMed Central

    2013-01-01

    Background Selective pressure from either the immune response or the use of nucleoside analogs in antiviral therapy could be driving the emergence of HBV mutants. Because of the overlap of the open reading frame (ORF) S for the HBsAg and ORF P for viral polymerase, rtM204I and rtM204V mutations in the polymerase would produce sI195M and sW196S in the HBsAg. The combined effects of immune-escaped mutations (sT118M, sG145K, sG145R) and drug-resistant mutations (rtM204I, rtM204V) on the antigenicity profiles of HBsAg has not been widely explored. Methods To determine the combined effects of immune-escaped and drug-resistant mutants on the antigenicity profiles of HBsAg, recombinant plasmids encoding HBsAg double mutants were constructed using site-directed mutagenesis. The supernatant from each plasmid transfection was analyzed for HBsAg in the western-blotting and five of the most commonly used commercial ELISA kits in China. Results Western-blotting assay showed the successful expression of each HBsAg mutant. All five ELISA kits manifested similar avidity, which were demonstrated by the slope of the curves, for the sT118M mutant, and sT118M-rtM204I (sT118M-sI195M) and sT118M-rtM204V (sT118M-sW196S) double mutants, suggesting that drug-resistant YMDD mutants caused negligible losses in the antigenicity of immune-escaped sT118M HBsAg. In contrast, the presence of the rtM204I (sI195M) mutation, but not rtM204V (sW196S) in combination with the sG145K mutation significantly reduced the avidity of sG145K HBsAg. The rtM204I (sI195M) mutation also decreased the antigenicity profiles for sG145R HBsAg. Conclusions Drug-resistant mutations rtM204I (sI195M) and rtM204V (sW196S) caused significant reduction in antigenicity for the immune-escaped HBsAg mutants sG145K and sG145R, which may hamper HBV diagnosis and disease control from HBV blood-transfusion transmissions in China. The development of ELISA kits with a greater sensitivity for drug-resistant and immune-escaped HBsAg

  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. Hepatitis B Core IgM antibody (anti-HBcIgM) among hepatitis B Surface antigen (HBsAg) negative blood donors in Nigeria

    PubMed Central

    2011-01-01

    Background Transfusion associated Hepatitis B virus (TAHBV) continues to be a major problem despite mandatory screening for Hepatitis B surface Antigen (HBsAg). Presence of HBsAg is the common method for detecting hepatitis B infection. Unfortunately, this marker is not detected during the window period of the infection. Nigeria being a developing country cannot afford DNA testing of all collected units of blood which serve as the only possibility of achieving zero risk of transfusion associated HBV. Five different serological makers of hepatitis B virus (HBV) infection were therefore assessed to evaluate the reliability of using HBsAg marker alone in diagnosis of HBV infection among blood donors and to detect the serological evidence of the infection at the window period. This will preclude the possibility of transmitting hepatitis B through transfusion of Hepatitis B surface antigen (HBsAg) negative blood in Nigeria. Methods Between July and August 2009, 92 blood donors were enrolled for the study. The prevalence of 5 different markers of Hepatitis B virus infection was detected using Enzyme Linked Immunosorbent Assay (ELISA). Demographic factors were assessed during the study. Results HBsAg and its antibody (anti-HBs) was detected in 18 (19.6%) and 14(15.2%) of the 92 blood donors respectively. Anti-HBc IgM was found in 12(13.0%) of the 92 blood donors while Hepatitis B envelope antigen (HBeAg) and its antibody (anti-HBe) were detected in 4(8.9%) and 12(26.7%) respectively from 45 donors sampled. HBeAg is a marker of high infectivity and appears after HBsAg. At least one serological marker was detected in 30(32.6%) of the blood donors. Five (5.4%) of the 92 donors had anti-HBc IgM as the only serological evidence of hepatitis B virus infection. Conclusions The result of this study shows that five donors have anti-HBcIgM as the only serological evidence of HBV infection. Inclusion of anti-HBcIgM in routine screening of blood donors in Nigeria should be encouraged

  8. Failure to detect hepatitis B surface antigen (HBsAg) in certified shellfish from New England.

    PubMed Central

    Koff, R S; Connelly, L J

    1976-01-01

    In this study, hard- and soft-shell clams harvested during a one-year period from state approved (certified) clam beds in Maine, Massachusetts, and Rhode Island were tested for HBsAg by radioimmunoassay. HBsAg could not be detected in clam viscera. The role of bivalve mollusk ingestion in transmission of Hepatitis B remains speculative. PMID:1251953

  9. 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

  10. 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. PMID:26711310

  11. 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. PMID:26003704

  12. Assaying Carcinoembryonic Antigens by Normalized Saturation Magnetization

    NASA Astrophysics Data System (ADS)

    Huang, Kai-Wen; Chieh, Jen-Jie; Shi, Jin-Cheng; Chiang, Ming-Hsien

    2015-07-01

    Biofunctionalized magnetic nanoparticles (BMNs) that provide unique advantages have been extensively used to develop immunoassay methods. However, these developed magnetic methods have been used only for specific immunoassays and not in studies of magnetic characteristics of materials. In this study, a common vibration sample magnetometer (VSM) was used for the measurement of the hysteresis loop for different carcinoembryonic antigens (CEA) concentrations ( Φ CEA) based on the synthesized BMNs with anti-CEA coating. Additionally, magnetic parameters such as magnetization ( M), remanent magnetization ( M R), saturation magnetization ( M S), and normalized parameters (Δ M R/ M R and Δ M S/ M S) were studied. Here, Δ M R and Δ M s were defined as the difference between any ΦCEA and zero Φ CEA. The parameters M, Δ M R, and Δ M S increased with Φ CEA, and Δ M S showed the largest increase. Magnetic clusters produced by the conjugation of the BMNs to CEAs showed a Δ M S greater than that of BMNs. Furthermore, the relationship between Δ M S/ M S and Φ CEA could be described by a characteristic logistic function, which was appropriate for assaying the amount of CEAs. This analytic Δ M S/ M S and the BMNs used in general magnetic immunoassays can be used for upgrading the functions of the VSM and for studying the magnetic characteristics of materials.

  13. 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. PMID:26615803

  14. 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 §...

  15. 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 §...

  16. 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 §...

  17. 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 §...

  18. 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 §...

  19. 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

  20. 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

  1. 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.

  2. Development of hen antihepatitis B antigen IgY-based conjugate for ELISA assay

    PubMed Central

    Nafea, Najat Muayed; Sabbah, Majeed Arsheed; AL-Suhail, Raghad; Mahdavi, Amir Hossein; Asgary, Sedigheh

    2015-01-01

    Background: Chicken antibodies have many advantages to the mammalian antibodies and have several important differences against mammalian IgG with regard to their specificity and large-scale production. In this study, the production, purification, and HRP conjugation of polyclonal IgY against hepatitis virus surface antigen (HBsAg) were carried out. Materials and Methods: Single Comb White Leghorn hens were immunized intramuscularly with hepatitis B vaccine in combination with Freund's adjuvants. Blood and eggs were collected before and during ten weeks after the first immunization. Results: A highly purified of 180 KDa with specific activity of 200 mIU/ml was obtained by our purification protocol. One milligram of the purified IgY was labeled with horseradish peroxidase (HRP). Sandwich ELISA was used to determine the optimum titer of anti-HbsAg IgY-conjugate which was found to be 1:20. Conclusions: This study showed that laying hens can be used as an alternative source for production of polyclonal antibodies against HBsAg and anti-HBs IgY could be labeled with HRP enzyme and could subsequently be used successfully as secondary antibody in ELISA for detection of HBsAg in the patients sera. PMID:26015926

  3. 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

  4. An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection.

    PubMed

    Jahnmatz, Peter; Bengtsson, Theresa; Zuber, Bartek; Färnert, Anna; Ahlborg, Niklas

    2016-06-01

    The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs. PMID:26930550

  5. Oral vaccination of mice with Tremella fuciformis yeast-like conidium cells expressing HBsAg.

    PubMed

    Shin, Dong-Il; Song, Kyu-Seon; Park, Hee-Sung

    2015-03-01

    Tremella fuciformis yeast-like conidium (YLC) cells were transformed by co-cultivation with Agrobacterium cells harboring the hepatitis B surface antigen (HBsAg) gene construct under the control of the CaMV35S promoter. Integration of HBsAg DNA into the YLC genome was confirmed by PCR and dot-blot hybridization. Immunoblotting verified expression of the recombinant protein. Oral administration of YLC cells expressing HBsAg in mice significantly increased anti-HBsAg antibody titer levels using a double prime-boost strategy that combined parenteral and oral HBsAg boosters. PMID:25374008

  6. 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. PMID:22529122

  7. 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

  8. Detection of sugarcane yellow leaf virus by direct antigen coated enzyme-linked immunosorbent assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The L9 (34) orthogonal diagram was applied to optimize detection conditions of direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) for Sugarcane yellow leaf virus (SCYLV) in sugarcane. Statistic analyses indicated that 150 µL of SCYLV in juice and leaf crude extract antigens was the ...

  9. Cryptococcus neoformans meningitis with negative cryptococcal antigen: Evaluation of a new immunochromatographic detection assay

    PubMed Central

    Opota, O.; Desgraz, B.; Kenfak, A.; Jaton, K.; Cavassini, M.; Greub, G.; Prod'hom, G.; Giulieri, S.

    2014-01-01

    Detection of cryptococcal antigen in serum or cerebrospinal fluid allows cryptococcal meningitis diagnosis within few hours with >90% sensitivity. In an HIV-positive patient with Cryptococcus neoformans meningitis, initial antigen detection by immunoagglutination was negative. We thus evaluated a new immunochromatographic detection assay that exhibited a higher sensitivity. PMID:25755893

  10. Clinical comparison of two assays for rapid detection of cytomegalovirus early nuclear antigen.

    PubMed

    Woods, G L; Johnson, A M; Thiele, G M

    1990-03-01

    Three methods for detection of cytomegalovirus (CMV) in 218 clinical specimens were compared: (1) shell vial assay to detect the early nuclear antigen after incubation for 16 hours and 40 hours (Syva Company); (2) 24-well plate assay to detect the early nuclear antigen after incubation for 16 hours (DuPont); and (3) convention tissue cell culture. CMV was detected in 26 specimens (12%) by one or more of these methods. With the shell vial assay, 12 (46%) and 15 (58%) specimens were positive after incubation for 16 hours and 40 hours, respectively. CMV was detected in 17 specimens (65%) by the 24-well plate assay. There was no significant difference in the detection of CMV between these assays. CMV was identified by conventional tissue culture in 15 of 22 (68%) evaluable cultures after an average of 14.2 days. More specimens were positive by conventional culture than by the 16-hour shell vial assay (P = 0.035). For optimal detection of CMV in clinical specimens, both conventional tissue cell culture and an early antigen assay should be performed. The two early antigen assays evaluated in this study yielded comparable results. However, the 24-well plates are more easily manipulated, and the 24-well plate assay, as performed, was easier to interpret and more cost efficient. PMID:2155527

  11. Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24Gag

    PubMed Central

    Gesner, Marianne; Maiti, Mekhala; Grant, Robert; Cavrois, Marielle

    2016-01-01

    The fluorescence-linked antigen quantification (FLAQ) assay allows a fast quantification of HIV-1 p24Gag antigen. Viral supernatant are lysed and incubated with polystyrene microspheres coated with polyclonal antibodies against HIV-1 p24Gag and detector antibodies conjugated to fluorochromes (Figure 1). After washes, the fluorescence of microspheres is measured by flow cytometry and reflects the abundance of the antigen in the lysate. The speed, simplicity, and wide dynamic range of the FLAQ assay are optimum for many applications performed in HIV-1 research laboratories.

  12. Evaluation of solubilized herpes simplex virus membrane antigen by enzyme-linked immunosorbent assay.

    PubMed Central

    Jeansson, S; Forsgren, M; Svennerholm, B

    1983-01-01

    An antigen prepared by solubilization of membranes from herpes simplex virus (HSV)-infected cells with deoxycholate was evaluated by enzyme-linked immunosorbent assay. The deoxycholate-solubilized antigen, previously shown to contain all major HSV glycoproteins, was noninfectious and adsorbed easily and reproducibly to a polystyrene surface at pH 9.6. The deoxycholate-solubilized antigen provided an enzyme-linked immunosorbent assay of high sensitivity and reproducibility with complete correlation with complement fixation for the diagnosis of acute HSV infection. The correlation with neutralization and immunofluorescence for the presence or absence of anti-HSV activity was very good. Comparison with an HSV envelope preparation yielded results slightly in favor of the deoxycholate-solubilized antigen. The assay seems to be useful for demonstration of intrathecal production of antibody activity in HSV encephalitis. PMID:6315767

  13. Evaluation of Diagnos Malaria Stix test (antigen detection assay) for diagnosis of malaria.

    PubMed

    Khan, Haris M; Shujatullah, Fatima; Shahid, M; Raza, Adil; Malik, Ritu

    2010-06-01

    Malaria is one of the most common parasitic infection in India. The diagnosis largely depends on peripheral blood smear examination. Newer diagnostic methods like various antigen detection assays are now in use for prompt diagnosis and treatment. This study was done to determine the effectiveness of Diagnos Malaria Stix (antigen detection) assay in diagnosis of malaria. This involves detection of PfHRP-2 antigen and P.V. specific pLDH antigen. 162 patients with signs and symptoms of malaria included in the study. Leishman stained blood smear examination was done for all patients. Commercially available Diagnos Malaria Stix assay was used. Diagnos Malaria Stix showed sensitivity, specificity positive and negative predictive values of 100% each while Sensitivity, specificity, positive and negative predictive values of Leishman stained blood smear examination were 45.45%, 100%, 100% and 92% respectively. PMID:22471175

  14. Validation of a von Willebrand factor antigen enzyme-linked immunosorbent assay and newly developed collagen-binding assay

    PubMed Central

    Burgess, Hilary; Wood, Darren

    2008-01-01

    No single test is comprehensive enough to detect all of the variants of von Willebrand Disease (VWD), making determination of both concentration and function of von Willebrand Factor (VWF) important for an accurate diagnosis. The objective of the study was to validate a newly developed VWF collagen binding assay (VWF:CB) and VWF antigen enzyme-linked immunosorbent assay (ELISA) developed at the Ontario Veterinary College (OVC VWF:Ag). Linearity, sensitivity, and coefficients of variation were determined. The Asserachrom VWF:Ag ELISA was used as the reference assay for this study. Concordance correlation and Bland-Altman plots were used to evaluate agreement between both VWF:Ag assays. The VWF:CB accuracy was assessed by degree of association with the VWF:Ag assays, and the VWF:Ag to VWF:CB ratio. All assays were assessed for their ability to distinguish between VWD negative and VWD positive patients. Linearity, intra-assay coefficients of variation, and inter-assay coefficients of variation were acceptable for both the newly developed VWF:CB (R2 = 0.97, average CV = 4.4, and 15, respectively) and OVC VWF:Ag assays (R2 = 0.96, average CV = 7.9, and 5.9, respectively). Agreement between the OVC VWF:Ag assay and reference assay was excellent (ρc = 0.89), and although differences between assay results precluded interchangeable use of the assays, both successfully distinguished VWD positive and VWD negative dogs (P < 0.0001). The VWF:CB showed a strong association with both VWF:Ag assays (R2 = 0.86, 0.82) and VWF:Ag to VWF:CB ratios (≤ 1) were as expected. The excellent performance of both assays in this validation study confirm their reliability and potential for clinical application. PMID:19086374

  15. 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...

  16. 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

  17. 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. PMID:20023265

  18. Immunoradiometric assay for examination and quantitation of Brucella abortus-specific antibodies reactive with the antigen(s) used in the indirect hemolysis test.

    PubMed Central

    Tedder, T F; Hoffmann, E M

    1981-01-01

    An immunoradiometric assay was designed to quantitate antibodies which bind to Brucella abortus antigens adsorbed to bovine erythrocytes. This allowed examination of antibodies specific for B. abortus antigens detectable in the indirect hemolysis test for bovine brucellosis. Assay parameters were optimized for measuring antigen-specific immunoglobulin G1 (IgG1), IgG2, and IgM antibodies. The immunoradiometric assay allowed examination of binding interactions which occur during the indirect hemolysis test. Affinity-purified antibovine IgG1, IgG2, and IgM were used to detect specific bovine antibodies of these classes (and subclasses). The binding of the anti-immunoglobulins was linear as a function of immunoglobulin concentration. However, the binding of bovine antibodies of the different classes and subclasses to B. abortus antigen was nonlinear. Since B. abortus-specific antibodies of all classes and subclasses were present in the "standard serum" during the immunoradiometric assays, it is possible that the non-linearity was due to competition between antibodies for antigenic sites. IgG2 and IgM antibodies specific for B. abortus antigen(s) appeared to be capable of binding independently to antigen(s). However, the binding efficiencies of IgG1 antibodies changed as the ratio of antigenic sites to antibodies was increased. PMID:6793625

  19. Five-Antigen Fluorescent Bead-Based Assay for Diagnosis of Lyme Disease.

    PubMed

    Embers, Monica E; Hasenkampf, Nicole R; Barnes, Mary B; Didier, Elizabeth S; Philipp, Mario T; Tardo, Amanda C

    2016-04-01

    The systematically difficult task of diagnosing Lyme disease can be simplified by sensitive and specific laboratory tests. The currently recommended two-tier test for serology is highly specific but falls short in sensitivity, especially in the early acute phase. We previously examined serially collected serum samples fromBorrelia burgdorferi-infected rhesus macaques and defined a combination of antigens that could be utilized for detection of infection at all phases of disease in humans. The fiveB. burgdorferiantigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection ofB. burgdorferiantigen-specific IgG antibodies. Samples from Lyme disease patients and controls were used to determine the diagnostic value of this assay. Using this sample set, we found that our five-antigen multiplex IgG assay exhibited higher sensitivity (79.5%) than the enzyme immunoassay (EIA) (76.1%), the two-tier test (61.4%), and the C6 peptide enzyme-linked immunosorbent assay (ELISA) (77.2%) while maintaining specificity over 90%. When detection of IgM was added to the bead-based assay, the sensitivity improved to 91%, but at a cost of reduced specificity (78%). These results indicate that the rational combination of antigens in our multiplex assay may offer an improved serodiagnostic test for Lyme disease. PMID:26843487

  20. 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. PMID:26762619

  1. 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. PMID:1954783

  2. Time to seroconversion of HBsAg to anti-HBs in individuals who lost HBsAg during follow-up.

    PubMed

    Roushan, M R H; Mohammadpour, M; Baiany, M; Soleimani, S; Bijani, A

    2016-09-01

    To determine the time to appearance of antibody against hepatitis B surface antigen (anti-HBs) after clearance of hepatitis B surface antigen (HBsAg) in chronically infected individuals, we followed up 3963 cases with positive antibody against hepatitis B e antigen (anti-HBe) from 1991 to 2014. Of these, 101 (67 males, 34 females) lost HBsAg. These serocleared cases were checked every 6-month interval regarding HBsAg, anti-HBs, liver function tests, and liver sonography. Hepatitis B virus DNA was assessed at the time of seroclearance or the appearance of anti-HBs. The mean age of these patients at entry to this study was 34·4 ± 13 years. The mean follow-up duration until seroclearance of HBsAg was 6·6 ± 4·3 years. After the mean follow-up of 43·7 ± 45 months, anti-HBs appeared in 64 (63·4%) cases. The cumulative probabilities of anti-HBs appearance for 2, 5 and 10 years were 24·3%, 58% and 78·2%, respectively. The appearance of anti-HBs was associated with age ⩾35 years and seroclearance of HBsAg (hazard ratio 1·96, 95% confidence interval 1·32-3·38, P = 0·016) but not with sex. The results show that anti-HBs may develop in 78·2% of cases within 10 years of HBsAg clearance. Age ⩾35 years at HBsAg loss was associated with earlier development of anti-HBs. PMID:27268129

  3. 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

  4. 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

  5. Detection of influenza antigenic variants directly from clinical samples using polyclonal antibody based proximity ligation assays

    PubMed Central

    Martin, Brigitte E.; Jia, Kun; Sun, Hailiang; Ye, Jianqiang; Hall, Crystal; Ware, Daphne; Wan, Xiu-Feng

    2016-01-01

    Identification of antigenic variants is the key to a successful influenza vaccination program. The empirical serological methods to determine influenza antigenic properties require viral propagation. Here a novel quantitative PCR-based antigenic characterization method using polyclonal antibody and proximity ligation assays, or so-called polyPLA, was developed and validated. This method can detect a viral titer that is less than 1000 TCID50/mL. Not only can this method differentiate between different HA subtypes of influenza viruses but also effectively identify antigenic drift events within the same HA subtype of influenza viruses. Applications in H3N2 seasonal influenza data showed that the results from this novel method are consistent with those from the conventional serological assays. This method is not limited to the detection of antigenic variants in influenza but also other pathogens. It has the potential to be applied through a large-scale platform in disease surveillance requiring minimal biosafety and directly using clinical samples. PMID:25546251

  6. 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

  7. Recombinant antigen-based enzyme-linked immunosorbent assay for diagnosis of Baylisascaris procyonis larva migrans.

    PubMed

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Ndao, Momar; Kazacos, Kevin R

    2011-10-01

    Baylisascaris larva migrans is an important zoonotic disease caused by Baylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although a B. procyonis excretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinant B. procyonis antigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis of Baylisascaris larva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinical Baylisascaris larva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies to Toxocara infection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology of Baylisascaris larva migrans by ELISA. PMID:21832102

  8. Demonstration of immunoglobulin M class antibodies to Toxoplasma gondii antigenic component p35000 by enzyme-linked antigen immunosorbent assay.

    PubMed Central

    Lindenschmidt, E G

    1986-01-01

    On the basis that 89% of 48 acute-phase toxoplasmosis patients showed immunoglobulin M (IgM) class antibodies to the 35,000-molecular-weight antigenic component (p35000) of Toxoplasma gondii, as demonstrated by IgM immunoblotting, the antigen was purified by sucrose gradient centrifugation and enzyme labeled for use in an enzyme-linked antigen immunosorbent assay (ELA) for the demonstration of IgM class antibodies to the p35000 component. The ELA showed a specificity of 96% with 139 serum specimens at a serum dilution of only 1:5. The test serologically detected 73 symptomatic acute-phase toxoplasmosis patients; 64 were positive in the 19S IgM indirect immunofluorescent-antibody test, and 9 were negative, although they showed IgM antibodies to p35000, as demonstrated by IgM immunoblotting. Also, the ELA turned out to be independent of IgM rheumatoid factors in six acute-phase toxoplasmosis serum specimens. PMID:3536996

  9. An enzyme-linked immunosorbent assay using detergent-soluble Plasmodium vivax antigen for seroepidemiological surveys.

    PubMed

    González-Cerón, L; Rodríguez, M H

    1991-01-01

    An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Plasmodium vivax parasites in human sera was developed using P. vivax-infected erythrocytes from local malarious patients in southern Mexico. Infected cells were concentrated using a discontinuous Percoll gradient and detergent-soluble antigens obtained using Triton X100. The use of detergent and the addition of protease inhibitors to the antigen preparation ensured high sensitivity and reproducibility of the assay. No cross reactions were observed in sera immune to other protozoan, helmintic and bacterial infections, although some cross reactivity was seen in P. falciparum immune sera. A strong correlation between antibody titre values obtained by the ELISA and those obtained using an IFAT was observed. In a small field trial, carried out in a village where malaria transmission occurs, both ELISA and IFAT produced similar seroepidemiological profiles with regard to frequency of positive antibody titres and their distribution among the different age groups of the population. PMID:1949138

  10. Wicking assay for the rapid detection of Rift Valley fever viral antigens in mosquitoes (Diptera: Culicidae).

    PubMed

    Turell, M; Davé, K; Mayda, M; Parker, Z; Coleman, R; Davé, S; Strickman, D

    2011-05-01

    Rift Valley fever virus (RVFV) causes outbreaks of severe disease in domestic ungulates as well as humans in Africa. There is a logical concern that RVFV could be introduced into the Americas and cause significant health and economic damage based on the precedent of the introduction and spread of West Nile virus (WNV). Unfortunately, there are currently no licensed diagnostic assays available for RVFV in the Americas. In this work, we report on the ability of a novel dipstick assay, VectorTest RVFV antigen assay, modeled on the VecTest assay for WNV, to detect a RVFV-infected female within a pool of mosquitoes. The dipsticks provided results in <20 min, were easy to use, and did not require a laboratory with containment facilities. Although readily able to detect a mosquito with a disseminated RVFV infection, it only occasionally detected RVFV in a mosquito with a nondisseminated infection, and therefore may fail to detect some pools that actually contain one or more positive mosquitoes. The RVFV dipstick assay was highly specific and did not react with samples to which had been added yellow fever, West Nile, Venezuelan equine encephalitis, sandfly fever Naples, sandfly fever Sicilian, or sandfly fever Toscana viruses. The RVFV assay can provide a rapid, safe, easy-to-use assay to alert public health personnel to the presence of RVFV in mosquitoes. Results from this assay will allow a rapid threat assessment and the focusing of vector control measures in high-risk areas. PMID:21661324

  11. 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

  12. 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

  13. Is HBsAg quantification ready, for prime time?

    PubMed

    Chevaliez, Stéphane

    2013-12-01

    Despite the availability of an efficient hepatitis B vaccine, approximately 240 million individuals are chronically infected with hepatitis B virus worldwide. One-fourth of hepatitis B surface antigen (HBsAg)-positive patients will develop complications, such as cirrhosis or hepatocellular carcinoma, both major causes of liver-related deaths. Antiviral therapies, such as pegylated interferon alpha or nucleoside/nucleotide analogues, are effective in suppressing HBV DNA and reducing the subsequent risk of fibrosis progression, cirrhosis and hepatocellular carcinoma. HBsAg has proven to be a steady, reliable marker of chronic HBV carriage that can also be used to predict clinical outcomes. Three commercial enzyme immunoassays are now available for HBsAg quantification. A number of recent studies have shown clinical utility of HBsAg quantification in combination with HBV DNA levels to identify inactive carriers who need antiviral therapy and in interferon treated-patients in order to predict the virological response to pegylated interferon alpha. PMID:23932705

  14. The cryptococcal antigen lateral flow assay: A point-of-care diagnostic at an opportune time.

    PubMed

    Tang, Michele W; Clemons, Karl V; Katzenstein, David A; Stevens, David A

    2016-08-01

    Cryptococcal meningitis is a devastating HIV-related opportunistic infection, affecting nearly 1 million individuals and causing over 500 000 deaths each year. The burden of disease is greatest in sub-Saharan Africa and Southeast Asia, where cryptococcal disease is the most common cause of meningitis. Rapid, accurate and affordable diagnosis of cryptococcal disease has been lacking in many of the most heavily affected areas. Here, we review a point-of-care assay for cryptococcal disease, the dipstick-formatted cryptococcal antigen lateral flow assay (LFA) (IMMY, Norman, OK). In comparison to culture, the assay is 99.5% sensitive and 98% specific. In comparison to other commercially available tests for cryptococcal antigen, the LFA has equal or superior sensitivity and specificity in CSF, plasma and serum samples. We discuss potential applications for the use of the assay in resource-limited settings, including what is likely to be an important role of the LFA in screening for early cryptococcal infection before clinical disease and in evaluating pre-emptive treatment. PMID:25612826

  15. Detection of Giardia duodenalis antigen in coprolites using a commercially available enzyme-linked immunosorbent assay.

    PubMed

    Gonçalves, Marcelo Luiz Carvalho; Araújo, Adauto; Duarte, Rosemere; da Silva, Joaquim Pereira; Reinhard, Karl; Bouchet, Françoise; Ferreira, Luiz Fernando

    2002-01-01

    The objective of this experiment was to assess the utility of a commercially available enzyme-linked immunosorbent assay (ELISA) kit for diagnosis of giardiasis in archaeological human remains. The kit, a monoclonal antibody assay, is used to detect the presence of Giardia-specific antigen 65 (GSA65) in human faeces. We utilized the assay in ancient faecal material. The material included desiccated faeces found in mummies or in archaeological sites, and sediments from latrines. A total of 83 specimens, previously examined microscopically for parasites, were examined. The ELISA detected 3 positive samples, dated to about 1200 AD, 1600 AD and 1700 AD. The ELISA was superior to direct observation. It was possible to identify G. duodenalis cysts by direct microscopy in only one of these samples. The results did not show cross-reactivity between this protozoan and helminths. The use of ELISA to detect G. duodenalis coproantigen could help the diagnosis of giardiasis in ancient human remains. PMID:12625140

  16. 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)

  17. 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

  18. Coexistence of HBsAg and HBsAb in a difficult-to-treat chronic hepatitis B: loss of HBsAg with entecavir plus tenofovir combination

    PubMed Central

    2014-01-01

    Background Some reports have documented the coexistence of Hepatitis B surfage Antigen (HBsAg) and anti-HBsAg antibodies (HBsAb) in patients with chronic hepatitis B (CHB), often in the absence of amino acid substitutions in the HBsAg sequences of the Hepatitis B Virus (HBV) genome able to explain an immunological escape variant. HBV genome has a very compact coding organization, with four partially overlapping open reading frames (ORFs). Because the reverse transcriptase region (rt) of HBV polymerase overlaps the HBsAg ORF, it is possible that some mutations in the HBsAg region correspond to mutations in the rt ORF, conferring resistance to current antiviral therapies. This unique case explores the response to antiviral therapies of a CHB with concurrent HBsAg and HBsAb positivity, and analyse the clinical implications of possible mutations in rt and HBsAg ORFs. Case presentation Here we describe the case of a 59 year-old Italian man suffering from Hepatitis B envelope Antigen (HBeAg) positive CHB with concurrent HBsAb positivity. By ultra-deep pyro-sequencing (UDPS) technique, mutations conferring immunological escape or resistance to antiviral therapies were found neither in HBsAg nor in HBV rt ORFs, respectively. The patient was unsuccessfully treated with interferon, adefovir monotherapy and adefovir plus entecavir combination. Surprisingly, during entecavir plus tenofovir combination, anti-HBe seroconversion and HBsAg loss were observed, while the titer of HBsAb persisted. Conclusions Concurrent HBsAg/HBsAb positivity in active CHB is a clinical and virological dilemma. In this setting, there are not consistent data about the response to conventional therapies and the immunological balance between host and virus remains so far unexplained. This is, to our knowledge, the first case described of a CHB with HBsAg/HBsAb positivity, wild type for clinically relevant mutations in HBsAg and rt ORFs, successfully treated with a combination of nucleot(s)ide analogues

  19. Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay.

    PubMed Central

    Anderson, L J; Tsou, C; Parker, R A; Chorba, T L; Wulff, H; Tattersall, P; Mortimer, P P

    1986-01-01

    Acute-phase serum from a patient with aplastic crisis provided sufficient human parvovirus B19 to make a monoclonal antibody against B19 and to develop antigen and immunoglobulin M (IgM) and IgG antibody detection enzyme-linked immunosorbent assays (ELISAs). The indirect capture antibody method was used for all three assays. Antigen was detected in 8 of 29 sera drawn within 2 days of onset of illness from patients with aplastic crisis. These sera had high titers of virus by electron microscopy and DNA hybridization and had no detectable B19 antibody. Antigen was not detected in serum specimens that had low titers of B19 DNA and had B19 antibody. With the IgM ELISA, we detected B19 IgM in over 85% of clinical cases of aplastic crisis and fifth disease and less than 2% of controls. The prevalence of B19 IgG antibodies increased with age. Approximately 2% of children less than 5 years of age and 49% of adults greater than 20 years of age had B19 IgG antibodies. The B19 antibody ELISAs are sensitive and specific tests to detect B19 infections. PMID:3021807

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

    PubMed

    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 2,000 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 1,000 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. PMID:6497105

  1. 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.

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

    PubMed

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

    2013-09-13

    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(78) and Pro(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 (NH4)2SO4 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. PMID:23969156

  3. 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

  4. Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

    PubMed

    López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

    2009-09-01

    A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used. PMID:19737754

  5. Comparison of various assay systems for prostate-specific antigen standardization.

    PubMed

    Kuriyama, M; Akimoto, S; Akaza, H; Arai, Y; Usami, M; Imai, K; Tanaka, Y; Yamazaki, H; Kawada, Y; Koiso, K

    1992-12-01

    To avoid confusion between serum prostate-specific antigen (PSA) values among various assay systems, clinical studies on the possibility of conversion among detection values were performed. The assay kits used for the PSA comparisons were MARKIT-F PA, MARKIT-M PA, EIKEN PA, PA test WAKO, Ball ELSA PSA, E-Test Tosoh II PA, PROS-CHECK PSA, DELFIA PSA and TANDEM-R PSA. Using each kit, the standards attached to each assay system were detected, and 142 sera samples from benign hypertrophies or prostate cancers were assayed for serum PSA values. By detecting the standards for each kit, slopes were obtained which were almost identical to those obtained from original assay system. The coefficients of correlation among the PSA detection systems, using patients' sera, were very high, and linear regression lines were also obtained. The results suggest that almost identical serum PSA values may be detected either by multiplying by a coefficient to bring it to the standard or using the conversion formula. PMID:1283993

  6. Extensive multiple test centre evaluation of the VecTest malaria antigen panel assay.

    PubMed

    Ryan, J R; Davé, K; Collins, K M; Hochberg, L; Sattabongkot, Jetsumon; Coleman, R E; Dunton, R F; Bangs, M J; Mbogo, C M; Cooper, R D; Schoeler, G B; Rubio-Palis, Y; Magris, M; Romer, L I; Padilla, N; Quakyi, I A; Bigoga, J; Leke, R G; Akinpelu, O; Evans, B; Walsey, M; Patterson, P; Wirtz, R A; Chan, A S T

    2002-09-01

    To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for > 15 weeks in dry conditions up to 45 degrees C and in humid conditions up to 37 degrees C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors. PMID:12243234

  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. Detecting polysaccharide antigen of Neisseria meningitidis group C in cerebrospinal fluid by dot-ELISA assay.

    PubMed

    Correia Barbosa, S F; Alkmin, M G; Landgraf, I M

    2000-06-01

    Cerebrospinal fluid (CSF) samples from 210 patients (200 with clinical evidence of bacterial meningitis, 10 with other clinical neurologic disease) were tested by a Dot-ELISA assay for detection of polysaccharide antigen of N. meningitidis group C. CSF samples were treated with EDTA 0.1 M, at pH 7.5 and heated to 90>C for 10 min. Polyclonal antiserum was purified by use of ethanol fractionation. The results were compared to those using bacterial culture (BC), latex agglutination (LA), counterimmunoelectrophoresis (CIE), and direct microscopy (DM) methods. Test results showed a correlation of 93.3%, 94.3%, 91.0% and 69.5% respectively, and sensitivity of 0.947 and specificity of 0.930. This study suggests that the dot-ELISA assay of CSF is a useful alternative technique for the diagnosis of group C meningitis. PMID:10934498

  9. TLR8 agonists stimulate newly recruited monocyte-derived cells into potent APCs that enhance HBsAg immunogenicity

    PubMed Central

    Du, Jun; Wu, Zhiyuan; Ren, Shurong; Wei, Yong; Gao, Meihua; Randolph, Gwendalyn J.; Qu, Chunfeng

    2011-01-01

    We previously reported that synthetic or natural Toll-like receptor (TLR) 7/8 agonists present within dead cells enhanced cell-associated antigen presentation both in vitro and in vivo. Here, we investigated the immunopotency of different chemically synthesized TLR7/8 agonists, Resiquimod, Gardiquimod, CL075, and CL097, on HBsAg immunogenicity. These agonists stimulated inflammatory monocyte-derived cells to become potent antigen-presenting dendritic cells (DCs), which augmented HBsAg specific T cell proliferation after they were conditioned with HBsAg. The TLR8 agonist CL075 and the TLR7/8 dual agonist CL097 showed more potent effects than the TLR7 agonist. Compared with alum adjuvant, when HBsAg mixed with CL075 was injected intramuscularly into mice, more monocyte-derived DCs carried antigens into draining lymph nodes and spleens. Specific Abs, particularly IgG2a, were significantly increased, and more IL-5 and IFN-γ were produced by splenocytes and intrahepatic immunocytes in mice that received HBsAg mixed with CL075 and CL097. These results suggest that TLR8 agonists are good candidates to enhance recombinant HBsAg immunogenicity to induce specific humoral and cellular immune responses. PMID:20637759

  10. Discordant diagnostic results due to a hepatitis B virus T123A HBsAg mutant.

    PubMed

    Osiowy, Carla; Kowalec, Kaarina; Giles, Elizabeth

    2016-07-01

    HBsAg immunoassay results are occasionally discordant among primary and confirmatory assays or with respect to other markers of HBV infection. Such discordance has been observed repeatedly in Canada with samples having a mutation at HBsAg codon 123 (sT123A). Detection of recombinant expressed HBsAg protein having either sT123 or sA123 was evaluated with one manual and six automated HBsAg immunoassays. The recombinant mutant HBsAg was non-reactive by Abbott AxSYM, while the Abbott ARCHITECT Quantitative and Qualitative II, ADVIA Centaur, and VITROS ECi detection signal was reduced compared with the wild-type protein, approaching the assay cut-off for certain assays, dependent upon the level of protein. The Roche Elecsys and manual immunoassays detected both wild-type and mutant proteins comparatively. The sT123A mutation leads to loss of detection by immunoassays commonly used in Canadian diagnostic laboratories, which may produce misleading results and diagnoses. PMID:27133305

  11. Avidity of immunoglobulin G antibody response to the different antigenic fractions of soluble Schistosoma mansoni adult worm antigen preparation (SWAP) using avidity immunoblotting assay.

    PubMed

    Tawfeek, Gihan M; Hameed, Dina M Abdel; el-Hoseiny, Laila M

    2004-08-01

    The detection of IgG antibodies against the different SWAP antigenic fractions plus the determination of their avidity in avidity immunoblotting assay using 6 M urea wash, presents a novel alternative for the characterization of optimal antigenic markers for acute and chronic phases of Schistosoma mansoni infection and for vaccine development. Human serum samples from 25 acute schistosomiasis patients, 20 chronic cases and 15 normal healthy controls were analysed by IgG avidity enzyme linked immunosorbent assay (ELISA) and IgG avidity immunoblotting assay. Using avidity ELISA, a pronounced overlap of avidity index values was observed between acute and chronic infections with a range of uncertainty (0.86-1) which was encountered in both groups. Using avidity immunoblotting assay, antigenic bands at >116, 84, 48, 40 & 34 KDa were exclusive for the acute phase. From these bands, 34 KDa was recognized mostly by low-avidity antibodies and showed a high sensitivity (96%) and specificity (100%) making it an optimal marker for the acute phase. 40 KDa band was recognized mostly by high-avidity antibodies even during acute infection. Bands of 80, 70, 42, 36, 30 & 26 KDa were exclusive for the chronic phase. Only 70 KDa band was recognized by high-avidity antibodies yet, with low sensitivity (35%), that limits its use as an optimal marker for the chronic infection. Meanwhile, 70 and 40 KDa bands, recognized by high-avidity antibodies, are considered as potential vaccine antigen candidates. PMID:15287177

  12. Comparison of simian and human cytomegalovirus reactivities in an enzyme-linked immunospecific assay: effect of antigen preparation on cross-reactive antigens.

    PubMed Central

    Tinghitella, T J; Swack, N; Baumgarten, A; Hsiung, G D

    1982-01-01

    Simian cytomegalovirus was substituted for human cytomegalovirus in an enzyme-linked immunoassay. Unlike the indirect immunofluorescence assay which demonstrates a two-way cross-reactivity, only one-way cross-reactivity was observed. Altering the method of simian antigen preparation gave some insight other this different reactivity. PMID:6288573

  13. Antigen

    MedlinePlus

    An antigen is any substance that causes your immune system to produce antibodies against it. This means your immune ... and is trying to fight it off. An antigen may be a substance from the environment, such ...

  14. HIV-1 derived peptides fused to HBsAg affect its immunogenicity.

    PubMed

    Gonzalez, Minerva Cervantes; Kostrzak, Anna; Guetard, Denise; Pniewski, Tomasz; Sala, Monica

    2009-12-01

    The hepatitis B virus (HBV) surface small antigen (HBsAg) self-assembles into virus-like particles (VLPs). HBsAg-based VLPs constitute a powerful vector for heterologous immunogenic peptides to develop a safe vaccine delivery system. HBV and the human immunodeficiency virus type 1 (HIV-1) are frequently associated in infection. An HIV-1 class I polyepitope was designed for an HIV-1/HBV vaccine prototype based on HBsAg VLPs. Invariable peptides from the original HIV-1 polyepitope were here permutated to study the influence of epitope order on HIV-1/HBV VLP immunogenicity. Anti-HIV-1 cellular responses were statistically comparable among polyepitope variants. Nevertheless, delivered HIV-1 polyepitopes impacted anti-HBsAg carrier immunogenicity in a polyepitope-specific manner. For a given set of epitopes, the choice of epitope order in polyepitopes is strategic to control immune responses towards HBsAg VLPs used as carrier of foreign immunogenic peptides. PMID:19766153

  15. [Comparison of 3 assay kits of prostate specific antigen in serum of prostatic cancer].

    PubMed

    Akimoto, S; Akakura, K; Shimazaki, J

    1988-12-01

    The serum prostate specific antigen (PA) of the patients with prostatic cancer were determined with 3 assay kits, the Diagnostic Products Cooperation (DPC) kit, the Eiken kit and the Dainippon Pharmaceutical Co. (MARKIT F) kit. The first 2 assay kits involve radioimmunoassay and the latter EIA. For comparison, prostatic acid phosphatase (PAP) and gamma-seminoprotein (gamma-Sm) were determined using an Eiken kit and Chugai kit. Efficiency of the DPC kit, Eiken kit and the MARKIT F kit for untreated prostatic cancer was 26, 25 and 36%, respectively. The PA level measured using the Eiken kit and the MARKIT F kit both well correlated to the PAP level, but with the DPC kit correlation was slightly low. The PA level measured using the 3 different kits correlated poorly with the gamma-Sm level. The PA values obtained with 3 different assays from patients with prostatic cancer were highly correlated, but showed great differences in the values measured. When the standards used in the DPC kit were analyzed by the Eiken kit, the DPC standards as measured by the Eiken kit had only about half of their assigned values. The same standards were analyzed by the MARKIT F kit, the standards yielded measured values about one third of their assigned values. When the standards used in the MARKIT F kit were analyzed by the Eiken kit, the MARKIT F standards yielded measured values about 2.5 fold of their assigned values. The differences between the values obtained with the 3 assay kits presented a serious problem in clinical use of PA. Standardization of these assay kits will be awaited. PMID:2467543

  16. Production of monoclonal antibodies to hepatitis B surface and core antigens, and use in the detection of viral antigens in liver biopsies.

    PubMed Central

    Tedder, R. S.; Guarascio, P.; Yao, J. L.; Lord, R. B.; Eddleston, A. L.

    1983-01-01

    Hybridomas secreting monoclonal antibodies to HBsAg and HBcAg were prepared from immunized mice. An antibody capture radioimmunoassay was used to detect and select appropriate hybrids for propagation and cloning. The advantages of this assay were discussed. The resulting monoclonal antibodies were compared with conventional polyclonal antisera for the detection of virus antigens in liver tissue and found to give excellent results. Images Plate 2 Plate 1 PMID:6337208

  17. 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.

  18. 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.

  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. PMID:26675461

  20. 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

  1. Evaluation of Recombinant Antigen-Based Assays for Diagnosis of Bullous Autoimmune Diseases

    PubMed Central

    D'Agosto, G.; Latini, A.; Carducci, M.; Mastroianni, A.; Vento, A.; Fei, P. Cordiali

    2004-01-01

    The diagnosis of autoimmune bullous diseases is based on clinical observation and on the presence of autoantibodies directed to molecules involved in the adhesion systems of the skin. Immunofluorescence assays are the currently accepted method for detection of autoantibodies; such assays depend greatly on the skill of operators and are difficult to standardize. Recombinant desmoglein-1 (Dsg1), Dsg3, and BP180 peptides, the main autoantigens in pemphigus or bullous pemphigoid, have been used to develop new quantitative enzyme immunoassays (EIA) for the detection of specific antibodies. The present study was undertaken to evaluate the sensitivity and specificity of these immunoassays and to determine the correlation between the results and the clinical aspects of diseases. Serum samples from patients with pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, or mucous membrane pemphigoid, from healthy individuals, and from patients with unrelated autoimmune conditions were tested. Anti-desmoglein reactivity was detected in all the patients with pemphigus and in none of the controls. Patients with the more benign form of cutaneous disease had anti-Dsg1 antibodies, while patients with deeper cutaneous lesions or with mucosal involvement had anti-Dsg3 reactivity also, or exclusively. The BP180-based assay was positive for 66.6% of patients with bullous pemphigoid and for none of the patients with mucous membrane pemphigoid, and no reactivity was detected in the control sera. In conclusion, the anti-Dsg1 and anti-Dsg3 assays are useful in the diagnosis of pemphigus and provide information on the clinical phenotype of the disease. However, the sensitivity of EIA for detection of autoantibodies in bullous pemphigoid should be improved by the use of additional antigens or epitopes. PMID:15242953

  2. 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.

  3. 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

  4. Use of a new hypersensitive assay for the detection of prostate specific antigen in prostate cancer.

    PubMed

    Arai, Y; Onishi, H; Oishi, K; Takeuchi, H; Yoshida, O

    1993-04-01

    We have developed a new hypersensitive enzyme immunoassay for prostate specific antigen (PSA) based on the (MARKIT-M PA) assay but employing a two-hour incubation of the primary monoclonal antibody. The analytical sensitivity has been determined at 0.2 ng/ml, calculated as the mean+three standard deviations of the zero calibrator. Serum PSA was measured at least one month after radical prostatectomy (nine patients) or cystoprostatectomy (six patients). Based on the PSA levels of these patients, the recommended PSA cut-off level indicative of residual disease after radical prostatectomy was 0.4 ng/ml. Increasing (> 0.4 ng/ml) PSA levels preceded recurrence by eight months in a patient who developed bone metastasis after radical prostatectomy. In two patients treated with endocrine therapy, increasing PSA levels also preceded clinical evidence of progression by between eight and nine months. The study suggests that the newly developed sensitive PSA assay allows for the identification of patients with disease progression and the early commencement of adjuvant treatment. PMID:7685834

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

    PubMed

    Alcorn, S W; Pascho, R J

    2000-05-01

    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 Renibacterium 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. PMID:10826838

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

    PubMed Central

    Omar, Noorsharmimi; Loh, Qiuting; Tye, Gee Jun; Choong, Yee Siew; Noordin, Rahmah; Glökler, Jörn; Lim, Theam Soon

    2014-01-01

    G-Quadruplex (G-4) structures are formed when G-rich DNA sequences fold into intra- or intermolecular four-stranded structures in the presence of metal ions. G-4-hemin complexes are often effective peroxidase-mimicking DNAzymes that are applied in many detection systems. This work reports the application of a G-rich daunomycin-specific aptamer for the development of an antibody-antigen detection assay. We investigated the ability of the daunomycin aptamer to efficiently catalyze the hemin-dependent peroxidase activity independent of daunomycin. A reporter probe consisting of biotinylated antigen and daunomycin aptamer coupled to streptavidin gold nanoparticles was successfully used to generate a colorimetric readout. In conclusion, the daunomycin aptamer can function as a robust alternative DNAzyme for the development of colorimetric assays. PMID:24379042

  7. Interactions of antigen-sensitized liposomes with immobilized antibody: a homogeneous solid-phase immunoliposome assay.

    PubMed

    Ho, R J; Huang, L

    1985-06-01

    Dioleoyl phosphatidylethanolamine (DOPE) does not form stable bilayer liposomes at room temperature and neutral pH. However, stable unilamellar liposomes could be prepared by mixing DOPE with a minimum of 12% of a haptenated lipid, N-(dinitrophenylaminocaproyl)-phosphatidylethanolamine (DNP-cap-PE). When the liposomes bound to rabbit anti-DNP IgG that had been adsorbed on a glass surface, lysis of the liposome occurred with the release of the contents into the medium as judged by the fluorescence enhancement of an entrapped self-quenching dye, calcein. On the other hand, incubation of the same liposomes with glass surfaces coated with normal rabbit IgG had little effect. In addition, free anti-DNP IgG induced aggregation of the liposomes but did not cause any dye release. Liposomes composed of dioleoyl phosphatidylcholine (DOPC) and DNP-cap-PE did not lyse when added to the glass surfaces coated with either anti-DNP IgG or normal IgG. A likely mechanism for liposome lysis is that the DNP-cap-PE laterally diffuse to the contact area between the liposome and the glass. Binding of the haptenated lipid with the immobilized and multivalent antibody trap the haptenated lipids in the contact area. As a result of lateral phase separation, lipids may undergo the bilayer to hexagonal phase transition, leading to the leakage of the entrapped dye. Because both the free hapten and the free antibody inhibited the liposome leakage, this process could be used to assay for the free hapten or antibody. We have shown that inhibition assays performed by using this principle can easily detect 10 pmol of free DNP-glycine in 40 microliter. Furthermore, by substituting human glycophorin A, a transmembrane glycoprotein, for the lipid hapten, we have demonstrated that this assay system is also applicable to detect protein antigen with a sensitivity of sub-nanogram level. PMID:3886794

  8. 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

  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. PMID

  10. Development of Ss-NIE-1 Recombinant Antigen Based Assays for Immunodiagnosis of Strongyloidiasis

    PubMed Central

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

    2015-01-01

    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

  11. 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

  12. Immunodiagnosis of fascioliasis using sandwich enzyme-linked immunosorbent assay for detection of Fasciola gigantica paramyosin antigen

    PubMed Central

    Abou-Elhakam, Hany Mohamed Adel; Bauomy, Ibraheem Rabia; El Deeb, Somaya Osman; El Amir, Azza Mohamed

    2013-01-01

    Background: Many immunological techniques have been developed over years using the different Fasciola antigens for diagnosis of parasitic infection and to replace the parasitological techniques, which are time consuming and usually lack sensitivity and reproducibility. Materials and Methods: In this study, Fasciola gigantica paramyosin (Pmy) antigen was early detected in cattle sera using sandwich enzyme-linked immunosorbent assay (ELISA), to evaluate the Pmy antigen performance in diagnosis. This work was conducted on 135 cattle blood samples, which were classified according to parasitological investigation into, healthy control (30), fascioliasis (75), and other parasites (30) groups. Results: The sensitivity of Sandwich ELISA was 97.33%, and the specificity was 95%, in comparison with parasitological examination, which recorded 66.66% sensitivity and 100% specificity, respectively. Conclusions: It was clear that the native F. gigantica Pmy is considered as a powerful antigen in early immunodiagnosis of fascioliasis, using a highly sensitive and specific sandwich ELISA technique. PMID:23961441

  13. Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model

    PubMed Central

    2012-01-01

    Background Antigen-specific CD8+ cytotoxic T lymphocytes represent potent effector cells of the adaptive immune response against viruses as well as tumours. Therefore assays capable at exploring the generation and function of cytotoxic T lymphocytes represent an important objective for both clinical and experimental settings. Methods Here we show a simple and reproducible assay for the evaluation of antigen-specific CD8+ cytotoxic T lymphocytes based on a LysiSpot technique for the simultaneous determination of antigen-specific IFN-γ production and assessment of tumor cytolysis. The assay was developed within an experimental model of colorectal carcinoma, induced by the colorectal tumor cell line DHD-K12 that induces tumors in BDIX rats and, in turn, elicits a tumor- specific immune response. Results Using DHD-K12 cells transfected to express Escherichia coli β-galactosidase as target cells, and by the fine setting of spot colours detection, we have developed an in vitro assay that allows the recognition of cytotoxic T lymphocytes induced in BDIX rats as well as the assessment of anti-tumour cytotoxicity. The method highlighted that in the present experimental model the tumour antigen-specific immune response was bound to killing target cells in the proportion of 55%, while 45% of activated cells were not cytotoxic but released IFN-γ. Moreover in this model by an ELISPOT assay we demonstrated the specific recognition of a nonapeptide epitope called CSH-275 constitutionally express in DHD-K12 cells. Conclusions The assay proved to be highly sensitive and specific, detecting even low frequencies of cytotoxic/activated cells and providing the evaluation of cytokine-expressing T cells as well as the extent of cytotoxicity against the target cells as independent functions. This assay may represent an important tool to be adopted in experimental settings including the development of vaccines or immune therapeutic strategies PMID:22296726

  14. 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. PMID:15031525

  15. 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

    2015-01-01

    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. PMID:26709095

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

    PubMed Central

    Sun, Yuning; Sun, Caibo; Zhang, Endong

    2015-01-01

    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/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. PMID:26709095

  17. 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

  18. Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Baylisascaris procyonis Larva Migrans ▿

    PubMed Central

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Ndao, Momar; Kazacos, Kevin R.

    2011-01-01

    Baylisascaris larva migrans is an important zoonotic disease caused by Baylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although a B. procyonis excretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinant B. procyonis antigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis of Baylisascaris larva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinical Baylisascaris larva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies to Toxocara infection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology of Baylisascaris larva migrans by ELISA. PMID:21832102

  19. Immunoblot assays using recombinant antigens for the detection of Mycoplasma hyopneumoniae antibodies.

    PubMed

    Subramaniam, S; Frey, J; Huang, B; Djordjevic, S; Kwang, J

    2000-07-01

    The 36kDa L-lactate dehydrogenase (LDH) and a 29kDa partial fragment of an ABC transporter ATP-binding protein analogue/multidrug resistance protein homologue (PR2) of Mycoplasma hyopneumoniae were tested for their potential as diagnostic antigens. Recombinant LDH was genetically engineered to contain six histidine residues at its C-terminal end, expressed in Escherichia coli and purified to a high degree using Ni(2+)-chelate affinity chromatography. A partial 262 amino acid segment representing the C-terminal end of the PR2 protein was cloned as a glutathione S-transferase (GST) fusion protein, expressed in E. coli and purified by urea extraction. Purified recombinant LDH-6xHis and PR2-GST were then reacted with pig sera in immunoblot assays. Our immunoblots showed that both proteins detected anti-M. hyopneumoniae antibodies in field and experimentally infected pig sera but not in any of the SPF control sera. The two proteins were specific for M. hyopneumoniae as they did not react with sera of pigs infected with the closely related Mycoplasma flocculare and Mycoplasma hyorhinis which are frequently isolated in pigs but are not of particular concern. PMID:10865156

  20. 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

  1. 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.

  2. Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

    PubMed Central

    2012-01-01

    Background HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. Method A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. Results The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. Conclusions When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3–4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV. PMID

  3. Investigation of cross-reactions against Trichinella spiralis antigens by enzyme-linked immunosorbent assay and enzyme-linked immunoelectrotransfer blot assay in patients with various diseases.

    PubMed Central

    De-la-Rosa, J L; Alcantara, P; Correa, D

    1995-01-01

    Data regarding cross-reactions against Trichinella spiralis in humans are scarce and controversial. For this reason, we tested serum samples from patients with typhoid fever, brucellosis, toxoplasmosis, amoebiasis, cysticercosis, trichocephaliasis, ascariasis, and onchocerciasis against an antigenic extract of T. spiralis infective larvae in an enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunoelectrotransfer blot (EITB) assay. All except one serum sample from the group of patients with onchocerciasis were negative in the ELISA; in the EITB assay, only faint bands were observed with the samples from patients with onchocerciasis and ascariasis and negative results were obtained with the samples from patients with other diseases. In conclusion, cross-reactions were found only in the groups of patients with other nematode infections and were of very low magnitude, most of them virtually negative. PMID:7719905

  4. Serodiagnosis of syphilis by enzyme-linked immunosorbent assay with purified recombinant Treponema pallidum antigen 4D.

    PubMed

    Radolf, J D; Lernhardt, E B; Fehniger, T E; Lovett, M A

    1986-06-01

    An enzyme-linked immunosorbent assay (ELISA) for syphilis has been developed that detects IgG antibody to purified recombinant Treponema pallidum surface antigen 4D. The 4D ELISA was capable of detecting 25 ng of 4D antigen-specific antibody. Neither 172 nonsyphilitic sera nor 20 false-positive sera in the Venereal Disease Research Laboratory test reacted in the 4D ELISA. The sensitivity of the 4D ELISA was comparable to that of the adsorbed fluorescent treponemal antibody test in primary, secondary, and latent disease. Most sera from patients with yaws or pinta were also reactive, a result indicating that a 4D antigen-like molecule also exists in the closely related pathogenic treponemes Treponema pertenue and Treponema carateum. PMID:3517186

  5. Evaluation of various plastic microtiter plates with measles, toxoplasma, and gamma globulin antigens in enzyme-linked immunosorbent assays.

    PubMed Central

    Shekarchi, I C; Sever, J L; Lee, Y J; Castellano, G; Madden, D L

    1984-01-01

    Seventeen lots of microtiter plates which differed in lot, batch, plastic type, or manufacturer were evaluated as solid-phase carriers in enzyme-linked immunosorbent assays for antibodies to measles, toxoplasma, and human gamma globulin. Most plates of polystyrene or polyvinyl chloride were found to give acceptable binding. The final choice depended on the antigen to be attached. Variations in binding between lots, batches, and types of plastic were found. Well-to-well variation was found to be of greater statistical significance than edge effect and should be a consideration in selection of a plate lot for enzyme-linked immunosorbent assays. Lots of plates should be pretested by the investigator to determine whether there is good binding of the antigen to be used and whether there is low plate-to-plate and well-to-well variation. PMID:6199371

  6. 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

  7. 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

  8. 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...

  9. Sensitivity of the VecTest antigen assay for eastern equine encephalitis and western equine encephalitis viruses.

    PubMed

    Nasci, Roger S; Gottfried, Kristy L; Burkhalter, Kristen L; Ryan, Jeffrey R; Emmerich, Eva; Davé, Kirti

    2003-12-01

    VecTest assays for detecting eastern equine encephalitis virus (EEE) and western equine encephalitis virus (WEE) antigen in mosquito pools were evaluated to determine their sensitivity and specificity by using a range of EEE, WEE, St. Louis encephalitis virus (SLE), and West Nile virus (WN) dilutions as well as individual and pooled mosquitoes containing EEE or WEE. The EEE test produced reliable positive results with samples containing > or = 5.3 log10 plaque-forming units (PFU) of EEE/ml, and the WEE test produced reliable positive results with samples containing > or = 4.7 log10 PFU WEE/ml. Both assays detected the respective viral antigens in single virus-positive mosquitoes and in pools containing a single positive mosquito and 49 negative specimens. The SLE and WN assays also contained on the dipsticks accurately detected their respective viruses. No evidence was found of cross reaction or false positives in any of the tests. The VecTest assays were less sensitive than the EEE- and WEE-specific TaqMan reverse transcriptase polymerase chain reaction and Vero cell plaque assay, but appear to be useful for detecting arboviruses in mosquito-based arbovirus surveillance programs. PMID:14710752

  10. 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

  11. 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.

  12. 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

  13. Comparison of vero cell plaque assay, TaqMan reverse transcriptase polymerase chain reaction RNA assay, and VecTest antigen assay for detection of West Nile virus in field-collected mosquitoes.

    PubMed

    Nasci, Roger S; Gottfried, Kristy L; Burkhalter, Kristen L; Kulasekera, Varuni L; Lambert, Amy J; Lanciotti, Robert S; Hunt, Ann R; Ryan, Jeffrey R

    2002-12-01

    Mosquitoes collected during the epidemic of West Nile virus (WN) in Staten Island, NY, during 2000 were identified to species, grouped into pools of up to 50 individuals, and tested for the presence of WN by using TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) to detect West Nile viral RNA, Vero cell plaque assay to detect infectious virus, and VecTest WNV/SLE Antigen Panel Assay. A total of 10,866 specimens was tested in 801 pools. Analysis of results indicated that TaqMan RT-PCR detected 34 WN-positive pools, more than either of the other techniques. The plaque assay detected 74% of the pools positive by TaqMan, and VecTest detected 60% of the pools positive by TaqMan. The VecTest assay detected evidence of West Nile viral antigen in 67% of the pools that contained live virus detected by plaque assay. A WN enzyme immunoassay performed similarly to the VecTest WN assay. Differences in performance were related to relative sensitivity of the tests. Infection rates of WN in Culex pipiens and Cx. salinarius calculated by the 3 techniques varied, but each estimate indicated a high infection rate in the population. Positive and negative attributes of each procedure, which may influence how and where they are used in surveillance programs, are discussed. PMID:12542186

  14. Cross-Reactivity Pattern of Asian and American Human Gnathostomiasis in Western Blot Assays Using Crude Antigens Prepared from Gnathostoma spinigerum and Gnathostoma binucleatum Third-Stage Larvae.

    PubMed

    Neumayr, Andreas; Ollague, Jose; Bravo, Francisco; Gotuzzo, Eduardo; Jimenez, Pedro; Norton, Scott A; Doanh, Pham Ngoc; Nawa, Yukifumi; Horii, Yoichiro; Nickel, Beatrice; Marti, Hanspeter

    2016-08-01

    Gnathostomiasis is a zoonotic parasitosis endemic in many Asian and some Latin American countries. Most human infections are caused by Gnathostoma spinigerum in Asia and Gnathostoma binucleatum in the Americas, and recently, imported cases have been increasing among travelers returning from endemic regions. Confirmation of the clinical diagnosis relies largely on serologic tests, with a G. spinigerum-antigen-based immunoblot currently being the diagnostic method of choice. However, we repeatedly experienced that sera from patients with clinically suspected American gnathostomiasis gave negative results in this assay. Therefore, we used homologous methods to prepare G. spinigerum- and G. binucleatum-antigen-based immunoblot assays, and evaluated the cross-reactivity of the two assays. The results show incomplete cross-reactivity between the two assays: the G. spinigerum-antigen-based immunoblot apparently only detects Asian gnathostomiasis caused by G. spinigerum, whereas the G. binucleatum-antigen-based immunoblot is apparently capable of detecting American as well as Asian gnathostomiasis. PMID:27325806

  15. Enzyme-linked immunosorbent assay for rubella immunoglobulin G: new method for attachment of antigens to microtiter plates.

    PubMed Central

    Skurrie, I J; Gilbert, G L

    1983-01-01

    Many of the enzyme-linked immunosorbent assay (ELISA) techniques previously described for detection of rubella-specific antibodies employ complex technology not available in routine diagnostic laboratories. The method described allows the use of commercially available rubella hemagglutination inhibition (HI) antigen. Passive adsorption of these antigens to plastic is variable, but with the use of albumin as a bridge, it is possible to attach the antigen reliably to the plastic wells. Over 1,500 sera were tested by both HI and ELISA techniques to detect the presence of rubella antibodies. These sera were selected with a bias towards those with low levels of rubella-specific antibody, since it has been demonstrated that it is in this range that discrepancies are more likely to occur between HI and ELISA techniques. In 99% of the sera tested, the results of both techniques were in agreement. On the basis of these results, the technique offers a useful alternative to the routine rubella HI test and other ELISA techniques which need sophisticated antigen preparations. PMID:6863497

  16. Enzyme-linked immunosorbent assay for detection of Salmonella typhi Vi antigen in urine from typhoid patients.

    PubMed

    Barrett, T J; Snyder, J D; Blake, P A; Feeley, J C

    1982-02-01

    Because typhoid fever continues to be a major cause of illness in many developing countries, there is a clear need for a sensitive and specific test that will permit rapid laboratory diagnosis of the disease. An enzyme-linked immunosorbent assay (ELISA) has recently been developed and tested, both in the laboratory and in a clinical situation, for its ability to detect Vi antigen in urine. The ELISA was capable of detecting as little as 1 ng of purified Vi antigen per ml in urine, compared with 100 ng/ml detectable by a previously tested coagglutination method. It could also detect antigen in urine diluted as much as 1:1,024 in normal urine. In tests of urine specimens from six stool culture-positive persons in a small typhoid outbreak in the United States, the ELISA detected antigen in specimens from four of the six patients. The ELISA also proved to be specific, giving no false-positive results for specimens from 50 persons who did not have typhoid fever. The apparent high sensitivity and specificity of this ELISA make it a promising test for rapid diagnosis of typhoid fever. PMID:7040446

  17. 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

  18. 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

  19. Genetic detection of Babesia bigemina from Mongolian cattle using apical membrane antigen-1 gene-based PCR assay.

    PubMed

    Sivakumar, Thillaiampalam; Altangerel, Khukhuu; Battsetseg, Badgar; Battur, Banzragch; Aboulaila, Mahmoud; Munkhjargal, Tserendorj; Yoshinari, Takeshi; Yokoyama, Naoaki; Igarashi, Ikuo

    2012-06-01

    We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established SpeI-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90 (33.8%) and 25 (9.4%) of the blood samples showed positive reactions for B. bigemina by the SpeI-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the SpeI-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina. PMID:22284301

  20. 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.

  1. 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. PMID:15634970

  2. Detection of enterotoxigenic Escherichia coli colonization factor antigen I in stool specimens by an enzyme-linked immunosorbent assay.

    PubMed Central

    Evans, D G; Evans, D J; Clegg, S

    1980-01-01

    An enzyme-linked immunosorbent assay (ELISA) was employed to detect and quantitate the fimbrial colonization factor antigen (CFA/I) of enterotoxigenic Escherichia coli in stool specimens obtained from adult cases of diarrhea in which CFA/I-positive E. coli was the known causative agent. The inhibition method, or blocking technique, was used. In this method, a standardized dilution of human anti-CFA/I serum was preincubated with dilutions of stool extract before transfer to CFA/I-coated microtiter plate wells, and then ELISA was performed with alkaline phosphatase-conjugated anti-human immunoglobulin. CFA/I purified from E. coli strain H-10407 (O78:H11) was used. Acute-phase diarrheal stool specimens were found to contain approximately 3.0 mg of antigen (mean value) per g stool, whereas control (CFA/I-negative) specimens contained insignificant amounts (less than 0.03 mg/g) of antigen. Also, CFA/I was detected in culture fluids of CFA/I positive enterotoxigenic E. coli belonging to a variety of serotypes and was undetectable in similar preparations from P-strains (spontaneous CFA/I-negative derivatives) of the same test cultures. Equivalent results were obtained in ELISA tests by using bacterial cells taken from isolated colonies grown on CFA agar. These results indicate that the ELISA technique will be useful for the diagnosis of diarrhea caused by CFA/I-positive enterotoxigenic E. coli. PMID:7031075

  3. Detection of group C rotavirus antigens and antibodies in animals and humans by enzyme-linked immunosorbent assays.

    PubMed Central

    Tsunemitsu, H; Jiang, B; Saif, L J

    1992-01-01

    Enzyme-linked immunosorbent assays (ELISAs) were developed to detect group (gp) C rotavirus antigens and antibodies. Both assays were confirmed to be specific for gp C rotavirus by using serogroup A, B, and C rotaviruses; hyperimmune antisera to these serogroups of rotaviruses; and paired serum specimens from animals infected with gp C rotaviruses. The ELISA for antigen detection reacted not only with porcine gp C rotaviruses but also with human and bovine gp C rotaviruses. Following experimental challenge of gnotobiotic pigs with porcine gp C rotavirus, the virus was found by ELISA in all diarrheic feces. A high prevalence of antibodies to gp C rotaviruses was detected in sera from adult pigs (93 to 97%) and cattle (47 to 56%) in the United States and Japan. However, no antibody to gp C rotavirus was detected in the sera (n = 20) of adult horses in the United States. In human sera from Hokkaido, Japan, 3% of children and 13% of adults possessed antibody to gp C rotaviruses. These results suggest that the ELISA that we developed may be useful for surveying gp C rotavirus infections in animals and humans. On the basis of serology, gp C rotavirus infections are common in pigs and cattle in the United States and Japan, but they occur at lower levels in humans from the Hokkaido area of Japan. PMID:1323577

  4. Quantification and characterization of specific T-cells by antigen-specific cytokine production using ELISPOT assay or intracellular cytokine staining.

    PubMed

    Letsch, Anne; Scheibenbogen, Carmen

    2003-10-01

    The novel generation of sensitive T-cell assays facilitates the direct quantitation and characterization of specific T-cell responses. Functional T-cell assays such as the ELISPOT assay and the intracellular cytokine cytometry (ICC) employ the antigen-specific induction of cytokines to detect specific T-cells on a single cell level. ICC has the advantage that the simultaneous phenotypic characterization of the antigen-specific T-cells is possible. There is evidence now from clinical cancer vaccination trials, that there is a relationship between the detection of vaccine-induced T-cells by cytokine-based assays and clinical responses. As these assays become increasingly relevant in clinical practice to suggest issues of assay validation and quality control become of major importance. PMID:12957572

  5. [Comparative assessment of the characteristics in various assay kits for prostate specific antigen].

    PubMed

    Akiyama, A; Yamamoto, S; Hokoishi, F; Ogawa, M; Aizawa, T; Matsumoto, T; Miki, M

    1993-12-01

    Six kinds of assay kits based on different detecting principle for PSA were evaluated. Polyclonal antibody was used in 2 kits (Eiken, Markit-F) and monoclonal antibody in 4 kits (Ball Elsa, Delfia, Markit-M, Tandem-R). To evaluate their characteristics, sera of 12 female patients, 5 prostatectomized patients and 2 high stage prostate cancer patients were measured by these kits. On the female sera the polyclonal assay kits yielded values higher than the defined minimal sensitive value, but the kits of monoclonal antibody detected nothing. Dilution test of sera with high PSA level showed satisfactory results in every kit but the values of 6 assays were different to each other. For monitoring the recurrence or recrudescence of the prostatic cancer, the monoclonal antibody kits may be preferable to the polyclonal antibody kits. These results showed that we must understand the characteristics of every assay kit which is used clinically. PMID:7508530

  6. 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

  7. 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

  8. Evaluation of enzyme-linked immunosorbent assay for detection of Salmonella typhi antigen and antibodies.

    PubMed

    el Fattah, M M; Hassan, E M; Shaheen, H; Awad, R A; Girgis, N

    1991-01-01

    Enteric fever is considered a major health problem in developing countries. The need for a rapid, accurate and conclusive method for diagnosis is important for adequate and proper treatment. The usefulness and reliability of the ELISA test in detection of S. typhi O antigen and specific IgG and IgM antibodies were assessed using sera obtained from 63 subjects clinically suspected to have enteric fever, 22 febrilenon-enteric subjects and 20 normal subjects. ELISA detection of S. typhi somatic antigen was positive in 75% of subjects with positive clot cultures. IgG and IgM antibodies to S. typhi lipopolysaccharide (LPS) were detected in sera from 83% and 88% of enteric fever subjects, respectively. While anti-LPS IgM was negative in all sera from febrile non-enteric subjects, 9% were positive for anti-LPS-IgG. The use of an ELISA for detection of anti-S. typhi LPS antibody in combination with the Widal test and/or the O antigen detection ELISA would provide a useful (95.8% sensitivity) adjunct to standard culture methods and allow for an earlier and more rapid diagnosis of enteric fever. PMID:1724780

  9. A miniaturized assay for influenza neuraminidase‐inhibiting antibodies utilizing reverse genetics‐derived antigens

    PubMed Central

    Sandbulte, Matthew R.; Gao, Jin; Straight, Timothy M.; Eichelberger, Maryna C.

    2009-01-01

    Background  Antibodies to neuraminidase (NA) contribute to protection during influenza virus infection, but NA inhibition (NI) titers are not routinely analyzed in vaccine trials. One reason is the cumbersome nature of the conventional thiobarbituric acid (TBA) NI assay, which uses chemical methods to quantify free sialic acid following incubation of NA with substrate in the presence of serum. In addition, the assay is complicated by the need to use virus of a hemagglutinin (HA) subtype novel to the host to detect NA‐specific antibodies only. Objectives  Our primary objectives were to miniaturize the colorimetric NI assay to a format suitable for quantitative analysis of large numbers of samples, and validate the specificity and sensitivity of the miniaturized format with ferret and human sera. An additional aim was to use reverse genetics to construct HA‐mismatched viral reagents bearing NA of recent influenza A vaccine strains and H6 HA. Results  Analysis of ferret antisera by the miniaturized assay demonstrated sensitivity and specificity comparable with the conventional assay. Similar increases in the NI titers in sera from vaccinated human volunteers were measured in miniaturized and conventional assays. Inactivated and live‐attenuated vaccines increased NI titers against a given subtype at approximately the same rate. Conclusions  The reagents and miniaturized format of the TBA method described here provide a platform for practical serological monitoring of functional antibodies against NA. PMID:21462400

  10. 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.

  11. Oral immunization of animals with transgenic cherry tomatillo expressing HBsAg

    PubMed Central

    Gao, Yi; Ma, Ying; Li, Mei; Cheng, Tong; Li, Shao-Wei; Zhang, Jun; Xia, Ning-Shao

    2003-01-01

    AIM: To investigate the expression of recombinant HBsAg (rHBsAg) in transgenic cherry tomatillo in order to explore the feasibility of producing HBV oral vaccine with cherry tomatillo by animal immune tests. METHODS: The recombinant plant expression vector containing HBsAg gene was constructed. Mediated with Agrobacterium tumefaciens, HBsAg gene was transferred into cotyledons of cherry tomatillo. Transformed cherry tomatillos were obtained through hygromycin delay-selection. Integrated DNA in transgenic cherry tomatillo was confirmed by hygromycin resistance selection, Gus detection, polymerase chain reaction (PCR) and dot blotting analysis. Antigenicity of rHBsAg was examined by ELISA and the immunogenicity of rHBsAg derived from transgenic cherry tomatillo tissues was confirmed by oral feed of transformed tissues to BALB/c mice primed with commercial HBV vaccines. Specific antibody titers in mice’s serum were examined by ELISA every week. RESULTS: By far, 10 positive lines of transgenic cherry tomatillos containing HBsAg gene were obtained. Among different organs of the same transgenic cherry tomatillo, level of rHBsAg expressed in leaves was the highest with the yield up to 300 ng/g fresh weight. And the rHBsAg expression level in fruits was about 10 ng/g fresh weight. In animal immune tests, oral delivery with transgenic tissues to mice primed with commercial vaccine instead of naive mice resulted in significant immune response. CONCLUSION: The result of this animal immune test indicated the rHBsAg derived from transgenic cherry tomatillo possessed normal immunogenicity. This work demonstrated the feasibility to generate oral immunogenic rHBsAg in transgenic cherry tomatillo, and would provide some experimental approach for the production of low-cost oral vaccines using transgenic cherry tomatillo in large scale. PMID:12717845

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

    PubMed Central

    Alfa, Michelle J; Sepehri, Shadi

    2013-01-01

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

  13. Evaluation of Rapid Blood Sample Collection in the Detection of Circulating Filarial Antigens for Epidemiological Survey by rWbSXP-1 Capture Assay

    PubMed Central

    Ansel Vishal, Lawrence; Nazeer, Y.; Ravishankaran, Rajendran; Mahalakshmi, Natarajan; Kaliraj, Perumal

    2014-01-01

    Background Lymphatic filariasis is a neglected tropical disease leading to profound disfiguring causing socio economic burden in the tropics. Current diagnosis strategies available during field surveys and epidemics are based on traditional microscopic detections and a few antigen/antibody assays. We have compared different sampling methodologies and standardized the highly sensitive and reliable rWbSXP-1 antigen detection assay to our new sampling methodology. Methodology Samples collected as serum, whole blood, whole blood on filter paper and whole blood on microscopic slides from patients belonging to various clinical groups of filariasis [endemic normal(EN), chronic pathology(CP), microfilaraemic(MF) and non-endemic normal(NEN)] were collected and standardized the rWbSXP-1 antigen detection assay using monoclonal antibody raised against rWbSXP-1 protein. The whole blood collected on microscopic slide based sampling method was employed in the field and the presence of circulating filarial antigen (CFA) was assessed using the rWbSXP-1 assay. Principal Findings The sampling methods were compared and no significant difference was observed for the detection of CFA (MF, P = 0.304, EN, P = 0.675, CP, P = 0.5698, NEN, P = 0.4494). Further the optimized sampling method was utilized to collect the 1106 samples from Polur, Tiruvannamalai. The rWbSXP-1 assay gave 98 antigen positive results whereas the microscopic method gave only 17. Conclusions Four sampling methodologies were analyzed and the new sampling methodology of whole blood collected on microscopic slide was found to be convenient for the detection of CFA using rWbSXP-1 antigen detection assay. The 1106 samples from Polur were collected using the new method. The rWbSXP-1 antigen assay perceived a 7.32% increased result which was read as false negatives on the conventional microscopic staining method. This new sampling methodology coupled with the rWbSXP-1 antigen assay can be used in

  14. 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

  15. Optical fiber SPR biosensor with sandwich assay for the detection of prostate specific antigen

    NASA Astrophysics Data System (ADS)

    Jang, Hyun Soo; Park, Kwang No; Kang, Chang Duk; Kim, Jun Pyo; Sim, Sang Jun; Lee, Kyung Shik

    2009-07-01

    We present a new, sensitive, few mode fiber (FMF) surface plasmon resonance (SPR) biosensor with a sandwich assay for the detection of PSA. The side-polished FMF biosensor does not need a polarizer and a thin high-index overlayer. The optical sensitivity of the SPR sensor was determined as 2.5 × 10 -6 RIU. In the SPR PSA sensor, the SPR signals were amplified by a factor of 6 in average over no secondary antibody, using the sandwich assay. The proposed FMF SPR biosensor has great potential for real-time analysis of immune reaction between biomolecules and the advantages of high-sensitivity and label-free detection.

  16. 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....

  17. IP-10 Is a Sensitive Biomarker of Antigen Recognition in Whole-Blood Stimulation Assays Used for the Diagnosis of Mycobacterium bovis Infection in African Buffaloes (Syncerus caffer).

    PubMed

    Goosen, Wynand J; Cooper, David; Miller, Michele A; van Helden, Paul D; Parsons, Sven D C

    2015-08-01

    African buffaloes (Syncerus caffer) are maintenance hosts of Mycobacterium bovis, the causative agent of bovine tuberculosis. They act as reservoirs of this infection for a wide range of wildlife and domestic species, and the detection of infected animals is important to control the geographic spread and transmission of the disease. Interferon gamma (IFN-γ) release assays (IGRAs) utilizing pathogen-derived peptide antigens are highly specific tests of M. bovis infection; however, the diagnostic sensitivities of these assays are suboptimal. We evaluated the diagnostic utility of measuring antigen-dependent interferon gamma-induced protein 10 (IP-10) release as an alternative to measuring IFN-γ levels. M. bovis-exposed buffaloes were tested using the Bovigam PC-EC and Bovigam PC-HP assays and a modified QuantiFERON TB-Gold (mQFT) assay. IP-10 was measured in the harvested plasma and was produced in significantly greater abundance in response to M. bovis antigens in Bovigam-positive than in Bovigam-negative animals. For each assay, using the Bovigam results as a reference, receiver operating characteristic curve analysis was done to determine diagnostically relevant cutoff values for IP-10. Thereafter, mQFT test results derived from measurement of IP-10 and IFN-γ were compared and a larger number of Bovigam-positive animals were detected using IP-10 as a diagnostic marker. Moreover, using IP-10, agreement between the mQFT assay and the Bovigam assays was increased, while the excellent agreement between the Bovigam assays was retained. We conclude that IP-10 is a sensitive marker of antigen recognition and that measurement of this cytokine in antigen-stimulated whole blood might increase the sensitivity of conventional IGRAs in African buffaloes. PMID:26108287

  18. IP-10 Is a Sensitive Biomarker of Antigen Recognition in Whole-Blood Stimulation Assays Used for the Diagnosis of Mycobacterium bovis Infection in African Buffaloes (Syncerus caffer)

    PubMed Central

    Goosen, Wynand J.; Cooper, David; Miller, Michele A.; van Helden, Paul D.

    2015-01-01

    African buffaloes (Syncerus caffer) are maintenance hosts of Mycobacterium bovis, the causative agent of bovine tuberculosis. They act as reservoirs of this infection for a wide range of wildlife and domestic species, and the detection of infected animals is important to control the geographic spread and transmission of the disease. Interferon gamma (IFN-γ) release assays (IGRAs) utilizing pathogen-derived peptide antigens are highly specific tests of M. bovis infection; however, the diagnostic sensitivities of these assays are suboptimal. We evaluated the diagnostic utility of measuring antigen-dependent interferon gamma-induced protein 10 (IP-10) release as an alternative to measuring IFN-γ levels. M. bovis-exposed buffaloes were tested using the Bovigam PC-EC and Bovigam PC-HP assays and a modified QuantiFERON TB-Gold (mQFT) assay. IP-10 was measured in the harvested plasma and was produced in significantly greater abundance in response to M. bovis antigens in Bovigam-positive than in Bovigam-negative animals. For each assay, using the Bovigam results as a reference, receiver operating characteristic curve analysis was done to determine diagnostically relevant cutoff values for IP-10. Thereafter, mQFT test results derived from measurement of IP-10 and IFN-γ were compared and a larger number of Bovigam-positive animals were detected using IP-10 as a diagnostic marker. Moreover, using IP-10, agreement between the mQFT assay and the Bovigam assays was increased, while the excellent agreement between the Bovigam assays was retained. We conclude that IP-10 is a sensitive marker of antigen recognition and that measurement of this cytokine in antigen-stimulated whole blood might increase the sensitivity of conventional IGRAs in African buffaloes. PMID:26108287

  19. Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep.

    PubMed Central

    Sugden, E A; Brooks, B W; Young, N M; Watson, D C; Nielsen, K H; Corner, A H; Turcotte, C; Michaelides, A; Stewart, R B

    1991-01-01

    The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity. Images PMID:1761688

  20. Biochemical and biophysical characterization of maize-derived HBsAg for the development of an oral vaccine.

    PubMed

    Shah, Shweta; Hayden, Celine A; Fischer, Maria E; Rao, A Gururaj; Howard, John A

    2015-12-15

    Although a vaccine against hepatitis B virus (HBV) has been available since 1982, it is estimated that 600,000 people die every year due to HBV. An affordable oral vaccine could help alleviate the disease burden and to this end the hepatitis B surface antigen (HBsAg) was expressed in maize. Orally delivered maize material induced the strongest immune response in mice when lipid was extracted by CO2 supercritical fluid extraction (SFE), compared to full fat and hexane-extracted material. The present study provides a biochemical and biophysical basis for these immunological differences by comparing the active ingredient in the differently treated maize material. Purified maize-derived HBsAg underwent biophysical characterization by gel filtration, transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-CD, and fluorescence. Gel filtration showed that HBsAg forms higher-order oligomers and TEM demonstrated virus-like particle (VLP) formation. The VLPs obtained from SFE were more regular in shape and size compared to hexane or full fat material. In addition, SFE-derived HBsAg showed the greatest extent of α-helical structure by far UV-CD spectrum. Fluorescence experiments also revealed differences in protein conformation. This work establishes SFE-treated maize material as a viable oral vaccine candidate and advances the development of the first oral subunit vaccine. PMID:26519888

  1. LSPR biomolecular assay with high sensitivity induced by aptamer-antigen-antibody sandwich complex.

    PubMed

    Guo, Longhua; Kim, Dong-Hwan

    2012-01-15

    Herein we demonstrate a sensitive approach for protein detection based on peak shifts of localized surface plasmon resonance (LSPR) induced by aptamer-antigen-antibody sandwich structures. The applicability of the proposed method is demonstrated using human α-thrombin as a model analyte. While the binding of thrombin to its specific receptor, thrombin binding aptamer (TBA) modified on Au nanorods (AuNRs), causes a measurable LSPR shift, a subsequent binding of an anti-thrombin antibody to the captured thrombin can exhibit a nearly 150% amplification in the LSPR response. This enhanced signal essentially leads to an improvement of limit of detection (LOD) by more than one order of magnitude. In addition, the use of TBA as thrombin recognition units makes the biosensor reusable. The feasibility of the proposed method was further exploited by the detection of thrombin in human serum, opening the possibility of a real application for diagnostics and medical investigations. PMID:22099957

  2. 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

  3. Development of Antigen Detection Assay for Diagnosis of Tuberculosis Using Sputum Samples

    PubMed Central

    Pereira Arias-Bouda, Lenka M.; Nguyen, Lan N.; Ho, Ly M.; Kuijper, Sjoukje; Jansen, Henk M.; Kolk, Arend H. J.

    2000-01-01

    The rising incidence of tuberculosis worldwide means an increasing burden on diagnostic facilities, so tests simpler than Ziehl-Neelsen staining are needed. Such tests should be objective, reproducible, and have at least as good a detection limit as 104 bacteria/ml. A capture enzyme-linked immunosorbent assay (ELISA) was developed for detection of lipoarabinomannan (LAM) in human sputum samples. As a capture antibody, we used a murine monoclonal antibody against LAM, with rabbit antiserum against Mycobacterium tuberculosis as a source of detector antibodies. The sensitivity of the capture ELISA was evaluated by using purified LAM and M. tuberculosis whole cells. We were able to detect 1 ng of purified LAM/ml and 104 M. tuberculosis whole cells/ml. LAM could also be detected in culture filtrate of a 3-week-old culture of M. tuberculosis. The culture filtrate contained approximately 100 μg of LAM/ml. The detection limit in sputum pretreated with N-acetyl-l-cysteine and proteinase K was 104 M. tuberculosis whole cells per ml. Thirty-one (91%) of 34 sputum samples from 18 Vietnamese patients with tuberculosis (32 smear positive and 2 smear negative) were positive in the LAM detection assay. In contrast, none of the 25 sputum samples from 21 nontuberculous patients was positive. This specific and sensitive assay for the detection of LAM in sputum is potentially useful for the diagnosis of tuberculosis. PMID:10834989

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

    SciTech Connect

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

    1988-11-01

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

  5. Chemoenzymatic Synthesis of a Type 2 Blood Group A Tetrasaccharide and Development of High-throughput Assays Enables a Platform for Screening Blood Group Antigen-cleaving Enzymes.

    PubMed

    Kwan, David H; Ernst, Sabrina; Kötzler, Miriam P; Withers, Stephen G

    2015-08-01

    A facile enzymatic synthesis of the methylumbelliferyl β-glycoside of the type 2 A blood group tetrasaccharide in good yields is reported. Using this compound, we developed highly sensitive fluorescence-based high-throughput assays for both endo-β-galactosidase and α-N-acetylgalactosaminidase activity specific for the oligosaccharide structure of the blood group A antigen. We further demonstrate the potential to use this assay to screen the expressed gene products of metagenomic libraries in the search for efficient blood group antigen-cleaving enzymes. PMID:25964111

  6. Hepatitis B Surface Antigen Quantity Positively Correlates with Plasma Levels of microRNAs Differentially Expressed in Immunological Phases of Chronic Hepatitis B in Children

    PubMed Central

    Winther, Thilde Nordmann; Heiberg, Ida Louise; Bang-Berthelsen, Claus Heiner; Pociot, Flemming; Hogh, Birthe

    2013-01-01

    Background and Aim Children with chronic hepatitis B (CHB) are at high risk of progressive liver disease. It is suggested that a newly-identified panel of 16 microRNAs is important in the pathogenesis of CHB in children. Subviral hepatitis B surface antigen (HBsAg) particles are produced in large excess over infectious virions. Interestingly, circulating HBsAg particles have been shown to carry microRNAs. A thorough characterisation of the identified microRNAs and HBsAg over time in plasma from children with CHB may provide useful information about the natural course of childhood CHB. Patients and Methods A cohort of 42 children with CHB was followed over time. Three to five blood samples were obtained from each child at minimum intervals of half a year; in total 180 blood samples. Plasma levels of the 16 microRNAs previously identified were analysed by quantitative real-time polymerase-chain-reaction. Plasma HBsAg was quantified using ARCHITECT® HBsAg assay. Results The presence of 14/16 plasma microRNAs in children with CHB was confirmed. All 14 microRNAs were significantly differentially expressed in different immunological phases of the disease. MicroRNA plasma levels were highest in immune-tolerant children, lower in immune-active children, and reached the lowest values in immune-inactive children, p<0.001. Plasma levels of four microRNAs decreased significantly over time in immune-tolerant and immune-active children whereas the microRNA plasma levels were stable in immune-inactive children, p<0.004. HBsAg quantity was positively correlated with plasma levels of 11/14 microRNAs, p<0.004. Conclusion This is the first study to characterise plasma microRNAs and HBsAg over time in children with CHB. Our data suggest that plasma levels of selected microRNAs and HBsAg are inversely correlated with immunological control of CHB in children. Further studies are, however, needed to advance the understanding of microRNAs and HBsAg in the pathogenesis of CHB in children

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

    PubMed Central

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

    2013-01-01

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

  8. 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

  9. Enzyme-Linked Immunosorbent Assay for Quantitating the Humoral Immune Response to the Colonization Factor Antigen of Enterotoxigenic Escherichia coli

    PubMed Central

    Clegg, Steven; Evans, Dolores G.; Evans, Doyle J.

    1980-01-01

    The enzyme-linked immunosorbent assay technique was used to quantitate, in milligrams per milliliter, anti-colonization factor antigen/I (CFA/I) immunoglobulin G (IgG) in acute- and convalescent-phase sera of individuals who experienced diarrhea associated with CFA/I-positive enterotoxigenic Escherichia coli. Purified CFA/I was used as antigen to coat polystyrene Microtiter plate wells for the determination of anti-CFA/I antibody. A reference anti-CFA/I IgG preparation was obtained by affinity chromatography of a high-titered serum with a CFA/I-Sepharose 4B column; IgG was the only class of immunoglobulin detectable in this serum as anti-CFA/I. Goat anti-human IgG conjugated to alkaline phosphatase was used in the enzyme-linked immunosorbent assay. Quantitation of IgG in the reference anti-CFA/I serum was achieved by comparison with a known sample of pure human IgG. Anti-CFA/I in test sera was quantitated by titration with CFA/I-coated Microtiter plate wells in the enzyme-linked immunosorbent assay, using a standard curve obtained with the reference anti-CFA/I serum. Anti-CFA/I IgG in paired sera was determined as percentage of total IgG by using the radial immunodiffusion technique to quantitate total IgG for each test serum. Diarrhea with isolation of CFA/I-positive enterotoxigenic E. coli was associated with a significant rise in serum anti-CFA/I IgG when these values were expressed as either milligrams of IgG per milliliter or as percentage of total IgG, although the response varied quantitatively and nonresponders were detected. None of the matched controls showed an anti-CFA/I IgG response. Further elucidation of the immune response to enterotoxigenic E. coli can now be accomplished by applying these methods to determine the class and specificity of immunoglobulins in external secretions such as saliva and intestinal contents. PMID:6103870

  10. 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

  11. A two-centre comparative evaluation of new automated assays for von Willebrand factor ristocetin cofactor activity and antigen.

    PubMed

    Stufano, F; Lawrie, A S; La Marca, S; Berbenni, C; Baronciani, L; Peyvandi, F

    2014-01-01

    von Willebrand disease (VWD) is caused by a quantitative and/or qualitative deficiency of the von Willebrand factor (VWF). The laboratory diagnosis of VWD is dependent on the measurement of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo). The aim of this study was to undertake a two-centre evaluation of two new automated VWF:Ag and VWF:RCo assays systems from Instrumentation Laboratory (Bedford, USA). Using the two new analytical systems that operated with different detection principles: immunoturbidimetric (TOP500 analyser) and chemiluminescent (AcuStar analyser), VWF:Ag and VWF:RCo levels were determined in samples from 171 healthy normal subjects, 80 VWD patients (16 type 1, 58 type 2 and 6 type 3) and 7 acquired von Willebrand syndrome patients. With commercial lyophilized normal and pathological plasmas VWF: Ag and VWF:RCo assays performed on both analysers exhibited low levels of inter-assay imprecision (AcuStar: CV% range 3.3-6.9; TOP500: CV% range 2.6-6.3). Samples from normal healthy subjects (range: VWF:Ag 44.6-173.9 IU dL(-1) ; VWF:RCo 43.1-191.5 IU dL(-1)) and patients (range: VWF:Ag <0.3-115.1 IU dL(-1) ; VWF:RCo <0.5-57.2 IU dL(-1)) showed a good correlation between the two VWF:Ag and VWF:RCo methods (rs = 0.92 and 0.82 respectively), with only a few inconsistent cases among the patients' samples evaluated. The chemiluminescent assays had a lower limit of detection for both VWF:Ag and VWF:RCo compared to immunoturbidimetric tests (0.3 IU dL(-1) vs. 2.2 IU dL(-1) and 0.5 IU dL(-1) vs. 4.4 IU dL(-1) respectively). The TOP500 and AcuStar VWF:Ag and VWF:RCo assays were precise and compare well between centres, making these systems suitable for the diagnosis of VWD in non-specialized and reference laboratories. PMID:24028703

  12. 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. PMID:17381718

  13. Evaluation of a Second Generation BOVIGAM Interferon Gamma (IFN-gamma) Assay with Alternative Antigens for Stimulation of Whole Blood Cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BOVIGAM®, a rapid laboratory assay, measures gamma interferon (IFN-gamma) production in whole blood samples after induction of a cell-mediated immune response (CMI) with M. bovis antigens. The test is widely used in the field and its excellent performance in TB eradication programs in many countries...

  14. 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

  15. BOVINE VIRAL DIARRHEA VIRUS PERSISTENTLY INFECTED AND ACUTELY INFECTED CALVES: ASSAYS FOR VIRAL INFECTIVITY, POLYMERASE CHAIN REACTION ANALYSIS, AND ANTIGEN DETECTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are numerous assays for bovine viral diarrhea virus (BVDV) detecting infectious virus, nucleic material, and antigen. Persistently infected (PI) and acutely/transiently infected calves with BVDV represent two different manifestations. Diagnostic test results impact on differentiation of PI o...

  16. A novel assay for typing Rh antigens in blood-stains using a lectin specific to the bisecting N-acetyl-D-glucosamine side chain of glycoprotein.

    PubMed

    Matsubara, K; Tanabe, K; Akane, A; Nakamura, H; Takahashi, S; Kimura, K

    1994-08-01

    A unique sandwich enzyme-linked immunosorbent assay for the determination of Rh antigens in blood stains has been developed using Rh antisera and phaseolus vulgaris E4/peroxidase conjugate (PHAE4/PO). The appropriate antiserum for detecting Rh C, c, D, E or e was coated on the inner surface of microplate wells, and the sample antigens from blood stains, solubilized with n-octyl-beta-D-glucopyranoside, were then placed in the wells. After washing the wells repeatedly, PHAE4/PO was added. Bound PHAE4/PO was detected by the development of colors using o-phenylenediamine/H2O2. All Rh antigens corresponding to the antisera were clearly detected using this technique. The detection limit expressed by sample dilution was more than 2 x 10(5) times (volume/dried blood weight) for the various antigens from the fresh 5 x 5 mm2 blood stain. Even when the blood stain samples were left beside a sunny window at room temperature for 2 months, Rh antigens were still detected. When the ABH, MN, P1, Kidd, Duffy and Lewis blood grouping systems were tested with similar ELISA procedures PHAE4 did not recognize any antigen. Since PHAE4 specifically recognizes and combines with the bisecting N-acetyl-D-glucosamine side chain, it was concluded that the glycoprotein was a component of all Rh antigens immune complexes. PMID:8046252

  17. [Usefulness of hyper sensitive PSA assay kits for determination on low range of prostate specific antigen in prostate cancer].

    PubMed

    Akimoto, S; Akakura, K; Ohki, T; Shimazaki, J; Kuriyama, M; Kawada, Y

    1995-02-01

    Prostate specific antigen (PSA) levels after total prostatectomy or radiation therapy to localized prostate cancer and also during endocrine therapy are within normal range. Therefore, it is necessary to use hyper sensitive PSA assay kits for early detection of relapse. The present study was undertaken to evaluate two hyper sensitive assay kits (Delfia kit, lower limit 0.1 ng/ml, Kabi Pharmacia Diagnostics Co. and Markit M kit, 0.5 ng/ml, Dainippon Pharmaceutical Co.) and to compare them with conventional PSA kit (Eiken Chemical Co., 1.0 ng/ml). Total of 291 sera were examined: patients consisted of 10 total prostatectomy+endocrine therapy, 9 radiation therapy+endocrine therapy, 5 radiation therapy alone and 44 endocrine therapy alone. Values of endocrine therapy alone were divided into two groups according to duration after start of treatment; more or less than 5 years. The following results were obtained. 1. In non-relapse patients after total prostatectomy+endocrine therapy and radiation+endocrine therapy, PSA showed under lower limit with hyper sensitive kit. On the contrary, conventional kit indicated more than 1.0 ng/ml. 2. Radiation therapy alone kept PSA in detectable range with hyper sensitive kit in spite of no sign of relapse. 3. In non-relapsed patients under endocrine therapy alone, long duration (more than 5 years after start of treatment) decreased PSA in non detectable values with hyper sensitive kits. In this case, conventional kit still showed PSA as more than 1 ng/ml. 4. Doubling time at relapse was estimated similar with Delfia kit and Markit M kit, and much longer with conventional kit. It is concluded that hyper sensitive kit is more useful to manage patients after therapy than conventional kit. PMID:7534843

  18. Evaluation of two Neospora caninum recombinant antigens for use in an enzyme-linked immunosorbent assay for the diagnosis of bovine neosporosis.

    PubMed

    Lally, N C; Jenkins, M C; Dubey, J P

    1996-05-01

    Neospora caninum is a recently described apicomplexan parasite which causes paralysis and death in dogs. Neospora parasites also cause abortion and neonatal morbidity in cattle, sheep, goats, and horses, and neosporosis is emerging as an important cause of bovine abortion worldwide. The purpose of this study was to identify N. caninum cDNA clones encoding antigens that would be useful for the immunodiagnosis of bovine neosporosis. Two N. caninum tachyzoite cDNA clones expressing antigens that were recognized by serum from naturally and experimentally infected cattle were identified. The DNA sequences of these clones were determined, and the inserts were subcloned into the plasmid expression vector pTrcHisB. Both recombinant antigens, expressed as fusion proteins with a His6 tag, were purified on a nickel-chelating affinity column and evaluated in separate enzyme-linked immunosorbent assays (ELISAs). Both recombinant antigen ELISAs were capable of distinguishing between sera from Neospora-infected cows and sera from uninfected control cows. Furthermore, both assays were able to detect an antibody response in animals that were experimentally inoculated with N. caninum. Neither antigen showed evidence of cross-reactivity with serum from animals inoculated with the closely related parasites Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, and Sarcocystis hirsuta. PMID:8705668

  19. Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

    PubMed Central

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

    2012-01-01

    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. PMID:22815145

  20. Antigen-capture enzyme-linked immunosorbent assays for detection of different H5 avian Influenza A virus.

    PubMed

    Chiu, Yi-Chung; Chu, Wen-Yu; Tsao, Zak; Wang, Ching-Ho

    2012-07-01

    Avian Influenza A virus (AIV) subtype H5 is divided into American and Eurasian lineages, according to hemagglutinin gene sequences. Although methods for detecting H5 AIVs have been described, no H5 strain-specific detection method has been reported. The purpose of the present study was to develop an antigen-capture enzyme-linked immunosorbent assay (ACE) to detect and differentiate between the American and the Eurasian H5 AIVs. Monoclonal antibodies (mAbs) against the HA fragment of a Eurasian H5N2 AIV were used as the capture antibodies as well as the detector antibodies after labeling with horseradish peroxidase to develop an ACE. One mAb was selected for detecting the American as well as the Eurasian H5 AIVs. The other mAb was used for detecting only the Eurasian H5N2 but not the American H5N2 AIVs, H6N1 AIVs, or Newcastle disease virus. The ACEs developed would be useful for detection and differentiation of H5 AIVs from the Eurasian and the American H5 AIVs. PMID:22621946

  1. An immune sandwich assay of carcinoembryonic antigen based on the joint use of upconversion phosphors and magnetic beads.

    PubMed

    Li, Yaohua; Wu, Zhengjun; Liu, Zhihong

    2015-06-21

    We herein report a sensitive and selective immunosensor for carcinoembryonic antigen (CEA) based on the joint use of upconversion phosphors (UCPs) and magnetic beads (MBs). UCPs as the signal probe were designed with a core-shell structure which provided a 40-fold enhancement of the luminescence intensity. Poly(acrylic acid) (PAA)-modified UCPs were covalently conjugated with the anti-CEA antibody (coating), and streptavidin functionalized magnetic beads were combined with another biotin-tagged anti-CEA antibody. With the assistance of a magnet, the as-formed immune sandwich in the presence of CEA can be readily separated from the assay matrix. The immunosensor showed a linear dynamic range for CEA within 0.05-20 ng mL(-1) in a buffered aqueous solution, and 0.1-20 ng mL(-1) in a human serum sample. The sensor was highly specific to CEA. Our results have suggested the potential application of the UCP-MB based immunoassay for CEA in clinical analysis. PMID:25882752

  2. 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

  3. Reactivity of heat-stable Leptospira antigenic preparation used in enzyme-linked immunosorbent assay for detection of antibodies in swine serum.

    PubMed

    Wasiński, B; Pejsak, Z

    2012-01-01

    Serology plays an important role in laboratory diagnosis of leptospirosis. Apart from the most often used microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA) seems to be useful especially in screenings of animal herds. The ELISA used for detection of antibodies against selected Leptospira serogroups in swine serum samples was investigated during the study. An essential element of this test is heat-stable antigenic preparation from cultures of Leptospira interrogans serovars Icterohaemorrhagiae, Pomona and L. borgpetersenii serovar Sejroe. The aim of the present study was to identify and analyze ELISA heat-stable antigen fractions playing a role in the reaction with leptospiral antibodies indicated in swine serum. Reactivity of the three-component antigenic preparation was compared in immunoblotting with reactivity of six heat-stable antigenic preparations made from the following single serovars: L. interrogans serovars Icterohaemorrhagiae, Pomona, Canicola, L. borgpetersenii serovars Sejroe, Tarassovi and L. kirshneri serovar Grippotyphosa. All antigenic preparations were submitted to SDS-PAGE and transferred to a nitrocellulose membrane using a semidry system. After the transfer, the membrane was incubated with diluted swine serum containing antibodies specific for one of the six above mentioned Leptospira serovars. For the three-component antigenic preparation and antigens prepared from single serovars the immunoblot revealed reaction of sera with fractions of the 20-26 kDa region and around the 14.5 kDa region. The investigated heat-stable Leptospira antigenic preparation contains fractions demonstrating serogroup- and species-specificity. Fraction 20-26 kDa showed serogroup-specific activity, whereas the fraction around 14.5 kDa showed species-specific activity. PMID:22708354

  4. 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

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

    2016-03-01

    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

  5. 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

  6. 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

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

    PubMed Central

    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

  8. 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

  9. Understanding early serum hepatitis D virus and HBsAg kinetics during pegylated interferon-alfa therapy via mathematical modeling

    PubMed Central

    Guedj, Jeremie; Rotman, Yaron; Cotler, Scott J.; Koh, Christopher; Schmid, Peter; Albrecht, Jeff; Haynes-Williams, Vanessa; Liang, Jake T.; Hoofnagle, Jay H.; Heller, Theo; Dahari, Harel

    2014-01-01

    There is little information on the early kinetics of hepatitis delta virus (HDV) and hepatitis B surface antigen (HBsAg) during interferon-α therapy. Here a mathematical model was developed and fitted to frequent HDV and HBsAg kinetic data from 10 patients during the first 28 weeks of pegylated-interferon-α2a (peg-IFN) therapy. Three patients achieved a complete virological response (CVR), defined as undetectable HDV 6 months after treatment stopped with loss of HBsAg and anti-HBsAg seroconversion. After initiation of therapy a median delay of 9 days (interquartile range IQR:[5;15]) was observed with no significant changes in HDV level. Thereafter, HDV declined in a biphasic manner, where a rapid first-phase lasting for 25 days (IQR:[23;58]) was followed by a slower or plateau second-phase. The model predicts that the main effect of peg-IFN is to reduce HDV production/release with a median effectiveness of 96% (IQR:[93;99.8]). Median serum HDV half-life (t1/2) was estimated to 2.9 days (IQR:[1.5;5.3]) with pretreatment production and clearance of about 1010 (IQR:[109.8-1010.8]) virions/day. None of the patients with flat 2nd phase in HDV achieved CVR. HBsAg kinetics of decline paralleled the second-phase of HDV decline consistent with HBsAg-productive-infected cells being the main source of production of HDV, with a median t1/2 of 135 days (IQR:[20-460]. The interferon lambda-3 polymorphism (rs12979860) was not associated with kinetic parameters. Conclusions Modeling results provide insights into HDV-host dynamics, the relationship between serum HBsAg levels and HBsAg-infected cells, IFN's mode of action and its effectiveness. The observation that a flat second phase in HDV and HBsAg kinetics was associated with failure to achieve CVR provides the basis to develop early stopping rules during peg-IFN treatment in HDV-infected patients. PMID:25098971

  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. PMID:26175067

  11. 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. PMID:19369434

  12. 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

  13. A comparative evaluation of assays for markers of activated coagulation and/or fibrinolysis: thrombin-antithrombin complex, D-dimer and fibrinogen/fibrin fragment E antigen.

    PubMed

    Boisclair, M D; Lane, D A; Wilde, J T; Ireland, H; Preston, F E; Ofosu, F A

    1990-04-01

    Measurements were made of levels of D-dimer in plasma and serum, thrombin-antithrombin complex (TAT) in plasma and fibrinogen/fibrin fragment E antigen (FgE) in serum in a normal healthy control group and in patients with a range of disorders associated with hypercoagulability. Levels were determined in 31 normal healthy controls, 30 patients with disseminated intravascular coagulation (DIC), 21 patients with deep venous thrombosis (DVT), 27 patients with myocardial infarction (MI), 26 patients with acute leukaemia and 56 patients with liver disease. Considering all subjects, significant correlations were established between the results of all assays. Notably high correlations (r greater than 0.9) were established between plasma and serum levels of D-dimer, between plasma levels of D-dimer and serum levels of FgE, and between serum levels of D-dimer and FgE. All assays showed very high discrimination (sensitivity) between the normal control group and patients with DIC (97-100%), but there were marked differences between the assays in sensitivity for DVT and MI. In general, the FgE assay was more sensitive than the D-dimer assay, whilst both the FgE and D-dimer assays were more sensitive than the TAT assay. The same trends were apparent in the capability of the assays to discriminate between the normal control group and patients with acute leukaemia and liver disease: disorders with an unknown prevalence of activation of coagulation/fibrinolysis. Our results indicated that measurements of fibrinogen/fibrin degradation products (FDPs) in serum were almost unaffected by artefacts. The data further suggested that the broad-spectrum FgE assay was better than the more specific D-dimer assay in detecting clinical hypercoagulability. Our study showed that, in the clinical conditions examined, FDPs were more effective markers of hypercoagulability than TAT. PMID:2189490

  14. 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

  15. IgG subclass composition of antibodies to HBsAg in circulating immune complexes from patients with hepatitis B virus infections.

    PubMed Central

    Rath, S; Devey, M E

    1988-01-01

    The IgG subclass of antibody associated with hepatitis B surface antigen (HBsAg) in circulating immune complexes (CIC) from patients with either acute or chronic hepatitis B virus (HBV) infections was measured using an isotype and antigen-specific ELISA. All patients were HBsAg positive but were negative for free anti-HBs antibody. The subclass of antibody associated with HBsAg in CIC in both groups was predominantly IgG1 and IgG4. This is in contrast to free anti-HBs in convalescent sera from patients recovering from HBV infection, which are highly restricted to IgG1 and IgG3. The finding of high levels of IgG4 antibodies in CIC suggest that CIC containing this subclass may be cleared less efficiently than CIC containing antibodies of other subclasses. Formation of these CIC may be an important factor in the progression of infection to chronicity and may also be involved in the antigen-specific immunosuppression seen in early acute and chronic HBV infections. PMID:3396217

  16. Characterization of monoclonal antibodies to human group B rotavirus and their use in an antigen detection enzyme-linked immunosorbent assay.

    PubMed Central

    Burns, J W; Welch, S K; Nakata, S; Estes, M K

    1989-01-01

    Three monoclonal antibodies (MAbs)--B5C9, B5E4, and B10G10--to human group B rotavirus, an agent implicated in epidemic outbreaks of diarrhea in the People's Republic of China, primarily in adults, were prepared. MAb reactivity was decreased when virus preparations were treated with EDTA, suggesting reactivity with the outer-capsid protein(s). Competition experiments suggested that these MAbs recognize overlapping epitopes within a single antigenic site. A simple antigen detection enzyme-linked immunosorbent assay (ELISA) specific for the human group B rotavirus was established by using these MAbs as capture antibodies. Fifteen clinical samples obtained from three epidemic areas in the People's Republic of China and previously shown by Chinese scientists to contain group B virus were all positive in the MAb capture antigen detection ELISA, whereas none of the 57 samples lacking the group B virus reacted in the test. The results suggest that this MAb capture antigen detection ELISA will be useful to identify outbreaks caused by the human group B rotavirus and to monitor possible spread of the virus. Images PMID:2536755

  17. Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

    PubMed

    López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

    2007-11-01

    An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen. PMID:17998551

  18. Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of an inflammatory response antigen in subclinical mastitic milk samples.

    PubMed Central

    Ball, H J; Finlay, D; Mackie, D P; Greer, D; Pollock, D; McNair, J

    1991-01-01

    A monoclonal antibody to a 23.5-kDa bovine inflammatory antigen present in high levels in mastitic milk has been used in an antigen-capture enzyme-linked immunosorbent assay (ELISA) to screen milk samples from herds of cattle for subclinical mastitis. The results from 20 herds with a total of 2,612 quarter samples are presented. Good correlation was observed between the ELISA level and the milk cell count (MCC). The results demonstrated an average of 5% false negatives (1.8% associated with isolates of Staphylococcus aureus and/or Streptococcus spp.) and 7.7% false positives for each herd in relation to mastitic (greater than 400,000 cells per ml) or nonmastitic (less than 400,000 cells per ml) MCCs. Images PMID:1761683

  19. Rapid determination of members of the family Enterobacteriaceae in drinking water by an immunological assay using a monoclonal antibody against enterobacterial common antigen.

    PubMed Central

    Hübner, I; Steinmetz, I; Obst, U; Giebel, D; Bitter-Suermann, D

    1992-01-01

    An immunological method for the detection of members of the family Enterobacteriaceae in drinking water was developed. The method was based on a sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody immunoglobulin G2a 898 against enterobacterial common antigen. The enterobacterial common antigen sandwich ELISA combined with selective preenrichment culture could be performed in only 24 h. Six hundred sixty-eight water samples from a variety of German public water supplies were screened to verify the effectiveness of the new method. Ninety-eight percent of the results obtained by the immunological method could be confirmed by conventional microbiological methods. The immunological method proved to be considerably faster and more specific and sensitive than the standard method specified by the German drinking water regulations. PMID:1444435

  20. Use of viral lysate antigen combined with recombinant protein in Western immunoblot assay as confirmatory test for serodiagnosis of severe acute respiratory syndrome.

    PubMed

    Guan, Ming; Chen, Hsiao Ying; Tan, Phuay Heng; Shen, Shuo; Goh, Phuay-Yee; Tan, Yee-Joo; Pang, Peow Hoon; Lu, Yang; Fong, Priscilla Yiquan; Chin, Daria

    2004-11-01

    A Western immunoblot assay for confirmatory serodiagnosis of severe acute respiratory syndrome (SARS) was developed utilizing viral lysate antigens combined with a recombinant nucleocapsid protein, GST-N (glutathione S-transferase-nucleocapsid) of the SARS coronavirus (SARS-CoV). The viral lysate antigens were separated by electrophoresis and transblotted onto nitrocellulose membranes. The resultant membrane was subsequently added with the GST-N recombinant protein at a specific location. The positions of bands corresponding to some of the structural proteins immobilized on the membrane were then located and verified with mouse or rabbit antisera specific to the respective proteins. The Western immunoblot assay was able to detect antibodies to SARS-CoV in all 40 serum specimens from SARS patients and differentiate the SARS-positive samples from those of the healthy donor or non-SARS patient controls (150 samples) when set criteria were followed. In addition, when the immunoblot was used to test samples considered falsely positive by an in-house-developed SARS-specific enzyme-linked immunosorbent assay, band patterns different from those with samples from SARS patients were obtained. PMID:15539520

  1. Structural comparison of O-antigen gene clusters of Legionella pneumophila and its application of a serogroup-specific multiplex PCR assay.

    PubMed

    Cao, Boyang; Tian, Zhenyang; Wang, Suwei; Zhu, Zhiyan; Sun, Yamin; Feng, Lu; Wang, Lei

    2015-12-01

    The Legionella pneumophila serogroups O1, O4, O6, O7, O10 and O13 are pathogenic strains associated with pneumonia. The surface O-antigen gene clusters of L. pneumophila serogroups O4, O6, O7, O10 and O13 were sequenced and analyzed, with the function annotated on the basis of homology to that of the genes of L. pneumophila serogroup O1 (L. pneumophila subsp. pneumophila str. Philadelphia 1). The gene locus of the six L. pneumophila serogroups contains genes of yvfE, neuABCD, pseA-like for nucleotide sugar biosynthesis, wecA for sugar transfer, and wzm as well as wzt for O-antigen processing. The detection of O-antigen genes allows the fine differentiation at species and serogroup level without the neccessity of nucleotide sequencing. The O-antigen-processing genes wzm and wzt, which were found to be distinctive for different for different serogroups, have been used as the target genes for the detection and identification of L. pneumophila strains of different O serogroups. In this report, a multiplex PCR assay based on wzm or wzt that diferentiates all the six serogroups by amplicon size was developed with the newly designed specific primer pairs for O1 and O7, and the specific primer pairs for O4, O6, O10, and O13 reported previously. The array was validated by analysis of 34 strains including 15 L. pneumophila O-standard reference strains, eight reference strains of other Legionella non-pneumophila species, six other bacterial species, and five L. pneumophila environmental isolates. The detection sensitivity was one ng genomic DNA. The accurate and sensitive assay is suitable for the identification and detection of strains of these serogroups in environmental and clinical samples. PMID:26415652

  2. In vitro antigen-induced antibody responses to hepatitis B surface antigen in man. Kinetic and cellular requirements.

    PubMed Central

    Cupps, T R; Gerin, J L; Purcell, R H; Goldsmith, P K; Fauci, A S

    1984-01-01

    In this report we define the parameters of the human immune response after immunization with hepatitis B vaccine. 2 wk after booster immunization, there is significant spontaneous secretion of antibody to hepatitis B surface antigen (anti-HBs IgG), which is not further augmented by stimulation with antigen or pokeweed mitogen (PWM). By 4 wk there is little spontaneous secretion of specific antibody, and low doses of antigen or PWM produce significant increases in the amount of anti-HBs IgG produced. By 8 wk the peripheral blood mononuclear cells are refractory to stimulation by antigen, but anti-HBs IgG is produced in response to PWM. 0.5 yr or more after the last immunization, some individuals will manifest an antigen-induced specific antibody response. This induction of anti-HBs IgG by hepatitis B surface antigen (HBsAg) is monocyte- and T cell-dependent. Note that there is a dichotomy in the T cell response to HBsAg. The specific antibody response is clearly T cell dependent, but no in vitro T cell proliferative response to HBsAG could be demonstrated in the immunized individuals. Although the precise reason for the absent proliferative response to HBsAg despite well-established humoral immunity has not been determined, there was no evidence to suggest nonspecific suppression by HBsAg or the presence of an adherent suppressor cell population. The ability to evaluate antigen-induced, antigen-specific responses to HBsAg will be useful in defining the unique interaction between the human immune response and this clinically important viral agent. PMID:6332826

  3. Efficacy evaluation of two synthetic lysine lipidated tripeptides as vaccine adjuvants against HBsAg.

    PubMed

    Sidiq, Tabasum; Khajuria, Anamika; Shafi, Syed; Ismail, Tabasum; Sampath Kumar, Halmathur; Kannappa Srinivas, Vellimedu; Krishna, Ella; Kamal Johri, Rakesh

    2013-04-01

    In the present investigation, adjuvant potential of two novel lipidated tripeptide lysine derivatives (KKSM and KKSMB) was evaluated using various in vitro and animal-derived models of humoral and cell-mediated immune events in response to hepatitis B surface antigen (HBsAg). The results were compared with alum adjuvanted with HBsAg. Both these molecules were found to stimulate anti-HBsAg IgG and neutralizing (IgG1 and IgG2a) antibody titres in mice sera. The two molecules stimulated the proliferation of T-lymphocyte sub-sets (CD4/CD8) as well as the production of soluble mediators of Th1 (IL-2 and IFN-γ) and Th2 response (IL-4) in spleen cell culture supernatant. Furthermore, the two lipidated tripeptides enhanced the CD4, CD8, CD3 and CD19 cell populations as well as CD4/CD8 derived IL-2, IL-4, IFN-γ and TNF-α in whole blood of treated mice. There was found to be the significant enhancement in the release of IL-12, IFN-γ and nitrite content in macrophage supernatant. Moreover, the two lipidated tripeptides enhanced the population of CD80 and CD86 in spleen-derived macrophages and did not show any hemolytic effect on rabbit RBCs. Taken together, these results suggest that both these molecules are the potent enhancers of anti-HBsAg immune response via augmenting Th1/Th2 response in a dose dependent manner. PMID:23474022

  4. Assessment of antiretroviral therapy by plasma viral load testing: standard and ICD HIV-1 p24 antigen and viral RNA (QC-PCR) assays compared.

    PubMed

    Kappes, J C; Saag, M S; Shaw, G M; Hahn, B H; Chopra, P; Chen, S; Emini, E A; McFarland, R; Yang, L C; Piatak, M

    1995-10-01

    To assess the utility of quantitative competitive-polymerase chain reaction (QC-PCR) measurements of plasma human immunodeficiency virus type 1 (HIV-1) RNA and other viral load markers for assessment of antiretroviral therapy, we used archived cryopreserved specimens from a randomized controlled clinical trial of 135 patients (CD4+ T cell count < or = 500/mm3), comparing zidovudine (500 mg/day) versus the nonnucleoside reverse transcriptase inhibitor L-697, 661 (50, 300, or 1,000 mg daily). We evaluated treatment-associated changes in plasma viral load by standard and immune complex-dissociated (ICD) HIV-1 p24 antigen assays, and, in a representative subset of patients (n = 46), by QC-PCR determination of virion-associated HIV-1 RNA. At baseline, HIV-1 RNA was quantifiable by QC-PCR in all patients tested (100%), whereas standard and ICD HIV-1 p24 antigen tests were positive (> or = 30 pg/ml) in 42% and 56%, respectively. All viral load parameters showed significant decreases from baseline within 1 week of initiation of zidovudine, as measured by standard p24 antigen assay, ICD p24 assay, and QC-PCR. At 1 week, patients treated with either 300 or 1,000 mg/day of L-697,661 showed significant decreases from baseline in plasma standard and ICD p24 antigen and QC-PCR-determined HIV-1 RNA levels. Whereas viral load decreases seen with zidovudine were sustained for the duration of treatment, plasma viral markers often returned to pretreatment levels despite ongoing L-697,661 treatment, with evidence of the emergence of drug-resistant virus. Whereas standard p24, ICD p24, and viral RNA levels changed similarly in response to treatment, the superior sensitivity and available dynamic range of plasma viral RNA assays like QC-PCR analysis provide an advantage for clinical monitoring of plasma viral load, allowing tracking of treatment-related changes even in patients with earlier stage disease and lower levels of viral load. PMID:7552477

  5. 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

  6. Sequencing of the Escherichia coli O22 O Antigen Gene Cluster and Detection of E. coli Serogroups O22 and O91 by Multiplex PCR Assays Targeting Virulence Genes and the wzy Gene in the Respective O-Antigen Gene Clusters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The O antigen gene cluster of Escherichia coli O22, an extraintestinal pathogenic E. coli (ExPEC) serogroup was sequenced, and eight open reading frames encoding O antigen sugar biosynthesis, transfer, and processing were identified. Multiplex PCR assays were developed targeting the wzx and wzy gen...

  7. Screening of Danish blood donors for hepatitis B surface antigen using a third generation technique.

    PubMed Central

    Wantzin, P; Nielsen, J O; Tygstrup, N; Soerensen, H; Dybkjaer, E

    1985-01-01

    The profit to be gained by testing Danish blood donors for hepatitis B surface antigen (HBsAg) with a third generation technique instead of the currently used immunoelectrophoresis was investigated by additional screening of 48 750 blood units by radioimmunoassay three weeks after donation. Twenty nine units were positive for HBsAg on radioimmunoassay (0.059%). Only six of these were found by immunoelectrophoresis (0.012%). Most of the 23 donors positive on radioimmunoassay and negative on immunoelectrophoresis were healthy carriers of HBsAg (20) or had asymptomatic chronic liver disease (two). One donor had acute hepatitis B. Fifteen of the 23 blood units were transfused. The 15 recipients were monitored biochemically and serologically for up to nine months. One recipient developed fulminant hepatitis B, three developed acute hepatitis B, and one became a healthy carrier of HBsAg. All these patients had received blood from healthy carriers of HBsAg. Two recipients were immunised against HBsAg, and in one patient no seroconversion was observed. The remaining recipients died soon after transfusion or were protected by antibodies to HBsAg that had been present before the transfusion. Testing of Danish blood donors using a third generation technique identified a substantial number of donors positive for HBsAg overlooked by immunoelectrophoresis. Most of these donors were healthy carriers of HBsAg. Blood taken from such carriers is highly infectious when transfused, probably because of the large amount of material transmitted. PMID:3929937

  8. 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

  9. 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

  10. 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

  11. Prevalence of HBsAg and knowledge about hepatitis B in pregnancy in the Buea Health District, Cameroon: a cross-sectional study

    PubMed Central

    2014-01-01

    Background Although infection with Hepatitis B Virus (HBV) remains a global public health problem, little is known about its epidemiology in pregnancy in sub-Saharan Africa. This study sought to determine the prevalence of, and identify factors associated with hepatitis B surface antigen (HBsAg) positivity among pregnant women in the Buea Health District (BHD) in rural Cameroon. We also assessed pregnant women’s knowledge about hepatitis B. Methods A cross-sectional, descriptive study was undertaken. Participants were evaluated using a structured questionnaire with clinical examination and were then screened for HBsAg using a commercial rapid diagnostic test. Assessment of knowledge was done using a hepatitis B basic knowledge summary score. Results Of the 176 pregnant women studied, 9.7% (95% CI: 5.7%, 15%) tested positive for HBsAg. None of the risk factors assessed was significantly associated with HBsAg positivity. The hepatitis B knowledge summary score ranged from 0 to 12 with a mean of 1.5 (SD = 3.14, median = 0, IQR = 0 to 0). Only 16% of participants had scores greater than 6/12. The knowledge summary score of the participants was associated with the educational level (p-value = 0.0037). Conclusion The high prevalence of HBsAg (9.7%) among women of child bearing age suggests that vertical transmission of HBV may be a public health problem in Buea Health District. Knowledge of HBV among pregnant women was poor. We recommend that all pregnant women ought to be routinely screened for HBV and that health education on HBV should be provided to pregnant women especially during antenatal visits. PMID:24965844

  12. HBsAg loss is not an ideal indicator for discontinuing treatment of HBeAg-negative chronic hepatitis B: a case report.

    PubMed

    Dong, Pei-Ling; Zhang, Xin; Zhao, Wen-Min; Ding, Hui-Guo

    2013-12-01

    We experienced a case of a 36-year-old married man who was found to be hepatitis B virus (HBV) positive at 23 years of age. His liver function was repeatedly abnormal in the past 13 years. In November 2007 he presented with fatigue. Laboratory tests showed serum alanine aminotransferase concentration 255.3 U/l, positive for hepatitis B surface antigen (HBsAg) and hepatitis B e antibody, HBV DNA 3.01 × 10(7) copies/ml; liver biopsy showed necroinflammatory scores 11 and fibrosis scores 4. After 20 weeks of treatment with Peg-IFN α-2b, laboratory tests showed HBV DNA <500 copies/ml and normal liver function. By week 52 of the treatment, HBsAg became negative. By week 92 of continuing treatment, HBsAb became weakly positive and Peg-IFN α-2b treatment was stopped. On follow-up, both HBsAg and HBsAb were negative 28 weeks after discontinuation of Peg-IFN α-2b. We then performed a second liver biopsy and histological examination revealed necroinflammtary scores 2 and fibrosis scores 2. We administered hepatitis B vaccine intramuscularly every 4 weeks combined with IFN α-1b 30 μg intramuscularly every other day. HBsAb was 244.8 IU/l at week 32 of this combined treatment. Follow-up showed that after discontinuation of the combined treatment HBsAb concentration declined rapidly but could be maintained above 100 IU/l by intermittent injections of hepatitis B vaccine. Findings from this case reveal that HBsAg loss may be not sufficient; however, HBsAg seroconversion together with maintenance of certain concentrations of HBsAb may be a better endpoint to HBV treatment. PMID:23783395

  13. Dynamic Characteristics of Serum Hepatitis B Surface Antigen in Chinese Chronic Hepatitis B Patients Receiving 7 Years of Entecavir Therapy

    PubMed Central

    Zhang, Xia-Xia; Li, Min-Ran; Xi, Hong-Li; Cao, Ying; Zhang, Ren-Wen; Zhang, Yu; Xu, Xiao-Yuan

    2016-01-01

    Background: The ultimate goal of hepatitis B treatment is hepatitis B surface antigen (HBsAg) seroclearance. Several factors have been suggested to be associated with the rate of HBsAg reduction in antiviral-naive or lamivudine therapy cohorts. However, there are few studies evaluating the factors during long-term entecavir (ETV) therapy. In the present study, we aimed to evaluate the factors to predict the outcome of ETV therapy for 7 years. Methods: A total of 47 chronic hepatitis B (CHB) patients treated with ETV monotherapy were included in this study. Liver biochemistry, hepatitis B virus (HBV) serological markers, serum HBV DNA, and HBsAg titers were tested at baseline, 3 months, 6 months, and yearly from 1 to 7. The associations between factors and HBsAg reduction were assessed using multivariate tests with repeated measure analysis of variance. Results: At baseline, serum HBsAg levels showed a positive correlation with baseline HBV DNA levels (r = 0.625, P < 0.001). The mean HBsAg titers after ETV treatment were significantly lower than the baseline titers (P ranges from 0.025 to 0.000,000,6). The HBsAg reduction rate during the 1st year was greater compared to after 1 year of treatment (P < 0.05). Multivariate test showed that hepatitis B e antigen (HBeAg) seroclearance and/or HBsAg reduction ≥0.5 log10 IU/ml at 6 months had a high negative predictive value (96.77%) for HBsAg seroclearance (P = 0.002, P = 0.012, respectively). Conclusions: The HBsAg reduction rate during the 1st year was greater than that after 1 year of treatment. Further, HBeAg status and HBsAg levels at month 6 are the optimal factors for the early prediction of HBsAg seroclearance after long-term ETV therapy in CHB patients. PMID:27064037

  14. Standardization of an enzyme linked immunosorbent assay (ELISA) for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus.

    PubMed

    de Rezende, N A; Dias, M B; Campolina, D; Chavéz-Olortegui, C; Amaral, C F

    1995-01-01

    The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antigens from toxic components of Tityus serrulatus scorpion venom was determined in patients stung by T. serrulatus before antivenom administration. Thirty-seven patients were classified as mild cases and 19 as moderate or severe cases. The control absorbance in the venom assay was provided by serum samples from 100 individuals of same socioeconomic group and geographical area who had never been stung by scorpions or treated with horse antisera. The negative cutoff value (mean + 2 SD) corresponded to a venom concentration of 4.8 ng/ml. Three out of the 100 normal sera were positive, resulting in a specificity of 97%. The sensitivity of the ELISA when all cases of scorpion sting were included was 39.3%. When mild cases were excluded, the sensitivity increased to 94.7%. This study showed that this ELISA can be used for the detection of circulating venom toxic antigens in patients with systemic manifestations following. T. serrulatus sting but cannot be used for clinical studies in mild cases of envenoming since the test does not discriminate mild cases from control patients. PMID:7569644

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

    PubMed

    Kim, Mi Na; Kim, Hyon Suk; Kim, Ja Kyung; Kim, Beom Kyung; Kim, Seung Up; Park, Jun Yong; Kim, Do Young; Ahn, Sang Hoon; Han, Kwang Hyub

    2016-09-01

    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

  16. 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

  17. Identification and expression of Babesia ovis secreted antigen 1 and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.

    PubMed

    Sevinc, Ferda; Cao, Shinuo; Xuan, Xuenan; Sevinc, Mutlu; Ceylan, Onur

    2015-05-01

    In order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5α cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis. PMID:25694531

  18. Identification and Expression of Babesia ovis Secreted Antigen 1 and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Cao, Shinuo; Xuan, Xuenan; Sevinc, Mutlu; Ceylan, Onur

    2015-01-01

    In order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5α cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis. PMID:25694531

  19. [Prostate specific antigen in patients with prostatic cancer--clinical usefulness of its measurement with sensitive enzyme immunoassay, TOSOH II PA assay].

    PubMed

    Shinka, T; Miyai, M; Sawada, Y; Ohkawa, T

    1994-10-01

    The TOSOH II PA (AIA-PACK PA in the U.S.A.) monoclonal immunoenzyme assay was used and the clinical usefulness of prostate specific antigen (PSA) was evaluated in 39 men with prostatic cancer and 32 men with benign prostatic hyperplasia (BPH) confirmed pathologically. We evaluated the lower limit of detection in this assay by the mean PSA level plue 3 standard deviations in 5 separate experiments with a zero control sera as well as the mean PSA level minus 3 standard deviations in 5 separate experiments with serial dilutions of a known concentration of PSA. PSA concentrations of 0 to 0.25 ng/ml could not be distinguished from the zero control. Therefore, we set the lower limit of detection for the assay as 0.25 ng/ml. At our laboratory, the normal standard value was determined by the mean PSA level plus 3 standard deviations in 79 healthy men as 2.3 ng/ml. Of patients that underwent radical cystoprostatectomy for bladder cancer, all had PSA levels less than 0.25 ng/ml, while only 6% had PAP levels less than 0.11 ng/ml, the lower limit of detection in the TOSOH II PAP assay. We compared TOSOH II PA with Markit PA, a commonly used assay in Japan. Although there was a close linear correlation (r = 0.90) between the TOSOH II PA and Markit PA, the difference of slope in the linear regression lines was great, it was thought due to anti-PSA antibodies in each assay recognizing different epitopes of PSA in the blood.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7527470

  20. 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

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

    PubMed

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

    2013-08-01

    Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals. PMID:23761660

  2. Serodiagnosis of pleuropneumonia using enzyme-linked immunosorbent assay with capsular polysaccharide antigens of Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7.

    PubMed Central

    Bossé, J T; Johnson, R P; Rosendal, S

    1990-01-01

    Capsular polysaccharide antigens of serotypes 1, 2, 5 and 7 of Actinobacillus pleuropneumoniae were used in enzyme-linked immunosorbent assays (ELISAs) to test sera from experimentally infected and field pigs. Specific reactions were found in sera of experimental pigs with antigens of serotypes 1, 5 and 7 whereas the serotype 2 antigen was cross-reactive. A 1:200 serum dilution was used for testing of 300 sera from 21 swine herds in southern Ontario. Cases of pleuropneumonia had occurred in 11 of these herds, but not in the others. The negative cut-off value was the mean optical density at 405 nm (OD405) + three standard deviations (SD) for 16 negative reference sera. Sera from four pigs naturally infected with Actinobacillus suis were tested and found to react to varying degrees with each of the antigens. Therefore a second cut-off value was determined as the mean OD405 + 2 SD for the A. suis sera. Sera which, in the ELISA produced OD readings above the latter cut-off were considered positive for antibodies to A. pleuropneumoniae; those which were lower than the former cut-off were considered negative. Readings between the two cut-off values may have been due to low positive titers or cross-reactivity, possibly with A. suis, and could not be used to predict pleuropneumonia. Of the pleuropneumonia-free herds, none had positive reactors to serotypes 5 or 7, whereas one and two herds had positive reactors to serotypes 1 and 2, respectively. Of the pleuropneumonia positive herds, six had positive reactors to serotype 1, one to serotype 2, four to serotype 5, and eight to serotype 7. PMID:2249177

  3. [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%. PMID:16933616

  4. One-step assay for the determination of free protein S antigen in plasma using real-time biospecific interaction analysis.

    PubMed

    Ravanat, C; Wiesel, M L; Schuhler, S; Dambach, J; Amiral, J; Cazenave, J P

    1998-06-01

    Real-time biospecific interaction analysis based on optical detection by surface plasmon resonance was used to develop an accurate one-step method for the direct measurement of free protein S in human plasma. This assay was validated, compared with classical immunological methods and shown to be suitable for the routine clinical diagnosis of protein S deficiency. The method relies on the specific capture of free protein S directly from plasma by a monoclonal antibody (mAb), 34G2, immobilized on a sensor chip surface. A calibration curve was established with serial dilutions of standard plasma (working range 5-50%) and a linear relationship was found to exist between the relative response in resonance units (RU) and the concentration of free protein S expressed as percentage plasma dilution (r = 0.99). The specificity of the assay was confirmed using purified human protein S and polyethylene glycol treated plasma. In addition, it could be demonstrated that no dissociation of C4b-BP-protein S complexes occurred under the chosen experimental conditions. The technique was reproducible with inter-assay, intra-assay and inter-sensor chip variation coefficients of 1.5-5.4%, 2-3.1% and 4.4-4.9%, respectively, as evaluated in two different plasma samples. Since all tests are automatic and long series of analyses can be performed with the same sensor chip, the method was applied to the determination of free protein S antigen in plasma from 20 normal blood donors and 38 thrombophilic patients. Results displayed excellent correlation with those of free protein S enzyme-linked immunosorbent assay (r = 0.99) and rocket immunoelectrophoresis of polyethylene glycol-treated plasma (r = 0.93). PMID:9690804

  5. 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

  6. RAPID ASSIGNMENT OF SWINE LEUKOCYTE ANTIGEN (SLA) HAPLOTYPES IN PEDIGREED HERDS USING A POLYMERASE CHAIN REACTION BASED ASSAY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present a simple assay to determine the SLA haplotypes of animals within two experimental herds of MHC defined miniature pigs. The Yucatan Miniature Pigs have four founder haplotypes (w, x, y, z) and one recombinant haplotype (q). The NIH Miniature Pigs have three founder haplotypes (a, c, d) an...

  7. 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

  8. 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. PMID:26335477

  9. 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

  10. 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. PMID:25424942

  11. Combined Antigen-Specific Interferon-γ and Interleukin-2 Release Assay (FluoroSpot) for the Diagnosis of Mycobacterium tuberculosis Infection

    PubMed Central

    Chesov, Dumitru; Lange, Christoph; Daduna, Franziska; Crudu, Valeriu; Preyer, Rosemarie; Ernst, Martin; Kalsdorf, Barbara

    2015-01-01

    Background To evaluate interleukin (IL)-2 and interferon (IFN)-γ secreting T-cells in parallel for the differentiation of latent infection with Mycobacterium tuberculosis infection (LTBI) from active tuberculosis. Methods Following ex-vivo stimulation of peripheral blood mononuclear cells (PBMC) with M. tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10, immune responses were assessed by enzyme-linked immunospot IFN-γ release assay (EliSpot-IGRA) and a novel dual cytokine detecting fluorescence-linked immunospot (FluoroSpot) in 18 patients with pulmonary tuberculosis, 10 persons with previously cured tuberculosis, 25 individuals with LTBI and 16 healthy controls. Results Correlation of IFN- γ+ spot-forming cells in EliSpot-IGRA and FluoroSpot were R2 = 0.67 for ESAT-6 and R2 = 0.73 for CFP-10. The number of IL-2- IFN- γ+ producing cells was higher in patients with tuberculosis compared with past tuberculosis (CFP-10-induced p = 0.0068) or individuals with LTBI (ESAT-6-induced p = 0.0136). A cutoff value of >16 CFP-10-induced IFN-γ+ secreting cells/200.000 PBMC in the EliSpot-IGRA discriminated with highest sensitivity and specificity (89% and 76%, respectively). However, overlap in cytokine responses precludes distinction between the cohorts on an individual basis. Conclusions Combined analysis of IFN-γ and IL-2 secretion by antigen specific T-cells does not allow a reliable differentiation between different states of M. tuberculosis infection in clinical practice. PMID:25785445

  12. Modified vaccinia Ankara expressing HIVA antigen stimulates HIV-1-specific CD8 T cells in ELISpot assays of HIV-1 exposed infants☆

    PubMed Central

    Slyker, Jennifer A.; Lohman, Barbara L.; Mbori-Ngacha, Dorothy A.; Reilly, Marie; Wee, Edmund G.-T.; Dong, Tao; McMichael, Andrew J.; Rowland-Jones, Sarah L.; Hanke, Tomas; John-Stewart, Grace

    2012-01-01

    Recombinant modified vaccinia virus Ankara expressing HIV-1 antigens (MVA.HIVA) was used in ELISpot assays to monitor HIV-1-specific T cell responses in infants. Responses to MVA.HIVA and HIV-1 peptides were examined in 13 infected and 81 exposed uninfected infants in Nairobi, Kenya. Responses to MVA.HIVA (38%) and peptide stimulation (38%) were similar in frequency (p = 1.0) and magnitude (mean 176 versus 385 HIVSFU/106, p = 0.96) in HIV-1 infected infants. In exposed uninfected infants, MVA.HIVA detected more positive responses and higher magnitude responses as compared to peptide. MVA.HIVA ELISpot is a sensitive method for quantification of HIV-1-specific CD8+ T cell responses in HIV-1 exposed infants. These results demonstrate the relevance of HIV-1 clade A consensus-derived immunogen HIVA for the viruses currently circulating in Nairobi. PMID:16043269

  13. Comparison of Fecal Antigen Detection Using Enzyme Linked Immunosorbent Assay With the Auramine Phenol Staining Method for Diagnosis of Human Cryptosporidiosis

    PubMed Central

    Jafari, Rasool; Maghsood, Amir Hossein; Safari, Marzieh; Latifi, Milad; Fallah, Mohammad

    2015-01-01

    Background: Fecal antigen detection using the enzyme linked immunosorbent assay (ELISA) and oocyst detection using auramine phenol (AP) staining methods, are told to be more sensitive compared to other conventional methods, for diagnosis of cryptosporidiosis. Objectives: The aim of the present study was to evaluate the antigen-detection capacity in the stool specimens using ELISA and oocyst detection by AP staining methods, for the diagnosis of human cryptosporidiosis. Materials and Methods: A total of 228 fecal samples were collected from residents of rural areas of Hamadan, West of Iran. Each fecal sample was divided into two parts, one kept frozen at -20˚C for Ag-capture ELISA and the other in 10% formalin for the AP staining method. Cryptosporidium Ag-detection ELISA procedure was performed according to the manual of the manufacturer. The preserved samples concentrated using the formalin-ether concentration technique were stained with AP and then investigated under florescent microscopy. Results: Eight (3.5%) and three (1.3%) out of 228 fecal samples were positive for Cryptosporidium infection by ELISA and AP staining methods, respectively. Cryptosporidium Ag-detection using ELISA showed an increased frequency of the infection, compared to the AP staining method (P = 0.062). Conclusions: For epidemiological studies and diagnostic purposes of the Cryptosporidium infection, especially in asymptomatic individuals, Ag-detection ELISA is an easy to perform and accurate method, compared to other conventional microscopic methods. PMID:25825642

  14. 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

  15. 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

  16. 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

  17. A comparison of two West Nile virus detection assays (TaqMan reverse transcriptase polymerase chain reaction and VecTest antigen assay) during three consecutive outbreaks in northern Illinois.

    PubMed

    Lampman, Richard L; Krasavin, Nina M; Szyska, Michael; Novak, Robert J

    2006-03-01

    Mosquitoes identified as female Culex (Culex) species, primarily mixtures or uniform batches of Culex pipiens and Culex restuans, were collected daily from gravid traps by 2 mosquito abatement districts (MADs) in Cook County, Illinois. From 2002 through 2004, batches (pools) of mosquitoes were tested by the MADs for West Nile virus (WNV) by using VecTest WNV antigen assays and the same samples were retested, usually within 1-2 wk, for WNV RNA by the TaqMan reverse transcriptase polymerase chain reaction (RT-PCR). There were 952 TaqMan-positive pools out of 3,953 pools over the 3 years, and about one half of that number were VecTest-positive. The difference between the 2 detection assays varied between and within years. The VecTest assays detected about 57% and 69% of the TaqMan RT-PCR-positive pools from Des Plaines Valley MAD and Northwest MAD in 2002, but only about 40% and 46% in 2003, and 36% and 55% in 2004, respectively. Based on a subset of the 2004 data, a linear relationship was found between VecTest detection of WNV and TaqMan cycle threshold between 18 and 28 cycles. A temporal decrease in the difference between the 2 assays was observed in 2003 and 2004, which we conjecture is due, at least partially, to a seasonal decline in the proportion of recently infected mosquitoes. This trend was not observed in 2002 because infection rates indicated a high likelihood of more than 1 infected mosquito per pool at the peak of transmission. Unlike a previous study, the 95% confidence intervals of infection rates based on the 2 detection methods did not always overlap. The highest infection rates occurred in 2002 when mean monthly temperatures were above average. PMID:16646326

  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. A novel flow cytometry single tube bead assay for quantitation of von Willebrand factor antigen and collagen-binding.

    PubMed

    Mina, Ashraf; Favaloro, Emmanuel J; Koutts, Jerry

    2012-11-01

    Deficiency of or defects in the plasma protein von Willebrand factor (VWF) lead to bleeding and von Willebrand disease (VWD), which may be congenital or acquired. VWD is considered the most common inherited bleeding disorder and laboratory testing for VWF level and activity is critical for appropriate diagnosis and management. We have designed and established a novel Flow Cytometry (FC) based method for measuring VWF antigen (VWF:Ag) and collagen binding (VWF:CB), together in the same tube and at the same time. The results of the novel FC method have been compared against existing reference methods using a range of normal and pathological material. Methods correlated well (VWF:Ag, r=0.866; VWF:CB, r=0.888) and generally permitted similar discrimination of quantitative versus qualitative VWD types (e.g. type 1 vs type 2A or 2B VWD). The novel procedure is expected to permit future streamlined performance of VWD screening, either using stand-alone FC systems or potentially incorporated into FC-capable automated blood cell and particle counters to allow for improved, automated and faster identification or exclusion of VWD. PMID:23014972

  20. Measurement of the free alpha subunit of human glycoprotein hormones by a monoclonal antibody-based immunoradiometric assay, and further exploration of antigenic sites on the choriogonadotropin molecule

    SciTech Connect

    Norman, R.J.; Haneef, R.; Buck, R.H.; Joubert, S.M.

    1987-07-01

    Three monoclonal antibodies were raised against the free alpha subunit of choriogonadotropin (hCG); each recognized a different antigenic site on the molecule. One (antibody 42) preferentially bound to the alpha subunit when it was coupled to the beta subunit as dimeric choriogonadotropin (hCG), thyrotropin (TSH), lutropin (LH), or follitropin (FSH). Antibody 71 showed some cross-reaction with intact FSH; antibody 75 was more specific for the alpha subunit. All were of low affinity (10(-7) to 10(-8) mol/L), but when combined in immunoradiometric assays (IRMAS) they proved to be as sensitive as current radioimmunoassays involving polyclonal antibodies. Advantages of the combination of antibody 75 bound to the solid phase and antibody 71 as the radiolabeled antibody were: detection limit of at least 0.1 micrograms/L; linear dilution of serum and urine; insignificant cross-reaction with intact hCG, allowing direct assay in pregnancy fluids; and a coefficient of variation less than 3% over the reference interval for nonpregnant women. There was 4% cross-reaction with intact FSH, suggesting that the epitopes recognized by nos. 71 and 75 are more exposed in FSH and that perhaps there is less folding in this molecule than in intact hCG.

  1. 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

  2. [Partially automated antigen determination and antibody detection with microtiter plates].

    PubMed

    Rapp, C; Weisshaar, C

    1993-01-01

    In addition to several conventional methods for the detection of red cell antigens, the use of microplates has various advantages either as a solid-phase assay (enzyme immunoassay) or as native microplate. Microplates may also be used for the detection of red cell antibodies in 'pooled-cell solid-phase assays' of the second generation and for antibody screening. Blood donors and patients are the two main fields which are to be examined in immunohematology. There are various advantages in using the microplate in blood group serology: (i) if there is hardware already available, like sample processors and microplate readers, the use of microplates in blood group serology reduces the costs even if the equipment has to be purchased for this purpose only; (ii) low quantities of reagents are used in microplate assays; (iii) the application of bar codes on tubes and microplates guarantees the most security in sample identification; (iv) it is possible to investigate blood samples selectively depending on the available software if antibody detection is done as the sixth test beside anti-HIV, anti-HCV, HBsAG, lues antibodies and ALT, and (v) recording of data will be easy if electronic data processing is used. PMID:7693246

  3. The prevalence of hepatitis B virus E antigen among Ghanaian blood donors

    PubMed Central

    Rufai, Tanko; Mutocheluh, Mohamed; Kwarteng, Kwaku; Dogbe, Elliot

    2014-01-01

    Hepatitis B viral infection is an important clinical problem due to its worldwide distribution and potential of adverse sequelae, including hepatocellular carcinoma (HCC). We studied the prevalence of hepatitis B virus ‘e’ antigen (HBeAg) among individuals determined to be hepatitis B virus (HBV) surface antigen-positive and analyzed the gender/age category associated with more active HBV infection and whether alteration in the levels of alanine aminotransferase could be associated with HBeAg positivity. A total of 150 prospective blood donors who tested positive for hepatitis B surface antigen (HBsAg) at the blood transfusion center of the Komfo Anokye Teaching Hosptital (KATH), Kumasi were randomly selected for the study. The serum samples were further tested for HBsAg and HBeAg using a lateral flow immunochromatographic assay. Twenty (20) individuals were found to be HBeAg-positive giving an overall prevalence of 13.3%, of which 18 (15.5%) were males and 2 (5.9%) were females. Our results also revealed that the prevalence of HBeAg was higher in patients between the age group of 10-20 years and appeared to decrease with increase in age. There was no statistical difference between the HBeAg positive and negative individuals with respect to alanine aminotransferase (ALT) levels. We show for the first time that approximately 1/10 of HBV-infected individuals are HBeAg positive in the Ashanti Region of Ghana, suggestive of active viral replication and liver-cell infectivity thereby contributing to an increased HBV-transmission pool within the Ghanaian population. PMID:25018803

  4. 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

  5. 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. German Clinical Trials Register: DRKS00005274. PMID:27016653

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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. PMID:23473776

  11. 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

  12. Optimization of immune responses induced by therapeutic vaccination with cross-reactive antigens in a humanized hepatitis B surface antigen transgenic mouse model.

    PubMed

    Bourgine, Maryline; Dion, Sarah; Godon, Ophélie; Guillen, Gerardo; Michel, Marie-Louise; Aguilar, Julio Cesar

    2012-08-15

    The absence of relevant animal models of chronic hepatitis B virus (HBV) infection has hampered the evaluation and development of therapeutic HBV vaccines. In this study, we generated a novel transgenic mouse lineage that expresses human class I and II HLA molecules and the hepatitis B surface antigen (HBsAg). HBsAg and hepatitis B core antigen (HBcAg) administered as plasmid DNAs and recombinant proteins, either alone or in combination, were evaluated as therapeutic vaccine candidates in this mouse model. Our results emphasize the importance of the route of administration in breaking HBsAg tolerance. Although immunizing the transgenic mice with DNA encoding homologous HBsAg was sufficient to induce CD8+ T-cell responses, HBsAg from a heterologous subtype was required to induce a CD4+ T-cell response. Importantly, only prime-boost immunization protocols that combined plasmid DNA injection followed by protein injection induced the production of antibodies against the HBsAg expressed by the transgenic mice. PMID:22591777

  13. 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

  14. 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

  15. Immunization of babies born to HBsAg positive mothers: An audit on the delivery and completeness of follow up in Norfolk and Suffolk, United Kingdom.

    PubMed

    Keeble, Stuart; Quested, Jane; Barker, Deborah; Varadarajan, Abina; Shankar, Ananda Giri

    2015-01-01

    Perinatal transmission of hepatitis B infection has increased in the UK over the last decade. Routine antenatal screening of pregnant mothers (based on HBsAg) provides an effective means to identify 'at risk' babies. Follow up of babies born to infected mothers involves 4 doses of vaccination and/or a single dose of HBIG at birth. In this study we report the outcome of follow up of babies born to infected mothers over a 5 y period. One hundred sixty-three babies born to HBsAg positive mothers were followed up to ascertain the completeness for immunization and serological testing. Vaccination completion was 99.4% (162 of babies) at birth (1st dose), 95.6% (152 babies) for the second dose (at 1st month), 94.3 % (148 babies) for the 3rd dose (at 2nd month) and 83.4% (106 babies) for the 4th dose (at 12 months). Additionally, at 12 months 29.9% (38) of babies had their blood tested serologically to ascertain infection status; all babies receiving antigen testing were HBsAg negative. The overall vaccination coverage was good, although there is scope to improve the coverage of 4th dose. However, the proportion of children who were serologically tested for surface antigen at 12 months was considerably lower and there is a greater need to test babies concurrently at the time of giving the 4(th) dose. The proposed dried blood spot testing which will be rolled out from September 2014 should address this issue. PMID:25876072

  16. Serological analysis by enzyme-linked immunosorbent assay using recombinant antigen LipL32 for the diagnosis of swine leptospirosis.

    PubMed

    Hartleben, Cláudia P; Leal, Fernanda M A; Monte, Leonardo G; Hartwig, Daiane D; Seixas, Fabiana K; Vasconcellos, Sílvio A; Brihuega, Bibiana; Dellagostin, Odir A

    2013-02-01

    Leptospirosis is an important global zoonotic disease caused by pathogenic Leptospira spp. species. Swine leptospirosis has a major economic impact because pigs are sources of animal protein and by-products. The signs of swine leptospirosis are abortion, stillbirth, birth of weak or ill piglets, appearing 14-60 days after infection. The reference method for diagnosis of leptospirosis is the microscopic agglutination test (MAT), in which serum samples are reacted with live antigen suspensions of leptospiral serovars. However, MAT is laborious and time consuming as a diagnostic procedure when dealing with a large number of samples; therefore, efforts are being made to develop novel, sensitive, and specific diagnostic tests for leptospirosis. In this study, a recombinant LipL32 based on enzyme-linked immunosorbent assay (rLipL32/ELISA) was evaluated as a screening test for the detection of pathogenic leptospiral-specific antibodies. A total of 86 swine serum samples tested by MAT were used to develop rLipL32/ELISA. Compared to positive and negative sera tested by MAT, rLipL32/ELISA showed 100 % sensitivity, 85.1 % specificity, and 91.86 % accuracy. No positive reaction for other bacterial diseases (enzootic pneumonia and brucellosis) was observed. The rLipL32/ELISA reported in this study is a specific, sensitive, and convenient test for the detection of antibodies against swine leptospiral infection and can be used as a rapid screening test in epidemiological surveys. PMID:23064970

  17. A Five-Country Evaluation of a Point-of-Care Circulating Cathodic Antigen Urine Assay for the Prevalence of Schistosoma mansoni

    PubMed Central

    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

    2013-01-01

    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. PMID:23339198

  18. Evaluation of an O antigen enzyme-linked immunosorbent assay for screening of milk samples for Salmonella dublin infection in dairy herds.

    PubMed Central

    Hoorfar, J; Lind, P; Bitsch, V

    1995-01-01

    Levels of antibodies to the O antigens (O:1,9,12) of Salmonella dublin were tested in 1355 serum, 1143 cow milk and 160 bulk milk samples from dairy herds using an enzyme-linked immunosorbent assay (ELISA). In order to define the background reaction, milk samples from all lactating cows and serum samples from 9 animals were collected in each of 20 salmonellosis-free herds located on the island of Bornholm, where cattle salmonellosis has not been reported. Similar samples were collected from all stalled animals in 10 herds with recent (< 6 months) outbreaks of salmonellosis located in Jutland, where salmonella infection is enzootic. Using herd history of salmonellosis, herd location and clinical status of the herds as criteria, the optimal cutoff in the milk ELISA was determined as being at least 5% of the samples having optical density > 0.5, resulting in herd sensitivity of 1.0 and herd specificity of 0.95. While none of the sera in the herds from Bornholm was ELISA positive, 2 herds had a few reactors in the milk ELISA. Using the same cutoff, all but 1 bulk milk sample from 150 herds on Bornholm was ELISA-negative, and all 10 salmonellosis-positive herds from Jutland were ELISA-positive. A significant correlation was found between ELISA reactions in milk and in serum of cows (34% and 32% respectively, rs = 0.69, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7648527

  19. Human Cytokine Genetic Variants Associated With HBsAg Reverse Seroconversion in Rituximab-Treated Non-Hodgkin Lymphoma Patients

    PubMed Central

    Hsiao, Liang-Tsai; Wang, Hao-Yuan; Yang, Ching-Fen; Chiou, Tzeon-Jye; Gau, Jyh-Pyng; Yu, Yuan-Bin; Liu, Hsiao-Ling; Chang, Wen-Chun; Chen, Po-Min; Tzeng, Cheng-Hwai; Chan, Yu-Jiun; Yang, Muh-Hwa; Liu, Jin-Hwang; Huang, Yi-Hsiang

    2016-01-01

    Abstract Hepatitis B virus (HBV) reactivation has been noted in HBV surface antigen (HBsAg)-seronegative patients with CD20+ B-cell non-Hodgkin lymphoma (NHL) undergoing rituximab treatment. Clinically, hepatitis flares are usually associated with the reappearance of HBsAg (reverse seroconversion of HBsAg, HBV-RS). It is unclear whether human genetic factors are related to rituximab-associated HBV reactivation. Unvaccinated HBsAg-seronegative adults (n = 104) with CD20+ NHL who had received rituximab-containing therapy without anti-HBV prophylaxis were enrolled. Eighty-nine candidate single nucleotide polymorphisms (SNPs) of 49 human cytokine genes were chosen and were analyzed using the iPLEX technique. Competing risk regression was used to identify the factors associated with HBV-RS. Participants had a median age of 66.1 years and 56.7% were male (n = 59). The anti-HBs and anti-HBc positivity rates were 82.4% and 94.1%, respectively, among patients for whom data were available (approximately 81%). A mean of 7.14 cycles of rituximab therapy were administered, and a total of 14 (13.4%) patients developed HBV-RS. Nine SNPs showed significant differences in frequency between patients with or without HBV-RS: CD40 rs1883832, IL4 rs2243248 and rs2243263, IL13 rs1295686, IL18 rs243908, IL20 rs1518108, and TNFSF13B rs12428930 and rs12583006. Multivariate analysis showed that ≥6 cycles of rituximab therapy, IL18 rs243908, and the IL4 haplotype rs2243248∼rs2243263 were independently associated with HBV-RS. The IL4 haplotype rs2243248∼rs2243263 was significantly associated with HBV-RS regardless of anti-HBs status. Polymorphisms in human cytokine genes impact the risk of rituximab-associated HBV-RS. PMID:26986131

  20. Overexpression of α2,3sialyl T-antigen in breast cancer determined by miniaturized glycosyltransferase assays and confirmed using tissue microarray immunohistochemical analysis

    PubMed Central

    Patil, Shilpa A.; Bshara, Wiam; Morrison, Carl; Chandrasekaran, E. V.; Matta, Khushi L.; Neelamegham, Sriram

    2014-01-01

    Glycan structure alterations during cancer regulate disease progression and represent clinical biomarkers. The study determined the degree to which changes in glycosyl transferase activities during cancer can be related to aberrant cell-surface tumor associated carbohydrate structures (TACA). To this end, changes in sialyltransferase (sialylT), fucosyltransferase (fucT) and galactosyltransferase (galT) activity were measured in normal and tumor tissue using a miniaturized enzyme activity assay and synthetic glycoconjugates bearing terminal LacNAc Type-I (Galβ1,3GlcNAc), LacNAc Type-II (Galβ1,4GlcNAc), and mucin core-1/Type-III (Galβ1,3GalNAc) structures. These data were related to TACA using tissue microarrays containing 115 breast and 26 colon cancer specimen. The results show that primary human breast and colon tumors, but not adjacent normal tissue, express elevated β1,3 galT and α2,3sialylT activity that can form α2,3sialylated Type-III glycans (Siaα2,3Galβ1,3GalNAc). Prostate tumors did not exhibit such elevated enzymatic activities. α1,3/4fucT activity was higher in breast, but not colon tissue. The enzymology based prediction of enhanced α2,3sialylated Type-III structures in breast tumors was verified using histochemical analysis of tissue sections and tissue microarrays. Here, the binding of two markers that recognize Galβ1,3GalNAc (peanut lectin and mAb A78-G/A7) was elevated in breast tumor, but not normal control, only upon sialidase treatment. These antigens were also upregulated in colon tumors though to a lesser extent. α2,3sialylated Type-III expression correlated inversely with patient HER2 expression and breast metastatic potential. Overall, enzymology measurements of glycoT activity predict glycan structure changes during cancer. High expression of the α2,3sialylated T-antigen O-glycans occur in breast tumors. A transformation from linear core-1 glycan to other epitopes may accompany metastasis. PMID:25142811

  1. A novel 57-kDa merozoite protein of Babesia gibsoni is a prospective antigen for diagnosis and serosurvey of canine babesiosis by enzyme-linked immunosorbent assay.

    PubMed

    Aboge, Gabriel Oluga; Jia, Honglin; Terkawi, Mohamad Alaa; Goo, Younkyoung; Kuriki, Ken; Nishikawa, Yoshifumi; Igarashi, Ikuo; Suzuki, Hiroshi; Xuan, Xuenan

    2007-10-21

    We isolated a novel single copy gene encoding a 57-kDa merozoite protein of Babesia gibsoni (BgP57). The nucleotide sequence of the cDNA was 2387 bp with an open reading frame (ORF) of 1644 bp encoding a 57-kDa predicted polypeptide having 547 amino acid residues. The recombinant BgP57 (rBgP57) without a predicted signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein. Western blotting showed that the corresponding native protein was 57-kDa, consistent with molecular weight of predicted mature polypeptide. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP57 detected specific antibodies in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with antibodies to B. canis sub-species and closely related apicomplexan parasites indicating that the rBgP57 was a specific antigen for B. gibsoni antibodies. The diagnostic performance of ELISA based on rBgP57 using 107 sera from B. gibsoni-naturally infected dogs was the same as the previously identified rBgP32 but performed better than the previously studied rBgP50. Although, seminested PCR detected higher proportions (82%) of positive samples than the ELISAs, the Mcnemar's chi-square test showed that there was no significant difference in relative effectiveness of rBgP57-ELISA and seminested PCR (chi(2)=2.70; P=0.1003) in identifying positive samples. The rBgP57-ELISA when used in combination with rBgP32-ELISA and rBgP50-ELISA appeared to improve sensitivity of the rBgP57-ELISA for detection of B. gibsoni antibodies. Overall, the rBgP57-ELISA and seminested PCR when used in combination, could improve epidemiological surveys and clinical diagnosis of B. gibsoni infection. PMID:17706873

  2. Characterization of hepatitis B virus surface antigen variability and impact on HBs antigen clearance under nucleos(t)ide analogue therapy.

    PubMed

    Velay, A; Jeulin, H; Eschlimann, M; Malvé, B; Goehringer, F; Bensenane, M; Frippiat, J-P; Abraham, P; Ismail, A M; Murray, J M; Combet, C; Zoulim, F; Bronowicki, J-P; Schvoerer, E

    2016-05-01

    For hepatitis B virus (HBV)-related chronic infection under treatment by nucleos(t)ide analogues (NUCs), HBsAg clearance is the ultimate therapeutic goal but very infrequent. We investigated how HBV envelope protein variability could lead to differential HBsAg clearance on NUCs. For 12 HBV genotype D patients receiving NUCs, six resolvers (HBsAg clearance) were compared to six matched nonresolvers (HBsAg persistence). PreS/S amino acid (aa) sequences were analysed with bioinformatics to predict HBV envelope antigenicity and aa covariance. To enrich our analyses on very rare resolvers, these were compared with other HBV genotype D strains in three characterized clinical cohorts including common chronically infected patients. The sT125M+sP127T combination was observed in four nonresolvers of six, corroborated by aa covariance analysis, associated with a lower predicted antigenicity than sT125T+sP127P. Concordant features within this HBV key functional domain, at positions 125 and 127, were reported from two of the three comparative cohorts. In our hands, a lower ELISA reactivity of HBV-vaccinated mice sera was observed against the sT125M mutant. In the S gene, 56 aa changes in minor variants were detected in non-resolvers, mainly in the major hydrophilic region, vs 28 aa changes in resolvers. Molecular features in patients showing HBsAg persistence on NUCs argue in favour of a different aa pattern in the HBV S gene compared to those showing HBsAg clearance. In nonresolvers, a decrease in HBs 'a' determinant antigenicity and more frequent mutations in the S gene suggest a role for the HBV envelope characteristics in HBsAg persistence. PMID:26742490

  3. Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen.

    PubMed

    Nourani, Sara; Ghourchian, Hedayatollah; Boutorabi, Seyed Mehdi

    2013-10-01

    An electrochemical immunosensor was developed for the detection of hepatitis B surface antigen (HBsAg). The biotinylated hepatitis B surface antibody was immobilized on streptavidin magnetic nanoparticles and used for targeting the HBsAg. By the addition of horseradish peroxidase conjugated with secondary antibody (HRP-HBsAb), a sandwich-type immunoassay format was formed. Aminophenol as substrate for conjugated HRP was enzymatically changed into 3-aminophenoxazone (3-APZ). This electroactive enzymatic production (3-APZ) was transferred into an electrochemical cell and monitored by cyclic voltammetry. Under optimal conditions, the cathodic current response of 3-APZ, which was proportional to the HBsAg concentration, was measured by a glassy carbon electrode. The immunosensor response was linear toward HBsAg in the concentration range from 0.001 to 0.015 ng/ml with a detection limit of 0.9 pg/ml at a signal/noise ratio of 3. PMID:23831477

  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. 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

  6. 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

  7. Hepatitis B surface antigen positive skin lesions. Two case reports with an immunoperoxidase study.

    PubMed

    Rosen, L B; Rywlin, A M; Resnick, L

    1985-12-01

    This study represents the first two case reports of skin lesions positive for hepatitis B surface antigen (HBsAg) with the immunoperoxidase technique. A 25-year-old man and a 64-year-old woman with serologic evidence of acute B viral hepatitis and concurrent skin lesions are presented. Immunoperoxidase study of the skin lesions for HBsAg revealed strong positive staining of squamous epidermal cells, eccrine sweat glands, and endothelial cells in the superficial papillary dermis. Immunoperoxidase staining for hepatitis B core antigen (HBcAg) was negative in both cases. Electron microscopy failed to reveal viral particles. PMID:3911798

  8. 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. PMID:24647111

  9. 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

  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. 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

  12. Double-Antigen Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis E Virus-Specific Antibodies in Human or Swine Sera ▿

    PubMed Central

    Hu, Wei Ping; Lu, Yang; Precioso, Nestor Amadeo; Chen, Hsiao Ying; Howard, Teresa; Anderson, David; Guan, Ming

    2008-01-01

    A new double-antigen sandwich-based enzyme-linked immunosorbent assay (ELISA) for the detection of total antibodies (immunoglobulin G [IgG] and IgM) specific for hepatitis E virus (HEV) was developed by utilizing well-characterized recombinant protein ET2.1 and its peroxidase-labeled counterpart. Our study showed that the ELISA detected all the positive patient samples (n = 265) regardless of whether they contained IgM or IgG antibodies, or both, while it maintained an excellent specificity of 98.8% with samples from various patient or healthy control groups (total number of samples, 424). The test had a detection limit for anti-HEV IgG antibodies that was equivalent to 62 mIU/ml of the international reference. Compared with the serological status of the specimens determined on the basis of tests performed at the individual collection sites, the testing outcome generated by the new ELISA had a good agreement of 99.3%, with a kappa value of 0.985. The positive predictive value and the negative predictive value for the new test reached 98.1% and 100%, respectively. This ELISA had a positive delta value of 4.836 and a negative delta value of 3.314 (where delta is a measure of the number of standard deviations by which the cutoff is separated from the mean of the sample groups) (N. Crofts, W. Maskill, and I. D. Gust, J. Virol. Methods 22:51-59, 1988), indicating that it had an excellent ability to differentiate the infected and noninfected cohorts. Furthermore, the new design enables the detection of antibodies not only in human samples but also in pig samples. Our preliminary data showed that the ELISA could detect seroconversion in samples from pigs at as early as 14 days postinoculation. The potential utility of detecting specific antibodies in pigs will be an added advantage for managing the disease, with suggested zoonotic implications. PMID:18495846

  13. AhpC, AhpD, and a secreted 14-kilodalton antigen from Mycobacterium avium subsp. paratuberculosis distinguish between paratuberculosis and bovine tuberculosis in an enzyme-linked immunosorbent assay.

    PubMed

    Olsen, I; Tryland, M; Wiker, H G; Reitan, L J

    2001-07-01

    Sera from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis (n = 56) and naturally (n = 4) and experimentally (n = 8) infected with Mycobacterium bovis were tested for the presence of antibodies against paratuberculosis antigens. An enzyme-linked immunosorbent assay (ELISA) was established based on absorption of M. avium subsp. paratuberculosis antigens on a hyperimmune antiserum against M. avium subsp. avium proteins in order to remove cross-reacting antigens. This absorbed-antigen ELISA recognized 66% of animals with paratuberculosis (37 of 56), while none of the animals with naturally occurring bovine tuberculosis (TB) had detectable antibodies. However, the animals with experimental bovine TB also responded in this ELISA. Similar results were found in a commercial ELISA, showing that neither of these tests was able to distinguish between paratuberculosis and bovine TB. The sera were further tested for antibody activities against purified AhpC and AhpD, which are proteins constitutively expressed by M. avium subsp. paratuberculosis, and against a secreted 14-kDa protein present in culture filtrates from the M. avium complex. Elevated antibody levels to AhpC, AhpD, and the 14-kDa antigen were found in 27% (13 of 48), 15% (7 of 48), and 27% (13 of 48), respectively, of the cattle with paratuberculosis. Together these ELISAs were positive with 35% (17 of 48) of the animals. None of the animals with bovine TB had detectable antibodies against any of the purified proteins despite their high levels of cross-reacting antibodies. These results show that purified specific antigens are needed to differentiate between paratuberculosis and bovine TB in ELISA. PMID:11427429

  14. 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 tissue 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 methodology is chosen ...

  15. 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...

  16. Hepatitis B surface antigen levels of cessation of nucleos(t)ide analogs associated with virological relapse in hepatitis B surface antigen-negative chronic hepatitis B patients

    PubMed Central

    Ge, Guo-Hong; Ye, Yun; Zhou, Xin-Bei; Chen, Li; He, Cong; Wen, Dan-Feng; Tan, You-Wen

    2015-01-01

    AIM: To investigate the virological relapse rate in hepatitis B e antigen (HBeAg)-negative patients after antiviral therapy discontinuation and analyze the factors associated with virological relapse. METHODS: Among patients diagnosed with chronic hepatitis B infection between May 2005 and July 2010, 204 were eligible for analysis. The Kaplan-Meier method and log-rank test were used to calculate the cumulative rate of relapse and compare cumulative relapse rates between groups. The Cox proportional hazards regression model was used to evaluate the predictive factor of virological relapse. RESULTS: The 2 and 1 year cumulative risks of virological relapse after antiviral therapy discontinuation were 79.41% (162/204) and 43.82% (71/162), respectively. Multivariate analysis revealed that only post treatment hepatitis B surface antigen (HBsAg) level was associated with virological relapse (P = 0.011). The cumulative risk of virological relapse was higher in the patients with HBsAg levels ≥ 1500 IU/L than in those with HBsAg levels < 1500 IU/L (P = 0.0013). The area under the curve was 0.603 (P = 0.033). The cutoff HBsAg value for predicting virological relapse was 1443 IU/L. CONCLUSION: We found that the virological relapse rate remained high after antiviral therapy discontinuation in the HBeAg-negative patients and that the post treatment HBsAg levels predicted virological relapse. PMID:26229407

  17. Usefulness of a new immunoradiometric assay of HCV core antigen to predict virological response during PEG-IFN/RBV combination therapy for chronic hepatitis with high viral load of serum HCV RNA genotype 1b.

    PubMed

    Sasase, Noriko; Kim, Soo Ryang; Kim, Ke Ih; Taniguchi, Miyuki; Imoto, Susumu; Mita, Keiji; Hotta, Hak; Shouji, Ikuo; El-Shamy, Ahmed; Kawada, Norifumi; Kudo, Masatoshi; Hayashi, Yoshitake

    2008-01-01

    We investigated the clinical usefulness of a new immunoradiometric (IRM) assay of hepatitis C virus (HCV) core antigen in predicting virological response during pegylated interferon plus ribavirin (PEG-IFN/RBV) combination therapy for chronic hepatitis with high viral loads of serum HCV RNA genotype 1b. Thirty-nine patients received a regimen of PEG-IFNalpha-2b (1.5 microg/kg/week s.c.) in combination with RBV (600-1,000 mg/day). Of the 39 patients, 18 (46.2%) achieved sustained virological response (SVR), 11 (28.2%) attained partial response (PR) and 10 (25.6%) showed no response (NR). Four weeks after the start of therapy, 1- and 2-log reductions in the amount of HCV core antigen were observed in 20 (2/10) and 0% (0/10) showing NR, 91 (10/11) and 63.6% (7/11) with PRs, and 88.9 (16/18) and 55.6% (10/18) of patients with SVR, respectively. The 1- and 2-log reductions 4 weeks after the start of therapy were not a defining condition for PR and SVR. The amount of HCV core antigen was significantly different between SVR and PR patients on days 1 and 7, and between patients with NR and SVR at all points of time. In conclusion, this new IRM assay is useful in predicting virological response during PEG-IFN/RBV therapy. PMID:18544951

  18. 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. PMID:24148389

  19. Human Leukocyte Antigens Influence the Antibody Response to Hepatitis B Vaccine.

    PubMed

    Jafarzadeh, Abdollah; Bagheri-Jamebozorgi, Masoome; Nemati, Maryam; Golsaz-Shirazi, Forough; Shokri, Fazel

    2015-06-01

    Hepatitis B virus (HBV) infection and its sequelae such as cirrhosis and hepatocellular carcinoma has remained a serious public health problem throughout the world. The WHO strategy for effective control of HBV infection and its complications is mass vaccination of neonates and children within the framework of Expanded Programme on Immunization (EPI). Vaccination with hepatitis B surface antigen (HBsAg) induces protective antibody response (anti-HBs ≥ 10 IU/L) in 90-99% of vaccinees. The lack of response to HBsAg has been attributed to a variety of immunological mechanisms, including defect in antigen presentation, defect in HBsAg-specific T and/or B cell repertoires, T-cell suppression, increase in the regulatory T cell count, lack of necessary help of T-cells for production of anti-HBs by B cells, defect in Th1 and/or Th2 cytokine production and selective killing of HBsAg-specific B-cells by human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes. The HLA complex plays an important role in many of these immunological processes. A variety of HLA class I, II, and III alleles and antigens have been reported to be associated with antibody response to HBsAg vaccination in different ethnic populations. Moreover, some HLA haplotypes were also associated with responsiveness to HBsAg. In this review the association of the HLA specificities with antibody response to hepatitis B (HB) vaccine is discussed. PMID:26546891

  20. Prevalence of HBsAg and high-risk behaviors in pregnant women rerring to Urban Health Centers in Isfahan province

    PubMed Central

    Ahmadi, Mahboubeh; Toghyani, Raheleh; Shahidi, Shahla; Izadi, Minoo; Merasi, Mohammad Reza; Agdak, Pejman; Meshkaty, Marjan; Nikkhahfard, Mojghan

    2011-01-01

    BACKGROUND: About 5% of the world population are carriers of the hepatitis B virus which is not the same in different areas of the world. Iran, with a rate of 2-3%, is among the countries with average prevalence. The aim of this study was to determine the prevalence of HBsAg and its associated factors in pregnant women who referred to urban health centers in Isfahan Province. METHODS: This was a descriptive study conducted on 1078 pregnant women who had referred to the urban health centers of Isfahan, Borkhar, Meymeh, Khomeini Shahr, Lenjan and Najaf Abad in 2009 in order to register their physical condition and receive pregnancy care. Random sampling method by quota was done. First, a questionnaire including demographic characteristics and history of high-risk behaviors in mothers and their husbands was completed. Then, a blood sample was taken and evaluated for the HB virus surface antigen. Finally, the data was analyzed using SPSS software, Chi-square, Fisher and Logistic Regression tests. RESULTS: HB virus surface antigen was traced in the serum of 0.5% of the participants. The average age of subjects was 26.1± 4.9 (mean±sd) years old and the average pregnancy number was 1.79 + 1.0 (mean±sd). Using Fisher's test as well as logistic regression test and based on the previous history of high-risk sexual behaviors and tattooing, a significant difference was observed between the two groups with positive and negative HBsAg (p ≤ 0.05). CONCLUSIONS: Instructing the target groups and close monitoring of the high-risk centers such as beauty shops, tattooing centers and etc, and also staff training on how to use disposable and sterilized equipments have to be accomplished at the right time. PMID:22039379

  1. 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

  2. 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

  3. Alkylglycerols Modulate the Proliferation and Differentiation of Non-Specific Agonist and Specific Antigen-Stimulated Splenic Lymphocytes

    PubMed Central

    Qian, Linxi; Zhang, Mingshun; Wu, Shengmei; Zhong, Yan; Van Tol, Eric; Cai, Wei

    2014-01-01

    Alkylglycerols (AKGs) are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed that oral AKGs administration significantly increased the immune response in mice. The aim of the present study was to investigate the in vitro immunomodulatory effect of AKGs on stimulating splenic lymphocyte responses. C57BL/6 mice were immunized with hepatitis B surface antigen (HBsAg). Splenic B cells were purified and stimulated with anti-BCR and anti-CD38. Meanwhile, splenic CD4+ T cells were purified and stimulated with anti-CD3 and anti-CD28. For antigen specific stimulation, the purified CD4+ T cells were cocultured with HBsAg -pulsed dendritic cells. The stimulated lymphocytes were treated with different concentrations of AKGs. The cell proliferation was assessed by [3H]-thymidine incorporation assay. The maturation of B cells was assessed by examining the germline (GL) transcription of IgG (γ1) mRNA expression, and the surface expressions of CD80/CD86 markers were examined by flow cytometry analysis. Th1/Th2 polarity was assessed by T-BET (Th1)/GATA-3 (Th2) flow cytometry assay and by characteristic cytokines ELISA assay (TNF-α and IFN-γ for Th1; IL-4 and IL-10 for Th2). It was found that AKGs significantly increased the BCR/CD38 -stimulated B cell proliferation. The T cell proliferation in response to CD3/CD28 or specific antigen stimulation was also increased by AKGs. The transcriptional level of IgG (γ1) and the expressions of CD80/CD86 molecules were markedly increased by AKGs in BCR/CD38 -stimulated B cells. Meanwhile, the results showed that AKGs increased the expression of T-BET transcriptional factor and the production of Th1 cytokines (TNF-α and IFN-γ) upon CD3/CD28 stimulation; whereas, levels of Th2 cytokines (IL-4 and IL-10) were decreased by AKGs. Our study demonstrated that AKGs can modulate immune responses by boosting the proliferation and maturation of murine lymphocytes in vitro. PMID

  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. Detection of endocervical anti-Chlamydia trachomatis immunoglobulin A in pregnant women by a rapid, 6-minute enzyme-linked immunosorbent assay: comparison with PCR and chlamydial antigen detection methods.

    PubMed Central

    Witkin, S S; Bongiovanni, A M; Inglis, S R

    1997-01-01

    There is a need for a rapid, uncomplicated, and inexpensive test for Chlamydia trachomatis infection in women. We evaluated the ability of a 6-min enzyme-linked immunosorbent assay (ELISA) that requires no laboratory equipment (IgA Rapid SeroTest; Savyon Diagnostics) to detect C. trachomatis immunoglobulin A (IgA) in the endocervices of 167 inner-city pregnant women and compared the results with DNA amplification (Amplicor PCR; Roche Diagnostics) and antigen detection (Chlamydiazyme; Abbott Laboratories) performed on the same women. Anti-C. trachomatis IgA was detected in the cervices of 32 women (19.2%). Samples from 23 women (13.8%) were PCR positive, while chlamydial antigen was present in 20 women (12.0%). There was only 1 sample (4.3%) that was positive by PCR but negative by ELISA; 10 samples were ELISA positive and PCR negative. In contrast, seven samples (30.4%) were PCR positive but Chlamydiazyme negative and four were Chlamydiazyme positive and PCR negative. Compared to PCR, the IgA ELISA had a sensitivity of 95.7%, a specificity of 93.1%, a positive predictive value of 68.8%, and a negative predictive value of 99.3%. The antigen assay had a sensitivity of only 69.6%, a specificity of 97.2%, a positive predictive value of 80.0%, and a negative predictive value of 95.2%. In high-risk groups where laboratory testing is not available, or where the patient might not return to obtain her testing result and be treated, the Rapid IgA SeroTest is a viable alternative for detection of cervical C. trachomatis in pregnant women. PMID:9196193

  6. 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

  7. Application of monoclonal antibodies in a rapid sandwich dot-enzyme linked immunosorbent assay for identification and antigen detection of Leptospira serovars.

    PubMed

    Sharma, Rashmi; Tuteja, Urmil; Khushiramani, Rekha; Shukla, Jyoti; Batra, H V

    2008-04-01

    Monoclonal antibodies (MAbs) were produced by fusing SP2/0 myeloma cells with spleen cells of BALB/c mice that were immunized with live whole cells of the four most prevalent Leptospira serovars--namely, autumnalis, australis, grippotyphosa, and icterohaemorrhagiae. A total of 26 MAbs (10 autumnalis, 5 australis, 4 grippotyphosa, and 7 icterohaemorrhagiae) were produced that showed specific, restricted, or broad cross-reactivity when tested with 19 standard pathogenic and 3 standard saprophytic serovars by MAT and dot-ELISA. Monoclonal antibodies like AT4 and AT5 against serovar autumnalis; AS1 and AS2 against serovar australis; GR1, GR3, and GR4 raised against serovar grippotyphosa; and also the MAbs IC3 to IC7 against serovar icterohaemorrhagiae were all usable as typing reagents in a rapid dot-ELISA. Selected MAbs were subsequently utilized in a rapid sandwich dot-ELISA for identification of Leptospira serovars as well as for antigen detection in experimentally infected mice and guinea pigs. Results of rapid sandwich dot-ELISA were compared with dark field microscopy, culture, and PCR in experimentally infected animals and sandwich dot-ELISA detected the presence of Leptospira antigen during the bacteremia stage in all experimental animals. Besides detecting antigens in animals infected with homologous serovars, the sandwich dot-ELISA employing pooled capture and revealing antibodies also detected Leptospira in the group of animals infected separately with the serovars australis and icterohaemorrhagiae. Results showed PCR to be a reliable and rapid test for demonstration of Leptospira in the plasma samples. The rapid sandwich dot-ELISA appeared more advantageous over PCR in being simple, rapid, field based, and economical. This method shows better promise of being used as a bedside test for routine diagnostic purposes. PMID:18642676

  8. Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

    PubMed Central

    2012-01-01

    Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing. PMID:23039947

  9. Design and expression of fusion protein consists of HBsAg and Polyepitope of HCV as an HCV potential vaccine

    PubMed Central

    Gholizadeh, Monireh; Khanahmad, Hossein; Memarnejadian, Arash; Aghasadeghi, Mohammad Reza; Roohvand, Farzin; Sadat, Seyed Mehdi; Cohan, Reza Ahangari; Nazemi, Ali; Motevalli, Fatemeh; Asgary, Vahid; Arezumand, Roghaye

    2015-01-01

    Background: Hepatitis C virus (HCV) infection is a serious public health threat worldwide. Cellular immune responses, especially cytotoxic T-lymphocytes (CTLs), play a critical role in immune response toward the HCV clearance. Since polytope vaccines have the ability to stimulate the cellular immunity, a recombinant fusion protein was developed in this study. Materials and Methods: The designed fusion protein is composed of hepatitis B surface antigen (HBsAg), as an immunocarrier, fused to an HCV polytope sequence. The polytope containing five immunogenic epitopes of HCV was designed to induce specific CTL responses. The construct was cloned into the pET-28a, and its expression was investigated in BL21 (DE3), BL21 pLysS, BL21 pLysE, and BL21 AI Escherichia coli strains using 12% gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the identity of expressed fusion protein was confirmed by Western blotting using anti-His monoclonal antibody and affinity chromatography was applied to purify the expressed protein. Results: The accuracy of the construct was confirmed by restriction map analysis and sequencing. The transformation of the construct into the BL21 (DE3), pLysS, and pLysE E. coli strains did not lead to any expression. The fusion protein was found to be toxic for E. coli DE3. By applying two steps inhibition, the fusion protein was successfully expressed in BL21 (AI) E. coli strain. Conclusion: The HBsAg-polytope fusion protein expressed in this study can be further evaluated for its immunogenicity in animal models. PMID:26682209

  10. 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

  11. Helicase Assays

    PubMed Central

    Wang, Xin; Li, Jing; Diaz, Jason; You, Jianxin

    2016-01-01

    Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.

  12. 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...

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. Treponema pallidum hemagglutination assay seroreactivity among healthy Indian donors and its association with other transfusion transmitted diseases

    PubMed Central

    Pahuja, Sangeeta; Gupta, Santosh Kumar; Pujani, Mukta; Jain, Manjula

    2014-01-01

    Background: The aim of the present study was to determine the prevalence of syphilis infection by Treponema pallidum hemagglutination assay (TPHA) among blood donors in Delhi and to study their correlation with other markers of transfusion transmitted infections such as hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B surface antigen (HBsAg) so as to establish the utility of TPHA over and above venereal diseases research laboratory test (VDRL), not only as a marker for testing T. pallidum infection, but also as a marker of high risk behavior. Materials and Methods: This prospective study was carried out in the Regional Blood Transfusion Centre, Lady Hardinge Medical College and associated Sucheta Kriplani Hospital, New Delhi for a period of 2 years. Donated blood was screened for TPHA seroreactivity along with screening for anti HIV I and II, anti-HCV, HBsAg by third generation enzyme-linked immunosorbent assay test. A total of 8082 serum samples of blood donors were collected from healthy blood donors in our blood bank. They were classified into two groups- test group and control group based on TPHA positivity. The co-occurrence of HBsAg, HIV and HCV infection were determined in TPHA positive blood donors (test group) in comparison with TPHA negative blood donors (control group). Results: We found the TPHA seroreactivity to be 4.4% in Delhi's blood donors. Nearly 8.2% (663/8082) of the donated blood had serological evidence of infection by at least one pathogen (syphilis/HIV/hepatitis B virus/HCV) and 6.63% (44/663) donors with positive serology had multiple infections (two or more). Quadruple infection was seen in one donor, triple infection was seen in three donors and double infection was seen in 40 donors. Prevalence of HIV seroreactivity was found to be statistically significant and HCV seroreactivity statistically insignificant in TPHA positive group in comparison to TPHA negative group. Discussion: In our study, the TPHA

  19. Development and clinical validation of a real-time PCR assay for PITX2 DNA methylation to predict prostate-specific antigen recurrence in prostate cancer patients following radical prostatectomy.

    PubMed

    Dietrich, Dimo; Hasinger, Oliver; Bañez, Lionel L; Sun, Leon; van Leenders, Geert J; Wheeler, Thomas M; Bangma, Chris H; Wernert, Nicolas; Perner, Sven; Freedland, Stephen J; Corman, John M; Ittmann, Michael M; Lark, Amy L; Madden, John F; Hartmann, Arndt; Schatz, Philipp; Kristiansen, Glen

    2013-03-01

    Prostate cancer is the most common cancer among men. The prospective discrimination of aggressive and clinically insignificant tumors still poses a significant and, as yet, unsolved problem. PITX2 DNA methylation is a strong prognostic biomarker in prostate cancer. Recently, a diagnostic microarray for prostate cancer prognosis based on PITX2 methylation has been developed and validated. Because this microarray requires nonstandard laboratory equipment, its use in a diagnostic setting is limited. This study aimed to develop and validate an alternative quantitative real-time PCR assay for measuring PITX2 methylation that can easily be established in clinical laboratories, thereby facilitating the implementation of this biomarker in clinical practice. A methylation cut-off for patient stratification was established in a training cohort (n = 157) and validated in an independent test set (n = 523) of men treated with radical prostatectomy. In univariate Cox proportional hazards analysis, PITX2 hypermethylation was a significant predictor for biochemical recurrence (P < 0.001, hazard ratio = 2.614). Moreover, PITX2 hypermethylation added significant prognostic information (P = 0.003, hazard ratio = 1.814) to the Gleason score, pathological T stage, prostate-specific antigen, and surgical margins in a multivariate analysis. The clinical performance was particularly high in patients at intermediate risk (Gleason score of 7) and in samples containing high tumor cell content. This assay might aid in risk stratification and support the decision-making process when determining whether a patient might benefit from adjuvant treatment after radical prostatectomy. PMID:23266319

  20. Human platelet antigen 1 (HPA 1) genotyping with 5' nuclease assay and sequence-specific primers reveals a single nucleotide deletion in intron 2 of the HPA 1a allele of platelet glycoprotein IIIa.

    PubMed

    Kjaer, Killie Mette; Jaegtvik, Sissel; Husebekk, Anne; Skogen, Bjorn

    2002-05-01

    We have established a 5' nuclease assay (5' NA) for human platelet antigen (HPA) 1a/b allelic discrimination. The assay is based on the simultaneous amplification and detection of the two targets in a one-tube system. The results are read optically, immediately after termination of the polymerase chain reaction (PCR), and no post-PCR processing is necessary. This genotyping procedure is less time-consuming and cheaper than our conventional sequence-specific primer PCR (SSP-PCR), which is run as a two-tube test, with verification of the results after electrophoresis in agarose gel. The reduction of analytical steps, simplification of the procedure and potential for automation were important advantages for our choice of system. This test system is more suitable for large-scale testing and fits better for our screening programme for HPA 1bb determination. DNA from 1093 individuals were tested in parallel with the SSP-PCR and the 5' NA. One thousand and ninety-one samples gave identical results in SSP-PCR and 5' NA. Upon repeated testing, two samples consistently came out as HPA 1bb in SSP-PCR and HPA 1ab in 5' NA. DNA sequencing revealed a defect located in an intronic area that corresponds to the consensus primer used for the SSP-PCR HPA 1a typing. PMID:11972525

  1. Oral immunization with hepatitis B surface antigen expressed in transgenic plants

    PubMed Central

    Kong, Qingxian; Richter, Liz; Yang, Yu Fang; Arntzen, Charles J.; Mason, Hugh S.; Thanavala, Yasmin

    2001-01-01

    Oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) derived from yeast (purified product) or in transgenic potatoes (uncooked unprocessed sample) was compared. An oral adjuvant, cholera toxin, was used to increase immune responses. Transgenic plant material containing HBsAg was the superior means of both inducing a primary immune response and priming the mice to respond to a subsequent parenteral injection of HBsAg. Electron microscopy of transgenic plant samples revealed evidence that the HBsAg accumulated intracellularly; we conclude that natural bioencapsulation of the antigen may provide protection from degradation in the digestive tract until plant cell degradation occurs near an immune effector site in the gut. The correlate of protection from hepatitis B virus infection is serum antibody titers induced by vaccination; the protective level in humans is 10 milliunits/ml or greater. Mice fed HBsAg-transgenic potatoes produced HBsAg-specific serum antibodies that exceeded the protective level and, on parenteral boosting, generated a strong long-lasting secondary antibody response. We have also shown the effectiveness of oral delivery by using a parenteral prime-oral boost immunization schedule. The demonstrated success of oral immunization for hepatitis B virus with an “edible vaccine” provides a strategy for contributing a means to achieve global immunization for hepatitis B prevention and eradication. PMID:11553782

  2. Impact of pooling on accuracy of hepatitis B virus surface antigen screening of blood donations.

    PubMed

    Novack, L; Sarov, B; Goldman-Levi, R; Yahalom, V; Safi, J; Soliman, H; Orgel, M; Yaari, A; Galai, N; Pliskin, J S; Shinar, E

    2008-08-01

    Expenditure on screening blood donations in developing countries can be reduced by testing donations in pools. This study evaluated serological screening in pools for hepatitis B virus (HBV) at the Israeli national blood bank and a hospital blood bank in Gaza, the Palestinian Authority. The accuracy of HBV surface antigen (HBsAg) enzyme immunoassay performed on pools of 3-24 samples was compared with individual tests. Delay in detecting positive samples due to dilution in pools and the possibility of antibody-antigen neutralization were analyzed. The sensitivity of pooled testing for HBsAg was 93-99%, prolonging the window period by 5 days (8.3%). Neutralization of HBsAg by hepatitis B surface antibodies (anti-HBs) could be minimized by testing immediately after pooling. Serological testing for HBsAg in pools may be performed using manually created pools of up to six samples, with 5% loss in sensitivity and a risk of neutralization by anti-HBs present in the donor population. Pooling can therefore be considered as an option only in countries with a low prevalence of HBV. PMID:18486172

  3. 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.

  4. Low-Dose Adenovirus Vaccine Encoding Chimeric Hepatitis B Virus Surface Antigen-Human Papillomavirus Type 16 E7 Proteins Induces Enhanced E7-Specific Antibody and Cytotoxic T-Cell Responses

    PubMed Central

    Báez-Astúa, Andrés; Herráez-Hernández, Elsa; Garbi, Natalio; Pasolli, Hilda A.; Juárez, Victoria; zur Hausen, Harald; Cid-Arregui, Angel

    2005-01-01

    Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (106 infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen-specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8+-mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein. PMID:16188983

  5. 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. PMID:25259405

  6. Intrahepatic distribution of hepatitis B virus antigens in patients with and without hepatocellular carcinoma

    PubMed Central

    Safaie, Parham; Poongkunran, Mugilan; Kuang, Ping-Ping; Javaid, Asad; Jacobs, Carl; Pohlmann, Rebecca; Nasser, Imad; Lau, Daryl TY

    2016-01-01

    AIM: To study the intrahepatic expression of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in chronic hepatitis B patients with and without hepatocellular carcinoma. METHODS: A total of 33 chronic hepatitis B patients (mean age of 40.3 ± 2.5 years), comprising of 14 HBeAg positive and 19 HBeAg negative patients; and 13 patients with hepatitis B virus related hepatocellular carcinoma (mean age of 49.6 ± 4.7 years), were included in our study. Immunohistochemical staining for HBcAg and HBsAg was done using standard streptavidin-biotin-immunoperoxidase technique on paraffin-embedded liver biopsies. The HBcAg and HBsAg staining distributions and patterns were described according to a modified classification system. RESULTS: Compared to the HBeAg negative patients, the HBeAg positive patients were younger, had higher mean HBV DNA and alanine transaminases levels. All the HBeAg positive patients had intrahepatic HBcAg staining; predominantly with “diffuse” distribution (79%) and “mixed cytoplasmic/nuclear” pattern (79%). In comparison, only 5% of the HBeAg-negative patients had intrahepatic HBcAg staining. However, the intrahepatic HBsAg staining has wider distribution among the HBeAg negative patients, namely; majority of the HBeAg negative cases had “patchy” HBsAg distribution compared to “rare” distribution among the HBeAg positive cases. All but one patient with HCC were HBeAg negative with either undetectable HBV DNA or very low level of viremia. Intrahepatic HBcAg and HBsAg were seen in 13 (100%) and 10 (77%) of the HCC patients respectively. Interestingly, among the 9 HCC patients on anti-viral therapy with suppressed HBV DNA, HBcAg and HBsAg were detected in tumor tissues but not the adjacent liver in 4 (44%) and 1 (11%) patient respectively. CONCLUSION: Isolated intrahepatic HBcAg and HBsAg can be present in tumors of patients with suppressed HBV DNA on antiviral therapy; that may predispose them to cancer development

  7. Inhibition of the HCV core protein on the immune response to HBV surface antigen and on HBV gene expression and replication in vivo.

    PubMed

    Zhu, Wenbo; Wu, Chunchen; Deng, Wanyu; Pei, Rongjun; Wang, Yun; Cao, Liang; Qin, Bo; Lu, Mengji; Chen, Xinwen

    2012-01-01

    The hepatitis C virus (HCV) core protein is a multifunctional protein that can interfere with the induction of an immune response. It has been reported that the HCV core protein inhibits HBV replication in vitro. In this study, we test the effect of the HCV core gene on the priming of the immune response to hepatitis B surface antigen (HBsAg) and on the replication of HBV in vivo. Our results showed that the full-length HCV core gene inhibits the induction of an immune response to the heterogeneous antigen, HBsAg, at the site of inoculation when HCV core (pC191) and HBsAg (pHBsAg) expression plasmids are co-administered as DNA vaccines into BALB/c mice. The observed interference effect of the HCV core occurs in the priming stage and is limited to the DNA form of the HBsAg antigen, but not to the protein form. The HCV core reduces the protective effect of the HBsAg when the HBsAg and the HCV core are co-administered as vaccines in an HBV hydrodynamic mouse model because the HCV core induces immune tolerance to the heterogeneous HBsAg DNA antigen. These results suggest that HCV core may play an important role in viral persistence by the attenuation of host immune responses to different antigens. We further tested whether the HCV core interfered with the priming of the immune response in hepatocytes via the hydrodynamic co-injection of an HBV replication-competent plasmid and an HCV core plasmid. The HCV core inhibited HBV replication and antigen expression in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, the mouse models of acute and chronic hepatitis B virus infections. Thus, the HCV core inhibits the induction of a specific immune response to an HBsAg DNA vaccine. However, HCV C also interferes with HBV gene expression and replication in vivo, as observed in patients with coinfection. PMID:23024803

  8. 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

  9. Enzyme-linked immunosorbent assays for the detection of equine antibodies specific to a recombinant Fasciola hepatica surface antigen in an endemic area.

    PubMed

    Arias, María Sol; Piñeiro, Pablo; Hillyer, George V; Francisco, Iván; Cazapal-Monteiro, Cristiana Filipa; Suárez, José Luis; Morrondo, Patrocinio; Sánchez-Andrade, Rita; Paz-Silva, Adolfo

    2012-02-01

    The utility of an enzyme-linked immunosorbent assay to determine the sensitization against the trematode Fasciola hepatica in horses from an endemic area (NW Spain) was assessed. Blood samples were collected from 536 horses and tested against a 2.9-kDa recombinant surface protein (FhrAPS) to estimate the presence of IgG antibodies. Data were analysed regarding several intrinsic (age, gender and breed) and extrinsic factors (aptitude and housing). The farm size (number of horses/farm) was also considered. Sixty percent (95% CI 56, 64) of the horses were positive to the FhrAPS-ELISA, with a significantly higher seroprevalence in the mares (67%). Foals reached the lowest percentage of sensitization against the trematode (12%), and a significant positive correlation between the seroprevalence of fasciolosis and the age of the horses was established. When considering all the factors together, the seroprevalence of fasciolosis was initially classified into two groups (nodes) regarding the age of the horses. The node composed of the horses older than 1 year was then divided into two other clusters according to their gender. The mares were finally classified and grouped into two nodes regarding their breed. We concluded that the FhrAPS-ELISA is very useful for the demonstration of specific equine IgG antibodies against F. hepatica. An elevated risk of exposition to this trematode in horses maintained in endemic areas was proven. The possible role of horses as reservoirs for F. hepatica infections is discussed. PMID:21847600

  10. 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

  11. Seroepidemiology of Toxoplasma in a coastal region of Haiti: multiplex bead assay detection of immunoglobulin G antibodies that recognize the SAG2A antigen.

    PubMed

    Priest, J W; Moss, D M; Arnold, B F; Hamlin, K; Jones, C C; Lammie, P J

    2015-02-01

    Toxoplasma gondii is a globally distributed parasitic protozoan that infects most warm-blooded animals. We incorporated a bead coupled with recombinant SAG2A protein into our Neglected Tropical Disease (NTD) multiplex bead assay (MBA) panel and used it to determine Toxoplasma infection rates in two studies in Haiti. In a longitudinal cohort study of children aged 0-11 years, the infection rate varied with age reaching a maximum of 0·131 infections/year in children aged 3 years [95% confidence interval (CI) 0·065-0·204]. The median time to seroconversion was estimated to be 9·7 years (95% CI 7·6-∞). In a cross-sectional, community-wide survey of residents of all ages, we determined an overall seroprevalence of 28·2%. The seroprevalence age curve from the cross-sectional study also suggested that the force of infection varied with age and peaked at 0·057 infections/year (95% CI 0·033-0·080) at age 2·6 years. Integration of the Toxoplasma MBA into NTD surveys may allow for better estimates of the potential burden of congenital toxoplasmosis in underserved regions. PMID:25600668

  12. Successful kidney transplantation from a hepatitis B surface antigen-positive donor to an antigen-negative recipient using a novel vaccination regimen.

    PubMed

    Singh, Gurmukteshwar; Hsia-Lin, Andrea; Skiest, Daniel; Germain, Michael; O'Shea, Michael; Braden, Gregory

    2013-04-01

    Transplanting a kidney from a hepatitis B surface antigen (HBsAg)-positive donor to an HBsAg-negative recipient who is naturally immune has been successful in countries endemic for hepatitis B virus (HBV). However, in most of these cases, the donors were deceased. We present a report of a successful HBsAg-discordant kidney transplantation in the United States; in this case, a living donor kidney was transplanted to a vaccinated recipient. The wife of a 58-year-old HBsAg-negative man volunteered to donate a kidney to her husband. She had chronic hepatitis B but undetectable HBV DNA. She tested positive for HBsAg and antibody to hepatitis B core antigen, but hepatitis B e antigen was undetectable. The recipient failed to develop an antibody response to 3 doses of intramuscular recombinant HBV vaccine given in consecutive months. Immunity was induced by using biweekly intradermal vaccine. However, antibody titer tapered to <10 mIU/mL over 14 months. An intramuscular booster vaccine resulted in a prolonged anamnestic response, allowing for successful living unrelated donor transplantation. During the 10 years since transplantation, the patient has continued to have normal liver function, with undetectable HBsAg and HBV DNA. Antibody titers to HBsAg slowly decreased to 5.8 mIU/mL during the 10 years. Transplant function has been well preserved. This approach to inducing long-term immunity for transplantation merits further study in the United States. PMID:23219109

  13. 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

  14. Bioencapsulation of the hepatitis B surface antigen and its use as an effective oral immunogen

    PubMed Central

    Hayden, Celine A.; Streatfield, Stephen J.; Lamphear, Barry J.; Fake, Gina M.; Keener, Todd K.; Walker, John H.; Clements, John D.; Turner, Debra D.; Tizard, Ian R.; Howard, John A.

    2012-01-01

    Hepatitis B remains a major global health problem despite the availability of a safe and effective vaccine. Segments of the population lack access to or respond poorly to the parenteral vaccine, perpetuating the infection-transmission cycle. A low cost, orally-delivered vaccine has the potential to alleviate many of these problems. Here we describe the expression of a bioencapsulated hepatitis B surface antigen (HBsAg) in maize and its immunogenicity, demonstrating for the first time a commercially feasible oral subunit vaccine production system for a major disease. This work surmounts previous barriers to plant-produced vaccines by expressing HBsAg at much higher levels and retaining antigen immunogenicity post-processing: factors which facilitated a robust immune response in mice without the need for an adjuvant. This method provides a practical solution to the delivery of a low-cost, stable oral vaccine. PMID:22406456

  15. Increased Hepatitis B surface antigen production by recombinant Aspergillus niger through the optimization of agitation and dissolved oxygen concentration.

    PubMed

    James, Emmanuel R; van Zyl, Willem H; Görgens, Johann F

    2007-05-01

    The capacity of the filamentous fungi Aspergillus niger to produce and assemble complex immunogenic viral proteins into virus-like particles (VLPs) in batch culture was enhanced by optimizing the bioprocessing parameters, agitation intensity and dissolved oxygen (dO(2)) concentration. Response surface methodology (RSM) and a two-factor-two-level central composite rotatable design (CCRD) were employed to evaluate the interactive response pattern between parameters and their optimum combination. The recombinant hepatitis B surface antigen (HBsAg) was used as a model VLP system to determine the effect of these parameters on biomass yield, fungal morphology, HBsAg production and bioreactor kinetics. The response surface model predicted optimum cultivation conditions at an agitation of rate of 100 rpm and a dO(2) concentration of 25%, obtaining highest intracellular membrane-associated HBsAg levels of [see text]. HBsAg production levels were increased tenfold compared to yields obtained in shake flask cultivation. Although hepatitis B VLPs mostly accumulated intracellularly, optimal bioreactor conditions resulted in significant HBsAg release in culture supernatant. These results compare favourably with other recombinant VLP systems in batch culture, and therefore, indicate a substantial potential for further engineering of the A. niger production system for the high level of intracellular and extracellular VLP production. PMID:17308907

  16. Hepatitis B Vaccination Coverage and Prevalence of Hepatitis B Surface Antigen Among Children in French Polynesia, 2014.

    PubMed

    Patel, Minal K; Le Calvez, Evelyne; Wannemuehler, Kathleen; Ségalin, Jean-Marc

    2016-06-01

    French Polynesia is considered to be moderately endemic for chronic hepatitis B virus infection, with an estimated 3% of the population having hepatitis B surface antigen (HBsAg). From 1990 to 1992, a 3-dose hepatitis B vaccination series was introduced into the routine infant immunization schedule in French Polynesia, including a birth dose (BD). In 2014, a nationally representative 2-stage cluster survey was undertaken to evaluate the impact of the vaccination program on HBsAg prevalence among school children (∼6 years of age) in Cours Préparatoire (CP). Documented vaccination data were reviewed for all eligible children; children with consent were tested for HBsAg with a rapid point-of-care test. In total, 1,660 students were identified; 1,567 (94%) had vaccination data for review and 1,196 (72%) participated in the serosurvey. Three-dose vaccination coverage was 98%, while timely BD coverage, defined as a dose administered within 24 hours of life, was 89%. Receipt of the second and third doses was often delayed, with 75% and 55% receiving a second and third dose within 1 month of the recommended age, respectively. No children tested positive for HBsAg. French Polynesia's vaccination program has achieved high coverage and an HBsAg seroprevalence of 0% (0-0.5%) among CP school children, but timeliness of vaccination could be improved. PMID:27001757

  17. Molecular Characterization of the Escherichia coli Serogroup O117, O126, and O146 O Antigen Gene Clusters and Development of PCR Assays Targeting Serogroup O117-, O126-, and O146-Specific DNA Sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The O antigen gene clusters of Escherichia coli serogroups O117, O126, and O146 were sequenced, and eleven, ten, and eleven open reading frames (ORFs) were identified, respectively. Genes required for O antigen sugar biosynthesis, sugar transfer and sugar processing in these O-antigen gene clusters...

  18. 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

  19. Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the Hepatitis B surface antigen

    PubMed Central

    2011-01-01

    Background A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification. Results High level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell. Conclusions It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells

  20. 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. PMID:27154596

  1. 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

  2. Hepatitis B virus antigens impair NK cell function.

    PubMed

    Yang, Yinli; Han, Qiuju; Zhang, Cai; Xiao, Min; Zhang, Jian

    2016-09-01

    An inadequate immune response of the host is thought to be a critical factor causing chronic hepatitis B virus (CHB) infection. Natural killer (NK) cells, as one of the key players in the eradication and control of viral infections, were functionally impaired in CHB patients, which might contribute to viral persistence. Here, we reported that HBV antigens HBsAg and HBeAg directly inhibited NK cell function. HBsAg and/or HBeAg blocked NK cell activation, cytokine production and cytotoxic granule release in human NK cell-line NK-92 cells, which might be related to the downregulation of activating receptors and upregulation of inhibitory receptor. Furthermore, the underlying mechanisms likely involved the suppression of STAT1, NF-κB and p38 MAPK pathways. These findings implicated that HBV antigen-mediated inhibition of NK cells might be an efficient strategy for HBV evasion, targeting the early antiviral responses mediated by NK cells and resulting in the establishment of chronic virus infection. Therefore, this study revealed the relationship between viral antigens and human immune function, especially a potential important interaction between HBV and innate immune responses. PMID:27341035

  3. Immunization against hepatitis B virus by mucosal administration of antigen-antibody complexes.

    PubMed

    McCluskie, M J; Wen, Y M; Di, Q; Davis, H L

    1998-01-01

    Antigen-antibody complexes have been shown to enhance immune responses against several antigens given by parenteral immunization. Herein, we have evaluated the potential of administering such immunostimulatory complexes by a mucosal route. Hepatitis B surface antigen (HBsAg) complexed with antibodies against HBsAg (anti-HBs) (HBsAg/Ab) was administered to BALB/c mice by intranasal inhalation. HBsAg by itself did not induce immune responses, whereas with HBsAg/Ab complexes, both systemic and mucosal immune responses were observed and these could be modulated by adjuvants. With HBsAg/Ab (1 or 10 microg), anti-HBs antibodies induced were predominantly of the IgG1 isotype (Th2-like). In contrast, anti-HBs induced by HBsAg/Ab plus cholera toxin (CT) or oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG) (1 microg each) were predominantly IgG2a (Th1-like). Results from this study indicate that HBsAg/Ab complexes can induce strong humoral immune responses when delivered by a noninvasive route, whether used alone or in combination with other mucosal adjuvants. PMID:10189191

  4. 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

  5. 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

  6. Dose-response association between hepatitis B surface antigen levels and liver cancer risk in Chinese men and women.

    PubMed

    Yang, Yang; Gao, Jing; Li, Hong-Lan; Zheng, Wei; Yang, Gong; Zhang, Wei; Ma, Xiao; Tan, Yu-Ting; Rothman, Nathaniel; Gao, Yu-Tang; Chow, Wong-Ho; Shu, Xiao-Ou; Xiang, Yong-Bing

    2016-07-15

    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), 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

  7. [Nature, character, occurrence, and demonstration of hepatitis B antigens (author's transl)].

    PubMed

    Müller, R

    1975-08-01

    The morphological, chemical and physical properties of HBAg suggest that the 42 nm component of the antigen, the Dane particle, represents the agent of viral hepatitis B. Its core contains a circular, double stranded DNA, a DNA polymerase and carried HBc-Ag. HBc-Ag is localized on the 21 nm particle, the tubular structures and the surface of the Dane particles. At least 8 different subdeterminants of HBs-Ag could be distinguished by means of specific animal anti-sera. HBs-Ag activity was demonstrated in almost all body fluids and excreta. The results of combined histologic, fluorescent and electronmicroscopic studies suggest ath HBc-Ag is localized in the liver cell nucleus and that HBs-Ag is found in the cysterna of the smooth endoplasmatic reticulum of the hepatocytes. The demonstration of HBs-Ag and the specific DNA polymerase in the serum indicate a hepatitis b virus infection with persistent reproduction of the agent, while demonstration of anti-HBs indicates that the infection has been overcome. The clinical importance importance of anti-HBc is controversial. PMID:52590

  8. Impact of correcting the lymphocyte count to improve the sensitivity of TB antigen-specific peripheral blood-based quantitative T cell assays (T-SPOT.®TB and QFT-GIT)

    PubMed Central

    van Zyl-Smit, Richard N.; Lehloenya, Rannakoe J.; Meldau, Richard

    2016-01-01

    Background The standardized blood-based TB antigen-specific T cell assay, T-SPOT.®TB, is ~10% more sensitive than QuantiFERON®-TB-GIT (QFT-GIT) in detecting presumed latent TB infection (LTBI). Whilst T-SPOT.®TB uses a fixed number of lymphocytes per well, QFT-GIT uses a fixed volume of blood (~1 mL). However, the person-to-person lymphocyte count can vary by 2 to 3 fold. We hypothesized that this variability could explain the reduced sensitivity of QFT-GIT. The findings could have potential implications for improving case detection. Methods T-SPOT.®TB was compared to QFT-GIT readouts before and after normalization of lymphocyte count (by adjusting the blood volume or lymphocyte enrichment within a fixed 1 mL volume) to an arbitrary value of 2.5×106 cells/mL. Within-test variability was evaluated to meaningfully interpret results. Results In patient-specific optimization experiments IFN-γ concentrations significantly increased when QFT-GIT positive samples were enriched with increasing concentrations of lymphocytes (1×106 vs. 2.5×106 cells/mL). However, for the group as a whole lymphocyte enrichment whilst maintaining a ~1 mL volume, compared to un-enriched samples, did not significantly increase IFN-γ [median (range): 0.03 (0–4.41) vs. 0.20 (0–2.40) IU/mL; P=0.64]. There was also no increase in IFN-γ readouts when QFT-GIT lymphocyte numbers were corrected (to 2.5×106 lymphocytes/mL) using volume adjustment. Interestingly, adjusted values were significantly lower than unadjusted ones [median (range): 0.02 (0–12.93) vs. 0.09 (0–14.23) IU/mL; P=0.008]. Conclusions In QFT-GIT negative subjects lymphocyte enrichment did not increase QFT-GIT positivity rates. The reduced clinical sensitivity of the QFT-GIT assay, compared to T-SPOT.®TB, is likely to be due to factors other than lymphocyte count alone. Further studies are required to clarify these findings. PMID:27076944

  9. 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. PMID:21217023

  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. 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. PMID:24141044

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. Hepatitis B surface antigen prevalence in pregnant women: A cross-sectional survey in Iran

    PubMed Central

    Shoghli, Alireza; Nabavi, Seyed Mahmood; Alavian, Seyed Moayed; Kolifarhood, Goodarz; Goya, Mohammad Mehdi; Namazi, Roshanak; Fallahnezhad, Mojtaba; Mohajeri, Mansor; Mousavinasab, Nouraldin; Zanjani, Rahim Sorouri; Saeini, Mohammad Reza; Jalilvand, Ahmad

    2014-01-01

    Background: Vertical transmission of hepatitis B virus (HBV) from infected mothers to their neonates is one of the most important routes of infection. The exact prevalence rate of HBV in Iranian pregnant mothers is not well known but based on different studies it is estimated between 0.35% and 6.5%. The aim of this study was to determine the seroprevalence of hepatitis B surface antigen (HBsAg) in pregnant women of selected provinces in Iran. Methods: At this cross-sectional study, seven provinces supposed to be of high and low prevalence of hepatitis B in the general population selected. Multistage sampling was used to enroll 5261 parturient women who attended the target provinces birth facilities, during January to March of 2011, were recruited to study. To determine the statistically significant mean and proportion differences, t-test and χ2 test were used, respectively. Results: Overall 1.2% was positive HBsAg of which 11% of them were hepatitis B e-antigen positive as well. The eastern and north eastern provinces were considerably higher in HBsAg seropositivity than the west and northwest of the country. Conclusions: In view of the considerable prevalence of hepatitis B in pregnant women, screening all pregnant women prioritizing the eastern and north-eastern provinces is strongly recommended. PMID:26622992

  19. 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:…

  20. Production of highly concentrated, heat-stable hepatitis B surface antigen in maize.

    PubMed

    Hayden, Celine A; Egelkrout, Erin M; Moscoso, Alessa M; Enrique, Cristina; Keener, Todd K; Jimenez-Flores, Rafael; Wong, Jeffrey C; Howard, John A

    2012-10-01

    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 fourfold higher than any previously reported lines. HBsAg expressed in maize seeds was extremely heat stable, tolerating temperatures up to 55 °C for 1 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

  1. Reactivation of Hepatitis B with Reappearance of Hepatitis B Surface Antigen After Chemotherapy and Immune Suppression

    PubMed Central

    Palmore, Tara N.; Shah, Neeral L.; Loomba, Rohit; Borg, Brian B.; Lopatin, Uri; Feld, Jordan J.; Khokhar, Farooq; Lutchman, Glen; Kleiner, David E.; Young, Neal S.; Childs, Richard; Barrett, A. John; Liang, T. Jake; Hoofnagle, Jay H.; Heller, Theo

    2009-01-01

    Background & Aims Hepatitis B virus (HBV) infection may reactivate in the setting of immune suppression, although the frequency and consequences of HBV reactivation are not well known. We report six patients who experienced loss of serologic markers of hepatitis B immunity and reappearance of Hepatitis B surface antigen (HBsAg) in the serum due to a variety of acquired immune deficiencies. Methods Between 2000 and 2005, six patients with reactivation of hepatitis B were seen in consultation by the Liver Diseases Branch at the Clinical Center, National Institutes of Health. The course and outcome of these six patients were reviewed. Results All six patients developed reappearance of HBsAg and evidence of active liver disease after stem cell transplantation (n=4), immunosuppressive therapy (n=1) or change in HIV antiretroviral regimen (n=1) despite having antibody to HBsAg (anti-HBs) or antibody to hepatitis B core antigen (anti-HBc) without HBsAg before. All six patients developed chronic hepatitis B, two patients transmitted hepatitis B to their spouses, and one patient developed cirrhosis. The diagnosis of hepatitis B reactivation was frequently missed or delayed and often required interruption of the therapy for the underlying condition. None of the patients received antiviral prophylaxis against HBV reactivation. Conclusions Serologic evidence of recovery from hepatitis B infection does not preclude its reactivation after immunosuppression. Screening for serologic evidence of hepatitis B and prophylaxis of those with positive results using nucleoside analogue antiviral therapy should be provided to individuals in whom immunosuppressive therapy is planned. PMID:19577007

  2. 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

  3. 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

  4. Improved diagnostic performance of a commercial anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study tested the hypothesis that removal of maltose binding protein from recombinant antigen used for plate coating would improve the specificity of Anaplasma antibody competitive ELISA. Three hundred and eight sera with significant MBP antibody binding (=30%I) in Anaplasma negative herds was 1...

  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 Central

    Gordon, David; Kelley, Peter; Heinzel, Susanne; Cooper, Peter; Petrovsky, Nikolai

    2014-01-01

    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 PMID:25267153

  6. Antigenic sites in carcinoembryonic antigen.

    PubMed

    Hammarstrom, S; Shively, J E; Paxton, R J; Beatty, B G; Larsson, A; Ghosh, R; Bormer, O; Buchegger, F; Mach, J P; Burtin, P

    1989-09-01

    The epitope reactivities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen from 11 different research groups were studied using competitive solid-phase immunoassays. About 60% of all possible combinations of Mabs as inhibitors and as the primary binding antibody were investigated. The inhibition data were analyzed by a specially developed computer program "EPITOPES" which measures concordance and discordance in inhibition patterns between Mabs. The analysis showed that 43 of the 52 Mabs (83%) could be classified into one of five essentially noninteracting epitope groups (GOLD 1-5) containing between four and 15 Mabs each. The epitopes recognized by the Mabs belonging to groups 1 to 5 were peptide in nature. With one or two possible exceptions non-classifiable Mabs were either directed against carbohydrate epitopes (4 Mabs) or were inactive in the tests used. Within epitope groups GOLD 1, 4, and 5 two partially overlapping subgroups were distinguished. Mabs with a high degree of carcinoembryonic antigen specificity generally belonged to epitope groups GOLD 1 and 3. PMID:2474375

  7. HBsAg blocks TYPE I IFN induced up-regulation of A3G through inhibition of STAT3.

    PubMed

    Xu, Fengchao; Song, Hongxiao; Li, Na; Tan, Guangyun

    2016-04-22

    Interferon (IFN) is a regularly utilized therapeutic for the treatment of chronic hepatitis B and appears to induce superior HBeAg seroconversion comparing nucleos/tide analogs. However, the mechanisms underlying IFN inhibition of HBV replication, as well as poor responses to IFN are unclear. Apobec3G has been reported to be involved in regulating HBV replication. In this study, we investigated Apobec3G expression and regulatory pathways during HBV infection. We show that over-expression of A3G leads to inhibition of HBV replication. We also show that IFN induces a significant increase in A3G protein expression, which is associated with STAT3 activation. We further show that A3G expression in HBV patients is lower compared to non-infected controls, possibly by HBsAg which inhibits IFN induced A3G up-regulation in a dose dependent manner. This process is likely mediated through inhibition of STAT3-Ser727 phosphorylation. The results presented in this study indicate that STAT3 plays an important role in IFN-induced A3G production, and HBsAg may correlated with poor response to IFN treatment. PMID:27003258

  8. TH1 and TH2 responses are influenced by HLA antigens in healthy neonates vaccinated with recombinant hepatitis B vaccine.

    PubMed

    Jafarzadeh, Abdollah; Shokri, Fazel

    2012-12-01

    The immune response to hepatitis B surface antigen (HBsAg) is influenced by several factors, of which HLA antigens and balanced secretion of Th1/Th2 cytokines play important roles. The aim of this study was to evaluate the influence of HLA antigens on cytokine secretion by HBsAg-stimulated peripheral blood mononuclear cells (PBMC) from healthy neonates vaccinated with recombinant HBsAg. PBMCs were isolated from 48 Iranian neonates vaccinated with a recombinant HBV vaccine. The cells were stimulated in vitro with rHBsAg and the concentration of IL-4, IL-10, IL-12 and IFN-γ were quantitated in culture supernatant by sandwich ELISA. HLA typing was performed by microlymphocytotoxicity method. Significant diminished secretion of both Th1 (IFN-γ) and Th2 (IL-4, IL-10) cytokines was observed in HBsAg-stimulated PBMC from vaccinees expressing the HLA-DR7 compared to DR7 negative vaccinees. Similarly, lower production of these cytokines was also observed in vaccinees with DR7-DR53-DQ2, B7-DR7-DR53-DQ2 and A2-DR7-DR53-DQ2 haplotypes (p<0.05, p <0.005). While HBsAg-stimulated PBMC of DR13+ subjects produced lower levels of Th2-type cytokines (IL-4 and IL-10), those of HLA-B8+ or HLA-A9+ subjects produced higher levels of Th2-type cytokines. Cytokine secretion in response to PHA mitogen was not associated with a given HLA antigen or haplotype and was similarly represented in all groups of subjects irrespective of their HLA complex. These results indicate that HLA antigens may differentially influence cytokine secretion by HBsAg-specific T-cells of healthy neonates vaccinated with recombinant HB vaccine. This phenomenon may have an important implication for control of the immune response to HBsAg vaccine. PMID:23264407

  9. 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.

  10. Removing N-terminal sequences in pre-S1 domain enhanced antibody and B-cell responses by an HBV large surface antigen DNA vaccine.

    PubMed

    Ge, Guohong; Wang, Shixia; Han, Yaping; Zhang, Chunhua; Lu, Shan; Huang, Zuhu

    2012-01-01

    Although the use of recombinant hepatitis B virus surface (HBsAg) protein vaccine has successfully reduced global hepatitis B infection, there are still a number of vaccine recipients who do not develop detectable antibody responses. Various novel vaccination approaches, including DNA vaccines, have been used to further improve the coverage of vaccine protection. Our previous studies demonstrated that HBsAg-based DNA vaccines could induce both humoral and CMI responses in experimental animal models. However, one form of the the HBsAg antigen, the large S antigen (HBs-L), expressed by DNA vaccine, was not sufficiently immunogenic in eliciting antibody responses. In the current study, we produced a modified large S antigen DNA vaccine, HBs-L(T), which has a truncated N-terminal sequence in the pre-S1 region. Compared to the original HBs-L DNA vaccine, the HBs-L(T) DNA vaccine improved secretion in cultured mammalian cells and generated significantly enhanced HBsAg-specific antibody and B cell responses. Furthermore, this improved HBsL DNA vaccine, along with other HBsAg-expressing DNA vaccines, was able to maintain predominantly Th1 type antibody responses while recombinant HBsAg protein vaccines produced in either yeast or CHO cells elicited mostly Th2 type antibody responses. Our data indicate that HBsAg DNA vaccines with improved immunogenicity offer a useful alternative choice to recombinant protein-based HBV vaccines, particularly for therapeutic purposes against chronic hepatitis infection where immune tolerance led to poor antibody responses to S antigens. PMID:22844502

  11. 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

  12. Quantitative removal of hepatitis B viral antigens from serum by a monoclonal IgM coupled to a biocompatible solid-phase support.

    PubMed Central

    Marciniak, R A; Wands, J R; Bruns, R R; Malchesky, P S; Nose, Y; Haber, E

    1983-01-01

    We covalently linked to regenerated cellulose filters a high-affinity monoclonal IgM produced against epitopes that reside on hepatitis B viral surface antigen (HBsAg). Conditions were established whereby as much as 250 micrograms of anti-HBsAg IgM could be linked to 2-4 mg of regenerated cellulose acetate by using cyanogen bromide and trichloro-s-triazine coupling agents. The immunoreactivity of the monoclonal anti-HBsAg IgM was preserved, and quantitative binding studies with HBsAg suggests that more than one functional binding site on the IgM molecule was operative. The specificity of the monoclonal anti-HBsAg IgM was established by demonstrating that a nonspecific monoclonal IgM (against influenza hemagglutinin), when coupled to the filters under identical conditions, had no effect on removal of HBsAg from serum. Most importantly, the monoclonal anti-HBsAg IgM-coupled filters quantitatively removed low levels of HBsAg from serum; after the third pass through the filter, HBsAg was undetectable in the perfusate. Further, the stability of the covalent bond between the anti-HBsAg IgM and regenerated cellulose acetate was shown by the lack of detectable murine monoclonal anti-HBsAg IgM in filtered serum despite 50 passages through the filter. Thus, we have demonstrated that monoclonal IgM antibodies with predefined specificity, when coupled to a biocompatible solid-phase support, may serve as a high-affinity and specific immunoabsorbant for quantitative removal and recovery of viral antigens from human serum. By using this approach, specific removal and recovery of many other substances from serum or plasma would seem possible. Images PMID:6190181

  13. Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

    PubMed Central

    Jiménez, T; Díaz, A M; Zlotnik, H

    1990-01-01

    Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Mycobacteria spp., they have the potential for use as reagents in the purification of Nocardia antigens. Images PMID:2405017

  14. A lateral electrophoretic flow diagnostic assay

    PubMed Central

    Lin, Robert; Skandarajah, Arunan; Gerver, Rachel E.; Neira, Hector D.; Fletcher, Daniel A.

    2015-01-01

    Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a “lateral e-flow assay” and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings. PMID:25608872

  15. 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

  16. Serological response to in vitro-shed antigen(s) of Tritrichomonas foetus in cattle.

    PubMed Central

    Bondurant, R H; van Hoosear, K A; Corbeil, L B; Bernoco, D

    1996-01-01

    We developed a serological assay for detection of (l) an erythrocyte-adhering molecule(s) shed by the bovine venereal pathogen Tritrichomonas foetus and (II) serum antibodies to this antigen(s) in exposed cattle. Sera from exposed and unexposed cattle were tested for their ability to induce complement-mediated lysis of bovine erythrocytes that had been previously incubated overnight at room temperature in pH-adjusted supernatants of T. foetus culture media. Eight of 180 serum specimens from six groups of presumably unexposed cows or heifers showed a positive (> or = 1:2) hemolytic titer (specificity = 95.6%). Thirteen of 14 females in two experimentally infected groups showed a positive hemolytic titer following infection (sensitivity = 94%). In experimentally infected heifers, there was little correlation (r2 = 0.33) between serum hemolytic titers with respect to shed antigen and titers obtained in serum enzyme-linked immunosorbent assays in which whole T. foetus served as the antigen. Serum hemolytic titers rose 3 to 4 weeks sooner than did previously described vaginal mucus immunoglobulin G1 or immunoglobulin A titers with respect to whole-cell antigen or TF1.17 subunit antigen, respectively. Among 14 chronically infected bulls, only 6 (43%) showed a positive hemolytic titer. This study is the first, to our knowledge, to show a specific serological response in the host to an in vitro-shed antigen(s) of T. foetus and suggests a useful diagnostic test for potentially exposed herds. PMID:8807209

  17. 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

  18. Characterisation of Sarcoptes scabiei antigens.

    PubMed

    Hejduk, Gloria; Hofstätter, Katja; Löwenstein, Michael; Peschke, Roman; Miller, Ingrid; Joachim, Anja

    2011-02-01

    In pig herds, the status of Sarcoptes scabiei infections is routinely monitored by serodiagnosis. Crude antigen for ELISA is usually prepared from S. scabiei var. canis or other variations and may lead to variations in the outcome of different tests, making assay standardisation difficult. This study was performed to investigate the antigen profiles of S. scabiei, including differences between hydrophilic and more hydrophobic protein fractions, by Western blotting with sera from pigs with defined infection status. Potential cross-reactivity among S. scabiei (var. canis, suis and bovis), Dermatophagoides farinae and Tyrophagus putrescentiae was also analysed. Hydrophobic S. scabiei antigens were detectable in the range of 40-50 kDa, whilst the hydrophilic fraction showed no specific antigenicity. In the hydrophobic fractions of D. farinae and T. putrescentiae, two major protein fractions in a similar size range could be identified, but no cross-reactivity with Sarcoptes-positive sera was detectable. However, examination of the hydrophilic fractions revealed cross-reactivity between Sarcoptes-positive sera and both the house dust mite and the storage mite in the range of 115 and 28/38 kDa. Specific bands in the same range (42 and 48 kDa) could be detected in blots from hydrophobic fractions of all three tested variations of S. scabiei (var. canis, bovis and suis). These results show that there are considerable differences in mange antibody reactivity, including reactions with proteins from free-living mites, which may interfere with tests based on hydrophilic antigens. Further refinement of antigen and the use of specific hydrophobic proteins could improve ELISA performance and standardisation. PMID:20865427

  19. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  20. Regulation of T-cell function by antibodies: enhancement of the response of human T-cell clones to hepatitis B surface antigen by antigen-specific monoclonal antibodies.

    PubMed Central

    Celis, E; Zurawski, V R; Chang, T W

    1984-01-01

    Eight mouse monoclonal antibodies specific for hepatitis B surface antigen (HBsAg) were examined for their effects on the antigen-induced proliferative response and lymphokine production of human HBsAg-specific T-cell clones in vitro. While all specifically enhanced the T-cell proliferative response, antibodies of the IgG class were generally more effective than those of the IgM class. Both the divalent F(ab')2 and the monovalent Fab fragments of an IgG monoclonal antibody had no effects, indicating that the Fc portion of the antibody molecules was required. Since antigen-presenting cells bear surface receptors for the Fc of IgGs and fewer or none for that of IgMs, the above results also suggest that antibodies enhance the capture of antigens by antigen-presenting cells as a result of the binding of antigen-antibody complexes to the Fc receptors on these cells. In addition to potentiating the proliferation of the T-cell clones, antibodies also increased the antigen-induced production of interferon-gamma by these cells. The present in vitro studies suggest that antibodies may regulate immune responses and do so by enhancing antigen presentation and thus augmenting antigen-induced activation and clonal expansion of T cells. PMID:6436821

  1. 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

  2. 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

  3. 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

  4. Peginterferon add-on results in more HBsAg decline compared to monotherapy in HBeAg-positive chronic hepatitis B patients.

    PubMed

    Brouwer, W P; Sonneveld, M J; Xie, Q; Guo, S; Zhang, N; Zeuzem, S; Tabak, F; Zhang, Q; Simon, K; Akarca, U S; Streinu-Cercel, A; Hansen, B E; Janssen, H L A

    2016-06-01

    It is unknown whether peginterferon (PEG-IFN) add-on to entecavir (ETV) leads to more HBsAg decline compared to PEG-IFN monotherapy or combination therapy, and whether ETV therapy may prevent HBsAg increase after PEG-IFN cessation. We performed a post hoc analysis of 396 HBeAg-positive patients treated for 72 weeks with ETV + 24 weeks PEG-IFN add-on from week 24 to 48 (add-on, n = 85), 72 weeks with ETV monotherapy (n = 90), 52 weeks with PEG-IFN monotherapy (n = 111) and 52 weeks PEG-IFN + lamivudine (combination, n = 110) within 2 randomized trials. HBsAg decline was assessed at the end of PEG-IFN (EOP) and 6 months after PEG-IFN (EOF) discontinuation. Differences in baseline characteristics were accounted for using inversed probability of treatment weights. At EOP, a HBsAg reduction of ≥1log10 IU/mL was more frequently achieved for patients in the add-on or combination therapy arms (both 36%), compared to PEG-IFN mono (20%) or ETV (8%) (add-on vs PEG-IFN mono P = 0.050). At EOF, the HBsAg reduction ≥1log10 IU/mL was only sustained in patients treated with ETV consolidation (add-on vs combination and PEG-IFN mono: 40% vs 23% and 18%, P = 0.029 and P = 0.003, respectively). For add-on, combination, PEG-IFN mono and ETV, the mean HBsAg-level change at EOF was -0.84, -0.81, -0.68 and -0.33 log10 IU/mL, respectively (P > 0.05 for PEG-IFN arms). HBeAg loss at EOF was 36%, 31%, 33% and 20%, respectively (P > 0.05). PEG-IFN add-on for 24 weeks results in more on-treatment HBsAg decline than does 52 weeks of PEG-IFN monotherapy. ETV therapy may maintain the HBsAg reduction achieved with PEG-IFN. PMID:26403919

  5. Comparison of Real-Time PCR, Conventional PCR, and Galactomannan Antigen Detection by Enzyme-Linked Immunosorbent Assay Using Bronchoalveolar Lavage Fluid Samples from Hematology Patients for Diagnosis of Invasive Pulmonary Aspergillosis

    PubMed Central

    Sanguinetti, Maurizio; Posteraro, Brunella; Pagano, Livio; Pagliari, Gabriella; Fianchi, Luana; Mele, Luca; La Sorda, Marilena; Franco, Angelica; Fadda, Giovanni

    2003-01-01

    An iCycler iQ real-time PCR assay targeting 18S rRNA Aspergillus-specific sequences was developed for the diagnosis of invasive pulmonary aspergillosis (IPA). Positive findings were obtained for 18 of 20 (90%) bronchoalveolar lavage (BAL) fluid specimens from patients with probable or confirmed IPA and were obtained for none of the 24 BAL samples from patients with no clinical evidence of aspergillosis. These results were concordant with those of a nested PCR assay, which detected 90% of the patients with IPA, while galactomannan ELISA revealed positivity for 100% of these patients, suggesting that combined use of methods might improve the diagnosis of IPA. PMID:12904419

  6. Co-incubation with IL-18 potentiates antigen-specific IFN-γ response in a whole-blood stimulation assay for measurement of cell-mediated immune responses in pigs experimentally infected with Lawsonia intracellularis.

    PubMed

    Riber, Ulla; Boesen, Henriette Toft; Jakobsen, Jeanne T; Nguyen, Lien T M; Jungersen, Gregers

    2011-02-15

    The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in vitro assay for a direct read-out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evaluate for less severe or immunocompromising infections. Here we investigated the performance of the assay when recombinant co-stimulatory cytokines IL-12 and/or IL-18 were added along with Ag or PBS to cultures of whole-blood from pigs infected with Lawsonia intracellularis. In pigs recovering from a natural infection, addition of rIL-12 or rIL-18 alone increased the Ag-specific IFN-γ release while addition of both cytokines resulted in increased IFN-γ release also in PBS cultures. In analyses after experimental infections with L. intracellularis, significant increased levels of Ag-specific IFN-γ production were measured in Ag+rIL-18 cultures from infected pigs compared to the background response in PBS+rIL-18 control samples (p<0.01) or to Ag+rIL-18 cultures from non-inoculated control pigs (p<0.05). Flow cytometry identified two lymphocyte subsets as the Ag-specific IFN-γ producers. The highest IFN-γ production was by CD4(+)CD8(+) cells while a more numerous population of CD4(-)CD8(+) cells produced lower amounts of IFN-γ in response to rIL-18 and L. intracellularis Ag. PMID:20889217

  7. Rotavirus antigen test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003349.htm Rotavirus antigen test To use the sharing features on this page, please enable JavaScript. The rotavirus antigen test detects rotavirus in the feces. This ...

  8. Vaccine adjuvant ginsenoside Rg1 enhances immune responses against hepatitis B surface antigen in mice.

    PubMed

    Yuan, Ding; Yuan, Qin; Cui, Qianqian; Liu, Chaoqi; Zhou, Zhiyong; Zhao, Haixia; Dun, Yaoyan; Wang, Ting; Zhang, Changcheng

    2016-06-01

    The adjuvant effect of ginsenoside Rg1 on immune responses against hepatitis B surface antigen (HBsAg) in mice was investigated. Female BALB/c mice were subcutaneously injected with saline or HBsAg antigen with or without Rg1 on days 7 and 21. Samples were collected 2 weeks after the boosting for the detection of anti-HBsAg immunoglobulin G (IgG) isotypes in sera and gamma interferon (IFN-γ) and interleukin-4 (IL-4) produced in splenocytes. The innate and adaptive immune responses were measured in mice immunized as described above. The results showed that ginsenoside Rg1 had adjuvant properties in stimulating IgG, splenocyte proliferation, and mRNA expression of cytokines IFN-γ and IL-4, as well as the expression of cell surface marker TLR4 in the HBsAg-immunized mice. These results indicate that Rg1 enhances both Th1 (IgG2b and IFN-γ) and Th2 (IgG1 and IL-4) responses. In addition, the TLR4 signaling pathway is involved in the adjuvant activities of ginsenoside Rg1. PMID:27095502

  9. Leucocyte migration inhibitory factor assay in Nigerians with urinary schistosomiasis.

    PubMed

    Arinola, O G; Salimonu, L S

    1999-01-01

    Schistosome antigens (soluble egg antigen and adult worm antigen) and non-schistosome antigens (Bacille Calmette Guerin vaccine antigen and measles virus vaccine antigen) were used to assay for leucocyte migration inhibitory factors (LMIF) in Nigerian children with and without Schistosoma haematobium infection. The severity of S. haematobium infection was graded into light infection (1-49 eggs/ 10 ml urine) or heavy infection (more than or equal to 50 eggs/10 ml urine). The mean percentage migration indices were significantly reduced in heavily infected urinary schistosomiasis (USS) subjects compared with the controls or lightly infected subjects when non-antigens were used to stimulate LMIF production. When Schistosome antigens were used to stimulate LMIF production, there were no significant decreases in the % migration indices in heavily infected USS subjects compared with the light USS subjects. The mean percentage migration index was significantly increased when schistosome antigen was used to stimulate LMIF production in treated USS subjects compared with untreated USS subjects but the increase was not significant when non-schistosome antigen was used. The conclusion that could be drawn from this study is that LMIF assay using schistosome antigen(s) has epidemiological value in schistosomiasis. PMID:10504865

  10. 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. PMID:26079614

  11. 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...

  12. 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.

  13. In vitro and in vivo synthesis of the hepatitis B virus surface antigen and of the receptor for polymerized human serum albumin from recombinant human adenoviruses.

    PubMed Central

    Ballay, A; Levrero, M; Buendia, M A; Tiollais, P; Perricaudet, M

    1985-01-01

    We have developed an adenovirus vector to express foreign proteins under the control of the adenovirus E1a promoter. Two recombinant plasmids, harbouring either the S gene or the pre-S2 region and the S gene of hepatitis B virus under the control of the E1a promoter, were used to construct two recombinant adenoviruses. These two viruses direct the synthesis of hepatitis B virus surface antigen (HBsAg) particles during the time course of an infectious cycle. When the pre-S2 region is present in the constructed virus, the synthesis of particles carrying the receptor for polymerized human serum albumin (pHSA) is observed. Moreover, the inoculation of rabbits with this latter purified recombinant adenovirus elicits the production of antibodies that react with both HBsAg and pHSA receptor. Images Fig. 4. PMID:3004975

  14. Antigenic Distance Measurements for Seasonal Influenza Vaccine Selection

    PubMed Central

    Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2011-01-01

    Influenza vaccination is one of the major options to counteract the effects of influenza diseases. Selection of an effective vaccine strain is the key to the success of an effective vaccination program since vaccine protection can only be achieved when the selected influenza vaccine strain matches the antigenic variants causing future outbreaks. Identification of an antigenic variant is the first step to determine whether vaccine strain needs to be updated. Antigenic distance derived from immunological assays, such as hemagglutination inhibition, is commonly used to measure the antigenic closeness between circulating strains and the current influenza vaccine strain. Thus, consensus on an explicit and robust antigenic distance measurement is critical in influenza surveillance. Based on the current seasonal influenza surveillance procedure, we propose and compare three antigenic distance measurements, including Average antigenic distance (A-distance), Mutual antigenic distance (M-distance), and Largest antigenic distance (L-distance). With the assistance of influenza antigenic cartography, our simulation results demonstrated that M-distance is a robust influenza antigenic distance measurement. Experimental results on both simulation and seasonal influenza surveillance data demonstrate that M-distance can be effectively utilized in influenza vaccine strain selection. PMID:22063385

  15. Complete hepatitis B virus prophylaxis withdrawal in hepatitis B surface antigen-positive liver transplant recipients after longterm minimal immunosuppression.

    PubMed

    Lenci, Ilaria; Baiocchi, Leonardo; Tariciotti, Laura; Di Paolo, Daniele; Milana, Martina; Santopaolo, Francesco; Manzia, Tommaso Maria; Toti, Luca; Svicher, Valentina; Tisone, Giuseppe; Perno, Carlo Federico; Angelico, Mario

    2016-09-01

    Tailored approaches have been attempted to prevent hepatitis B virus (HBV) reinfection in antibodies against hepatitis B surface antigen (HBsAg)-positive liver transplantation (LT) recipients in order to minimize the use of hepatitis B immune globulin (HBIG) and nucleoside analogues (NAs). We report the results of complete HBV prophylaxis withdrawal after a follow-up of at least 6 years in LT recipients with undetectable serum HBV DNA and intrahepatic total HBV DNA and covalently closed circular DNA at LT. We included 30 HBsAg positive, hepatitis B e antigen-negative recipients, 6 with hepatitis C virus and 7 with hepatitis D virus coinfection, who had received HBIG plus NA for at least 5 years after LT. Stepwise HBIG and NA withdrawal was performed in two 6-month periods under strict monitoring of HBV virology. All patients underwent a clinical, biochemical, and virological follow-up at 3-6 month intervals. HBV recurrence (HBsAg seroreversion ± detectable HBV DNA) occurred in 6 patients: in 1 patient after HBIG interruption and in 5 after both HBIG and NA cessation. Only 3 patients required reinstitution of HBV prophylaxis because of persistent HBV replication, and all achieved optimal control of HBV infection and did not experience clinical events. The other who recurred showed only short-lasting HBsAg positivity, with undetectable HBV DNA, followed by spontaneous anti-HBs seroconversion. An additional 15 patients mounted an anti-HBs titer, without previous serum HBsAg detectability. At the end of follow-up, 90% of patients were still prophylaxis-free, 93.3% were HBsAg negative, and 100% were HBV DNA negative; 60% had anti-HBs titers >10 IU/L (median, 143; range, 13-1000). This small series shows that complete prophylaxis withdrawal is safe in patients transplanted for HBV-related disease at low risk of recurrence and is often followed by spontaneous anti-HBs seroconversion. Further studies are needed to confirm this finding. Liver Transplantation 22 1205

  16. Effects of mitogens and lymphokines on the regulation of the immune response to HBs antigen in vitro.

    PubMed

    Knöller, I; Hoffmann, U J; Werchau, H H; König, W

    1990-11-01

    We studied the immunoregulatory mechanisms of responsiveness and non-responsiveness to hepatitis B (HB) vaccine by analysing the influence of HB surface antigen (HBsAg) on lymphokine- or mitogen-stimulated peripheral lymphocytes from healthy volunteers. Stimulation with pokeweed mitogen (PWM) led to a reduced production of polyclonal IgG from responder cells compared to non-responder lymphocytes. PWM did not enhance the HBs-specific IgG production from responder lymphocytes when the cells were obtained at Day 10 after the last vaccination. A slight reduction of the proliferative response was observed when lymphocytes of non-responders were stimulated with phytohaemagglutinin (PHA) or concanavalin A (Con A). Production of HBs-specific antibodies was enhanced by incubating responder lymphocytes with interleukin-4 (IL-4). The HBs antigen itself did not modulate the expression of the CD23 B-cell differentiation antigen in unseparated lymphocytes. However, CD23 expression induced by low doses of IL-4 was markedly enhanced in an antigen-specific way. Our data indicate that HBs antigen enhances the lymphokine-induced CD23 expression, whereas the mitogen-induced CD23 expression is not affected. Lymphocytes obtained from non-responders exerted a reduced expression of CD25 surface antigen compared to responder lymphocytes. Exogeneous addition of IL-2 in the absence or presence of HBsAg induced a marked enhancement of the IL-2 receptor expression in responder lymphocytes. Furthermore, no significant modulation of CD25 expression was observed in non-responder lymphocytes. PMID:2148540

  17. 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

  18. Survey of both hepatitis B virus (HBsAg) and hepatitis C virus (HCV-Ab) coinfection among HIV positive patients

    PubMed Central

    2009-01-01

    Background HIV, HBVand HCV is major public health concerns. Because of shared routes of transmission, HIV-HCV coinfection and HIV-HBV coinfection are common. HIV-positive individuals are at risk of coinfection with HBV and HCV infections. The prevalence rates of coinfection with HBV and HCV in HIV-patients have been variable worldwide depending on the geographic regions, and the type of exposure. Aim This study aimed to examine HBV and HCV coinfection serologically and determine the shared and significant factors in the coinfection of HIV-positive patients. Methods This descriptive, cross-sectional study was carried out on 391 HIV-positive patients including 358 males and 33 females in Lorestan province, west Iran, to survey coinfection with HBsAg and anti-HCV. The retrospective demographic data of the subjects was collected and the patients' serums were analyzed by ELISA kits including HBsAg and anti-HCV. The collected data was analyzed with SPSS software (15) and Chi-square. Fisher's exact test with 5% error intervals was used to measure the correlation of variables and infection rates. Results The results of the study indicated that the prevalence of coinfection in HIV-positive patients with hepatitis viruses was 94.4% (370 in 391), out of whom 57 (14.5%) cases were HBsAg positive, 282 (72%) cases were anti-HCV positive, and 31 (7.9%) cases were both HBsAg and anti-HCV positive. Conclusion There was a significant correlation between coinfection with HCV and HBV and/or both among HIV-positive patients depending on different variables including sex, age, occupation, marital status, exposure to risk factors.(p < 0.001). PMID:19922624

  19. 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

  20. Hepatitis B Virus Genotypes Distribution with HBsAg Positive in the North of Iran (Mazandaran) During 2011-2014

    PubMed Central

    Mousavi, Tahoora; Haghshenas, Mohammad Reza; Rafiei, Alireza; Navaei, Reza Alizadeh; khah, Zahra Hosseini

    2014-01-01

    Background: HBV infection is a major global health problem and ten genotypes (A to J) and multiples subtypes of HBV have been identified, and they show some distinct geographic distributions. The available data on HBV genotype in Iran are very heterogeneous and limited. Therefore in this study, we tried to identify the HBV genotypes by using polymerase chain reaction. Methods: In this cross-sectional study, HBV- positive serum samples of 122 patients with chronic hepatitis from 2011 to 2014 were studied. HBV-DNA was extracted from plasma samples using QIAamp® MiniElute® Virus Spin Kit (Qiagen). Plasma samples from HBsAg positive were confirmed the presence of HBV nucleic acid and determined the genotypes of HBV genome by PCR using the DNA PCR kit (Cinagene) with Taq-DNA polymerase enzyme and type of specific primers. All samples were examined in the virology laboratory of Sari Medical School. Results: The mean age of patients were 45 ± 25 (range, 20 to 70) year that 70 (57.37%) patients were male and 52 (42.62%) were female. The majority of HBV positive patients had a major surgery (44% patients) and then 32% patients followed by intra familial of hepatitis B virus infected and 11% of HBV positive patients had a history of blood transfusions. In this study, 91(74.59%) had genotype D, 7(5.73%) genotype B and 24(19.67%) genotype D and B. Conclusion: This study indicates that the genotype D is the most frequent followed by the mixed genotypes D and B and genotype B in our region. Prevalence and incidence of HBV genotypes are with distributed among of areas and different genotypes may show different responses with antiviral therapy. PMID:25648402

  1. Prevalence of HBsAg mutants and impact of hepatitis B infant immunisation in four Pacific Island countries.

    PubMed

    Basuni, Ashraf A; Butterworth, Lesley; Cooksley, G; Locarnini, S; Carman, W F

    2004-07-29

    The prevalence rate of hepatitis B virus (HBV) infection in Pacific Island countries is amongst the highest in the world. Hepatitis B immunisation has been incorporated into national programmes at various times, often with erratic supply and coverage, until a regionally co-ordinated programme, which commenced in 1995 ensured adequate supply. The effectiveness of these programmes was recently evaluated in four countries, Vanuatu and Fiji in Melanesia, Tonga in Polynesia and Kiribati in Micronesia. That evaluation established that the programmes had a substantial beneficial impact in preventing chronic hepatitis B infection [Vaccine 18 (2000) 3059]. Several studies of hepatitis B vaccination programmes in endemic countries have identified the potential significance of surface gene mutants as a cause for failure of immunisation. In the study outlined in this paper, we screened infected children and their mothers for the emergence and prevalence of these variants in specimens collected from the four country evaluation. Although the opportunity for the emergence of HBV vaccine escape mutants in these populations was high due to the presence of a considerable amount of the virus in the population and the selection pressure from vaccine use, there were no "a" determinant vaccine escape mutants found. This suggests that vaccine escape variants are not an important cause for failure to prevent HBV transmission in this setting. Other HBsAg variants were detected, but their functional significance remains to be determined. The failure to provide satisfactory protection during such immunisation programmes reflects the need for achieving and sustaining high vaccine coverage, improving the timeliness of doses as well as improving 'cold-chain' support, rather than the selection of vaccine-escape mutants of HBV. PMID:15246613

  2. Bacterial antigen detection in body fluids: methods for rapid antigen concentration and reduction of nonspecific reactions.

    PubMed Central

    Doskeland, S O; Berdal, B P

    1980-01-01

    We sought procedures which would allow a rapid concentration in high yield of bacterial antigens from tissue fluids of patients and which could be applied also to protein-rich fluids like serum. Ethanol precipitation at a subzero temperature with albumin added as an antigen coprecipitant made it possible to achieve a more than 20-fold concentration of antigen in 15 min and a 200-fold concentration in 45 min. Heat-stable antigens could be concentrated from protein-rich fluids (like serum) after the sample had been deproteinized by boiling. Such heating (100 degrees C, 3 min) also liberated bacterial polysaccharides from antibody complexes and elminated the nonspecific interference of serum in enzyme-linked immunosorbent assay. PMID:7372801

  3. Chimeric Antigens of Toxoplasma gondii: Toward Standardization of Toxoplasmosis Serodiagnosis Using Recombinant Products

    PubMed Central

    Beghetto, Elisa; Spadoni, Andrea; Bruno, Luca; Buffolano, Wilma; Gargano, Nicola

    2006-01-01

    We have evaluated the diagnostic utility of six antigenic regions of the Toxoplasma gondii MIC2, MIC3, M2AP, GRA3, GRA7, and SAG1 gene products, assembled in recombinant chimeric antigens by genetic engineering, in order to replace the soluble, whole-cell tachyzoite extract in serological assays. Serum samples from 100 adults with acquired T. gondii infection and from 30 infants born to mothers with primary toxoplasmosis contracted during pregnancy, of whom 20 were congenitally infected, were included. Immunoglobulin G (IgG) and IgM antibodies against epitopes carried by chimeric antigens were measured by performing parallel enzyme immunoassays (recombinant enzyme-linked immunosorbent assays [Rec-ELISAs]), and the results obtained by standard commercial assays with the whole-cell Toxoplasma antigen and assays with the chimeric antigens were compared. Our results demonstrate that IgG and IgM Rec-ELISAs with individual chimeric antigens have performance characteristics comparable to those of the corresponding commercial assays. Furthermore, we show that IgM-capture assays based on chimeric antigens improve the ability to diagnose congenital toxoplasmosis postnatally compared with the ability to diagnose congenital toxoplasmosis by the use of standard assays. The use of recombinant chimeric antigens is effective in distinguishing T. gondii-infected individuals from T. gondii-uninfected individuals and shows that immunoassays based on recombinant products could provide the basis for standardized commercial tests for the serodiagnosis of toxoplasmosis. PMID:16757610

  4. Characterization of 'e' antigen associated with hepatitis B.

    PubMed

    Tedder, R S; Bull, F G

    1979-03-01

    Hepatitis B 'e' antigen (HBe) from the serum of a chronic carrier of HBsAg has been partially purified and characterized. It behaves as an acidic protein, pI 4.5--5.0, which is thermolabile and sulphydryl-sensitive. In serum it usually has a flotation density 1.3 g/cm3, but is sometimes found at density 1.15 g/cm3 because of its association with lipid. HBe from serum is polydisperse on gel filtration although most antigen is recovered with a nominal molecular weight of 3 x 10(5) Daltons. In contrast, in the presence of chaotropic ions, the bulk of serum HBe is found as a species of 3 X 10(4) Daltons previously detected in small amounts under non-dissociating conditions. This suggests that the larger material is formed by non-covalent association of the 3 X 10(4) Dalton species either with itself or other serum components. This would include IgG, although there is no evidence that HBe itself bears immunoglobulin determinants. Analysis of HBe precipitins by polyacrylamide gel electrophoresis under reducing and dissociating conditions suggests that its component polypeptide chains are about 1.7 X 10(4) Daltons. PMID:110505

  5. A Toolbox for Tuberculosis (TB) Diagnosis: An Indian Multi-Centric Study (2006-2008); Evaluation of Serological Assays Based on PGL-Tb1 and ESAT-6/CFP10 Antigens for TB Diagnosis

    PubMed Central

    Lagrange, Philippe H.; Thangaraj, Satheesh K.; Dayal, Rajeshwar; Deshpande, Alaka; Ganguly, Nirmal K.; Girardi, Enrico; Joshi, Beenu; Katoch, Kiran; Katoch, Vishwa M.; Kumar, Manoj; Lakshmi, Vemu; Leportier, Marc; Longuet, Christophe; Malladi, Subbalaxmi V. S.; Mukerjee, Deepali; Nair, Deepthi; Raja, Alamelu; Raman, Balambal; Rodrigues, Camilla; Sharma, Pratibha; Singh, Amit; Singh, Sarman; Sodha, Archana; Kabeer, Basirudeen Syed Ahamed; Vernet, Guy; Goletti, Delia

    2014-01-01

    Background The aim of this multi-centric prospective study in India was to assess the accuracy of a serological test as an additional tool for diagnosing active tuberculosis (ATB). In particular, an assay based on ELISA using a phenolic glycolipid (PGL-Tb1) or a fusion protein (ESAT-6/CFP10) was compared to the tuberculin skin test (TST) and the microbiological results according to HIV status. Methods Individuals with and without ATB and HIV infection were enrolled. Serology and TST results were analyzed per se and in combination with the microbiological data. Results Among the 778 ATB patients, 102 were HIV-infected, 316 HIV-uninfected and 360 had an HIV-unknown status. Of the 945 non-ATB subjects, 559 were at low risk (community adults) and 386 at high risk of M. tuberculosis exposure. Among those with ATB, the sensitivity of ELISA-PGL-Tb1 for ATB was higher than that of ELISA-ESAT-6/CFP10, both in HIV-infected (72.3% versus 63.7%, p = 0.29) and HIV-uninfected/HIV-unknown groups (40.5% versus 28.6%; p<0.0001), whereas the specificity was around 91% for both tests. Sensitivity for ATB increased when the results of the two ELISA were combined, reaching 75.5% in the HIV-infected and 50.9% in the group of HIV-uninfected/HIV-unknown ATB, with a significant decrease of the global specificity (83.9%). Analyzing the ELISA results with the microbiological results, we observed that the sensitivity of both serology tests was independent of the ATB patients' smear microscopy (SM) status and grade. Combining the results of SM with both ELISA, the detection of ATB patients significantly increased (p<0.0001), particularly in those with extrapulmonary TB (up to 45.1%) or HIV infection (up to 83.3%). No significant association was observed between TST and serology results. Conclusions In this prospective multi-centric study, the combination of two rapid tests, such as SM and serology, might be useful in detecting ATB, especially in HIV-infected patients. PMID:24797271

  6. Isolation and antigenicity of a 45-kilodalton Paracoccidioides brasiliensis immunodominant antigen.

    PubMed Central

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

    1992-01-01

    In the present study, we analyzed human antibody responses to Paracoccidioides brasiliensis cellular antigens by the immunoblot technique to identify specific cellular components and to investigate the existence of antigen profile differences among serological responses of paracoccidioidomycosis (PCM) patients. Among the 64 PCM serum samples analyzed, a relatively homogeneous immunoglobulin G response to P. brasiliensis antigens was observed. The polypeptide with a mass of 45 kDa was the most clinically important, since antibody to this antigen was detectable in 90.6% of PCM patients studied and the six individuals who did not produce antibody were either at the end of treatment or in the posttherapy period and had shown clinical recovery. These facts suggested that the presence of this antibody may be an indicator of active disease. The 45-kDa antigen was also the most specific antigen of the PCM humoral immune response, since it reacted with only 2 of 79 (2.5%) heterologous serum samples tested: 1 histoplasmosis case and 1 tuberculosis case. This polypeptide was isolated from gels by electroelution and, when tested by an immunoradiometric assay and immunoblotting, maintained its reactivity with PCM sera and also with anti-P. brasiliensis polyclonal antibodies raised in rabbits at the same sensitivity levels as those obtained in immunoblotting with a crude antigen. Since in our assays the 45-kDa polypeptide was the major P. brasiliensis antigen and seemed to be specific for PCM, its use in alternative diagnostic methods is promising, especially in patients suspected of having the juvenile clinical form of PCM often associated with negative double-immunodiffusion results. Images PMID:1612736

  7. 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. PMID:26342828

  8. 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

  9. Use of recombinant chimeric antigens for the serodiagnosis of Mycoplasma pneumoniae infection.

    PubMed

    Montagnani, F; De Paolis, F; Beghetto, E; Gargano, N

    2010-11-01

    In this paper, we have evaluated the diagnostic utility of three antigenic regions of the Mycoplasma pneumoniae P1, P30, and MPN456 gene products in order to replace the soluble, whole-cell bacterial extract in serological assays. Antigenic regions, being previously identified as B-cell epitopes, were used individually or assembled in a recombinant chimeric antigen by genetic engineering. Paired serum samples from 47 patients with M. pneumoniae infection and from 39 subjects with a clinical picture of atypical pneumonia but without a defined diagnosis of M. pneumoniae infection were included. Immunoglobulin G (IgG) antibodies against epitopes carried by recombinant antigens were measured by performing recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Rec-ELISA results were compared to those obtained by a commercial assay using the whole-cell Mycoplasma antigen. Our study demonstrates that all IgG Rec-ELISAs using recombinant antigens have better sensitivity with respect to the commercial assay. Furthermore, we show that the use of chimeric antigens improve the performance of the assays. The use of recombinant antigens is effective in distinguishing M. pneumoniae-infected patients from uninfected individuals and shows that immunoassays based on recombinant antigens could provide the basis for standardized commercial tests for the serodiagnosis of M. pneumoniae diseases. PMID:20632053

  10. 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. PMID:20521635

  11. 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

  12. 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

  13. Angiogenesis Assays.

    PubMed

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis. PMID:26608294

  14. Use of an enzyme-linked immunosorbent assay to measure antigenaemia during acute plague*

    PubMed Central

    Williams, James E.; Gentry, Mary K.; Braden, Carol A.; Leister, Flora; Yolken, Robert H.

    1984-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to measure concentrations of the specific F1 antigen of the plague bacillus in biological fluids. The assay employed a monoclonal antibody to capture the antigen. Sensitivity of the assay was 0.4 ng of F1 antigen. ELISA-inhibition was used to confirm the specificity of the reactions. This assay detected F1 antigen in two of ten sera from patients with acute bubonic plague and indicated that antigenaemia in man during plague may reach levels of 4-8 μg of F1 antigen per ml of serum. The probability for a correct serodiagnosis of plague was improved when the patients' sera were tested for both antibody and antigen. Two patients with antigenaemia did not have antibody, while two patients with antibody lacked antigenaemia. PMID:6380787

  15. 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...

  16. The correlation between serum HBsAg levels and viral loads depends upon wild‐type and mutated HBV sequences rather than the HBeAg/anti‐HBe status

    PubMed Central

    Liu, Mo‐Han; Chen, Qin‐Yan; Harrison, Tim J.; Li, Guo‐Jian; Li, Hai; Wang, Xue‐Yan; Ju, Yu; Yang, Jin‐Ye

    2015-01-01

    Despite several studies regarding the correlation between serum HBsAg titers and viral loads, the association remains uncertain. Eighty‐nine individuals were selected randomly from a Chinese cohort of 2,258 subjects infected persistently with hepatitis B virus (HBV) for cross‐sectional and longitudinal analysis. Viral loads of mutant HBV are lower than those of wild type HBV. The serum HBsAg titers correlate positively with viral loads in both HBeAg positive and negative subjects (r = 0.449, P = 0.013; r = 0.300, P = 0.018, respectively). No correlation between serum HBsAg titer and viral loads was found in any of the four phases of chronic HBV infection. The serum HBsAg titers correlate positively with viral loads in the group with wild type sequences of the PreS/S, basal core promoter (BCP), and preC regions of HBV(r = 0.502, P = 0.040). However, the correlation was not seen in the group with mutations in these regions (r = 0.165, P = 0.257). The correlation between HBsAg titers and viral loads was seen in individuals with wild type PreS/S sequences but not in the subgroup with BCP double mutations or PreC stop mutation, although their sequences in the preS/S regions were wild type. All these findings were confirmed by the longitudinal analysis. In conclusion, the correlation between serum HBsAg levels and viral loads may not differ between HBeAg positive and negative individuals but may depend on wild‐type or mutated genomic sequences. Therefore, HBsAg quantitation may be used as a surrogate for viral loads in only wild‐type HBV infections. J. Med. Virol. 87:1351–1360, 2015. © 2015 Wiley Periodicals, Inc. PMID:25879734

  17. 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

  18. Genetic and antigenic changes in porcine rubulavirus.

    PubMed

    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

  19. Cathepsin G: roles in antigen presentation and beyond

    PubMed Central

    Burster, Timo; Macmillan, Henriette; Hou, Tieying; Boehm, Bernhard O.; Mellins, Elizabeth D.

    2014-01-01

    Summary Contributions from multiple cathepsins within endosomal antigen processing compartments are necessary to process antigenic proteins into antigenic peptides. Cysteine and aspartyl cathepsins have been known to digest antigenic proteins. A role for the serine protease, Cathepsin G (CatG), in this process has been described only recently, although CatG has long been known to be a granule-associated proteolytic enzyme of neutrophils. In line with a role for this enzyme in antigen presentation, CatG is found in endocytic compartments of a variety of antigen presenting cells. CatG is found in primary human monocytes, B cells, myeloid dendritic cells 1 (mDC1), mDC2, plasmacytoid DC (pDC), and murine microglia, but is not expressed in B cell lines or monocyte-derived DC. Purified CatG can be internalized into endocytic compartments in CatG non-expressing cells, widening the range of cells where this enzyme may play a role in antigen processing. Functional assays have implicated CatG as a critical enzyme in processing of several antigens and autoantigens. In this review, historical and recent data on CatG expression, distribution, function and involvement in disease will be summarized and discussed, with a focus on its role in antigen presentation and immune-related events. PMID:19910052

  20. Cross-Reactivity of the PLATELIA CANDIDA Antigen Detection Enzyme Immunoassay with Fungal Antigen Extracts

    PubMed Central

    Rimek, Dagmar; Singh, Jagpal; Kappe, Reinhard

    2003-01-01

    We studied the specificity of the PLATELIA CANDIDA Ag enzyme immunoassay by using 130 isolates of 63 clinically relevant fungal species. Antigen extracts of seven Candida spp. (Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. lusitaniae, and C. tropicalis) repeatedly yielded positive reactions (>0.5 ng/ml). Geotrichum candidum and Fusarium verticillioides were found to yield borderline-positive reactions (0.25 to 0.50 ng/ml). Antigen preparations from the other 54 fungal species, including yeasts, molds, dermatophytes, and dimorphic fungi, did not cross-react in the assay. PMID:12843102

  1. The search for new antigenic targets in myasthenia gravis.

    PubMed

    Cossins, Judith; Belaya, Katsiaryna; Zoltowska, Katarzyna; Koneczny, Inga; Maxwell, Susan; Jacobson, Leslie; Leite, Maria Isabel; Waters, Patrick; Vincent, Angela; Beeson, David

    2012-12-01

    Around 80% of myasthenia gravis patients have antibodies against the acetylcholine receptor, and 0-60% of the remaining patients have antibodies against the muscle-specific tyrosine kinase, MuSK. Another recently identified antigen is low-density lipoprotein receptor-related protein 4 (Lrp4). To improve the existing assays and widen the search for new antigenic targets, we have employed cell-based assays in which candidate target proteins are expressed on the cell surface of transfected cells and probed with patient sera. These assays, combined with use of myotube cultures to explore the effects of the antibodies, enable us to begin to identify new antigenic targets and test antibody pathogenicity in vitro. PMID:23278587

  2. Hepatitis B Surface Antigen S Gene is an Effective Carrier Molecule for Developing GnRH DNA Immunocastration Vaccine in Mice.

    PubMed

    Han, Y G; Ye, W J; Liu, G Q; Jiang, X P; Ijaz, N; Zhao, J Y; Tesema, B

    2016-06-01

    Relatively molecular mass of GnRH antigens is small and hence needs to couple to a large carrier molecule to enhance its immunogenicity. This study investigated whether hepatitis B surface antigen S (HBsAg-S) gene can be used as an effective carrier molecule for developing GnRH DNA immunocastration vaccine. Two copies of human GnRH gene were fused with HBsAg-S gene for constructing a recombinant plasmid pVAX-HBsAg-S-2GnRH that coded for 27 kDa target fusion protein. Ten male mice were divided into two equal groups, treatment and control. The vaccine (50 μg/mice) prepared in saline solution was injected into male mice at weeks 0, 1, 2, 4 and 7 of the experiment. Vaccine's efficacy was evaluated in terms of GnRH-specific IgG antibody response, plasma testosterone levels, testicular weight and extent of the testicular tissue damage. The specific anti-GnRH antibody titre in vaccinated animals was significantly higher than in controls in only 4th week of immunization (p < 0.05). In addition, vaccinated animals showed lower testicular weight than those of the controls (p < 0.05). Spermatogenesis in seminiferous tubules in vaccinated animals was suppressed. In conclusion, in this study, the engineered plasmid to be used as a GnRH DNA vaccine induced antibody response and suppressed spermatogenesis in mice. This suggests that HBsAg-S gene can be an effective carrier molecule for developing GnRH DNA immunocastration vaccine when relatively molecular mass of the aimed antigens is small. PMID:27157596

  3. Comparison of Schistosoma mansoni Soluble Cercarial Antigens and Soluble Egg Antigens for Serodiagnosing Schistosome Infections

    PubMed Central

    Doenhoff, Mike; Aitken, Cara; Bailey, Wendi; Ji, Minjun; Dawson, Emily; Gilis, Henk; Spence, Grant; Alexander, Claire; van Gool, Tom

    2012-01-01

    A Schistosoma mansoni cercarial antigen preparation (cercarial transformation fluid – SmCTF) was evaluated for detection of anti-schistosome antibodies in human sera in 4 collaborating laboratories. The performance of SmCTF was compared with that of S. mansoni egg antigens (SmSEA) in an indirect enzyme-immunoassay (ELISA) antigen assay, the latter being used routinely in 3 of the 4 participating laboratories to diagnose S. mansoni and S. haematobium infections. In the fourth laboratory the performance of SmCTF was compared with that of S. japonicum egg antigens (SjSEA) in ELISA for detection of anti-S. japonicum antibodies. In all 4 laboratories the results given by SmCTF in ELISA were very similar to those given by the antigen preparation routinely used in the respective laboratory to detect anti-schistosome antibodies in human infection sera. In so far as the ELISA results from SmCTF are thus so little different from those given by schistosome egg antigens and also cheaper to produce, the former is a potentially useful new diagnostic aid for schistosomiasis. PMID:23029577

  4. 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. PMID:25604799

  5. Prevention of hepatitis B infection in newborns through mass screening and delayed vaccination of all infants of mothers with hepatitis B surface antigen.

    PubMed

    Schalm, S W; Mazel, J A; de Gast, G C; Heijtink, R A; Botman, M J; Bänffer, J R; Cerards, L J; Zwijnenberg, J; Fetter, W P; Nuijten, S M

    1989-06-01

    Beginning in 1982 all pregnant women undergoing prenatal routine blood analysis in three large city hospitals and one large rural area were tested for hepatitis B surface antigen (HBsAg). Infants of all HBsAg-positive mothers received hepatitis B immunoglobulin (HBIg), 0.5 mL/kg of body weight within two hours of birth and, after randomization, 10 micrograms of hepatitis B vaccine either at 0, 1, 2, and 11 months of age (schedule A) or at 3, 4, 5, and 11 months of age (schedule B). A second injection of HBIg (1 mL) was given to infants on schedule B at 3 months of age. Blood samples were obtained at 3, 6, 11, 12, 24, and 36 months. In a two-year period, 28,412 pregnant women were tested for HBsAg; screening efficiency varied between 85% and 98%. The overall prevalence of HBsAg was 0.8%, with a marked variation between urban centers (2.2%) and the rural area (0.3%). Vaccinations were received by 180 of 193 infants of HBsAg-positive mothers (90 on schedule A and 90 on schedule B). Concentrations of hepatitis B surface antibody less than 10 IU/L were observed in nine instances in five children from group A and in seven instances in six children from group B. Four hepatitis B viral infections (two HBsAg carriers, two who underwent antihepatitis B core seroconversions) were recorded in group A v one infection (antihepatitis B core seroconversion) in group B. The protective efficacy of the program (screening plus passive immunization and delayed vaccination) was 94%. The estimated cost of preventing one cae of hepatitis B infection in neonates was $3,000 (US currency).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2726331

  6. The pathogenesis of arthritis associated with acute hepatitis-B surface antigen-positive hepatitis. Complement activation and characterization of circulating immune complexes.

    PubMed Central

    Wands, J R; Mann, E; Alpert, E; Isselbacher, K J

    1975-01-01

    Circulating immune complexes were identified in cryoproteins isolated from serial samples of serum from six patients with acute viral hepatitis with and without arthritic symptoms. Cryoprecipitates were analyzed for the presence of hepatitis-B surface antigen (HBsAg) and hepatitis-B surface antibody (anti-HBs) by hemagglutination inhibition and hemagglutination. Complement components were detected by counter electrophoresis, and immunoglobulins were detected by gel diffusion. HBsAg, IgG, and IgM were identified in cryoprecipitates from all hepatitis patients, but were higher in concentration in patients with arthritis. Only cryoprecipitates from hepatitis patients with arthritis contained IgA and complement components C3, C4, and C5 as well as IgG and IgM, which disappear with resolution of the arthritis. The subtypes of IgG in these cryoprecipitates were predominantly the complement-fixing IgG1 and IgG3, HBsAg and anti-HBs were concentrated several-fold in the cryoprecipitates when compared to the serum concentration. Sequential studies in two patients demonstrated that the initial appearance of anti-HBs in the cryoprotein complex was associated with the detection in the complex of IgM suggesting a primary immune response to HBsAg. The C3 activator fragment (C3A) of the properdin complex was found in fresh serum obtained from three hepatitis patients with arthritis and not in uncomplicated hepatitis. The cryoprecipitable immune complexes from patients with arthritis converted C3PA in fresh normal sera to C3A in vitro whereas cryoprotein isolated from patients with uncomplicated hepatitis had no such effect. Thus, the transient appearance of circulating complement-fixing immune complexes in patients with the arthritis of acute hepatitis is associated with activation of both classical and alternate complement pathways and suggests that they play an important role in the pathogenesis of these serum sickness-like extrahepatic symptoms. Images PMID:1123429

  7. Field evaluation of alternative antigens in the Bovigam assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine tuberculosis (TB) was detected in a Holstein cow upon routine slaughter inspection in March 2010. Initial ante-mortem testing indicated that the herd of origin (~900 Holsteins, Colorado) had a relatively high within herd prevalence of bovine TB (11 - 25%). The owner agreed to additional TB te...

  8. Purification of genus-specific chlamydial antigen and its separation into several components by ion-exchange chromatography.

    PubMed Central

    Schmeer, N; Krauss, H

    1982-01-01

    Sodium deoxycholate-extracted genus-specific chlamydial antigen was purified from contaminating substances by ion-exchange chromatography with DEAE-sephacel, resulting in a decrease in the complement-fixing activity of the antigen, whereas the enzyme-linked immunosorbent assay activity increased. By successive elution with 0.5, 1.0, and 1.5 M acetate buffer at least three clearly separated components were consistently recovered in 14 trials. The identity of these components as genus-specific chlamydial antigen was demonstrated in enzyme-linked immunosorbent assay tests with specific antisera. The antigenic activity of these components was not diminished by prior treatment of the chlamydial particles with pronase. Antiserum prepared by immunization of rabbits with the antigenic component I of an egg-propagated antigen reacted predominantly with the antigenic components I and II of a cell culture-propagated antigen. PMID:7096557

  9. Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

    SciTech Connect

    Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J. )

    1990-09-01

    The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins.

  10. 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. PMID:27018820

  11. 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

  12. 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

  13. 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. PMID:26615808

  14. 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.

  15. Analysis of human tumor associated Thomsen-Friedenreich antigen

    SciTech Connect

    Samuel, J.; Noujaim, A.A.; MacLean, G.D.; Suresh, M.R.; Longenecker, B.M. )

    1990-08-01

    The Thomsen-Friedenrich (TF) antigen is a precursor structure of MN blood group antigens and is also expressed by about 90% of human carcinomas. The immunodominant group of TF antigen (beta-galactosyl(1-3)-alpha-N-acetylglactosamine) is present in cryptic form in normal RBC and is revealed by neuraminidase treatment. A murine monoclonal antibody (Mab 49H.8) developed against neuraminidase treated human RBC was reactive against a variety of human tumors. We have characterized the human tumor associated TF antigen detected by this antibody from a human transitional bladder carcinoma cell line (647V), a human colon adenocarcinoma cell line (LS174T), and a pleural effusion fluid of a breast adenocarcinoma patient (PE 89). A heterologous sandwich radioimmunoassay for TF antigen was developed using Mab 49H.8 as the catcher and 125I-peanut agglutinin as the probe. Detergent extracts of 647V and LS174T cells, media conditioned by culturing these cells, and PE 89 were shown to contain the antigen by this assay. The specificity of the antigen capture by Mab 49H.8 in this assay was demonstrated by its selective inhibition by nitrophenyl-beta-D-galactoside, phenyl-beta-D-galactoside, and a TF hapten. Preliminary studies on TF antigen in serum samples using this assay showed that about 53.7% of the carcinoma samples contained an antigen concentration greater than 200 units/ml whereas for 90.9% of the normal samples the antigen concentration was below 200 units/ml. These studies demonstrated that the TF antigen is shed by the tumor cells both in vitro and in vivo. The TF antigen was sensitive to treatment with alkali (0.1 M NaOH for 5 h at 37 degrees C) and periodate (10 mM sodium periodate for 1 h at room temperature), was resistant to acidic pH (50 mM acetate buffer, pH 4.5, for 5 h at 37 degrees C), and could be extracted with perchloric acid.

  16. 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.

  17. 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.

  18. Enzyme immunoassay for detection of Giardia lamblia cyst antigens in formalin-fixed and unfixed human stool.

    PubMed Central

    Stibbs, H H; Samadpour, M; Manning, J F

    1988-01-01

    An antigen-capture enzyme-linked immunosorbent assay employing rabbit and mouse antisera to Giardia lamblia cyst antigens was developed for the diagnosis of Giardia infection through detection of G. lamblia-specific stool antigens in cell-free aqueous eluates of human stool. This is the first report of the use of anti-cyst antibodies in an enzyme immunoassay for G. lamblia. The assay gave a positive result with 54 of 59 stools from patients with symptomatic, clinically diagnosed giardiasis, giving the test a sensitivity of 91.5%. A negative reading was obtained with all of 25 stools from G. lamblia-negative control patients. The assay could detect as few as 20 sonicated cysts added to control stool eluate. The assay was more sensitive to cyst-derived antigens than to trophozoite-derived antigens. With two exceptions, the assay gave a negative result with stools from patients infected with Entamoeba histolytica (seven), Cryptosporidium sp. (four), or Blastocystis hominis (seven) and thus appears to be specific for G. lamblia antigens. Storage of stool eluates for more than 6 months at 4 degrees C as unpreserved aqueous eluates or as formalinized eluates did not affect the ability of the assay to detect the giardial antigens. The enzyme-linked immunosorbent assay proved useful for monitoring the levels of G. lamblia-specific stool antigens in the stool of patients undergoing antigiardial chemotherapy. PMID:3183015

  19. 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.

  20. Determination of the serological sex-specific (Sxs) antigen ("H-Y antigen") in birds and mammals using high-titer antisera and a sensitive urease ELISA.

    PubMed

    Bradley, M P; Ebensperger, C; Wiberg, U H

    1987-08-01

    A rapid, sensitive, and reproducible enzyme-linked immunosorbent assay (ELISA), for the detection of the serological sex-specific (Sxs) antigen (formerly termed H-Y antigen; see Introduction), is described. This assay uses bovine testes extract as the solid phase antigen, and high-titer anti-Sxs antisera and a urease-conjugated anti rat-IgG as the first and second antibody respectively. The urea containing substrate causes a pH shift in a positive reaction, which in turn is visualized by the use of bromocresol purple as a pH indicator. The method, and some representative applications of it, are described in detail. PMID:3610155

  1. 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

  2. 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

  3. Lipid antigens in immunity

    PubMed Central

    Dowds, C. Marie; Kornell, Sabin-Christin

    2014-01-01

    Lipids are not only a central part of human metabolism but also play diverse and critical roles in the immune system. As such, they can act as ligands of lipid-activated nuclear receptors, control inflammatory signaling through bioactive lipids such as prostaglandins, leukotrienes, lipoxins, resolvins, and protectins, and modulate immunity as intracellular phospholipid- or sphingolipid-derived signaling mediators. In addition, lipids can serve as antigens and regulate immunity through the activation of lipid-reactive T cells, which is the topic of this review. We will provide an overview of the mechanisms of lipid antigen presentation, the biology of lipid-reactive T cells, and their contribution to immunity. PMID:23999493

  4. 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

  5. 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

  6. 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.

  7. Antigen smuggling in tuberculosis.

    PubMed

    Hudrisier, Denis; Neyrolles, Olivier

    2014-06-11

    The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells. PMID:24922567

  8. Challenge assay in vitro using lymphocyte blastogenesis for the contact hypersensitivity assay.

    PubMed

    Kashima, R; Okada, J; Ikeda, Y; Yoshizuka, N

    1993-10-01

    To confirm positivity in routine guinea pig studies, contact allergenicity was investigated by a challenge assay in vitro using a co-culture of autologous lymphocytes passed through a nylon-wool column and antigen-presenting cells (APCs) modified with or without antigen. Proliferation of the lymphocytes primed with ovalbumin and/or 2,4-dinitrochlorobenzene was antigen specific and dependent on the presence of APCs (peripheral blood monocytes, splenic macrophages and macrophages induced by liquid paraffin). For another nine haptens, primed lymphocytes proliferated significantly more than control lymphocytes; the stimulation index (SI; ratio between [3H]methylthymidine ([3H]TdR) incorporation of lymphocytes with antigen-modified APCs and [3H]TdR incorporation of lymphocytes with APCs not modified by antigen) was 1.6-4.8 in sensitized animals whereas it was about 1.0 in control animals. Sodium dodecyl sulfate did not cause lymphocyte proliferation. The SI value in vitro was correlated with both the positive rate in vivo (r = 0.736) and the mean response score in vivo (r = 0.645). Thus, it was possible to confirm that positivity in routine experiments was a true sign of allergy. A combination of this assay and short-term animal studies would provide an efficient assessment of the allergic potential of chemicals. PMID:8225135

  9. 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

  10. Duality of β-glucan microparticles: antigen carrier and immunostimulants.

    PubMed

    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

  11. Aspergillus antigen skin test (image)

    MedlinePlus

    The aspergillus antigen skin test determines whether or not a person has been exposed to the mold aspergillus. It is performed by injecting an aspergillus antigen under the skin with a needle. After 48 ...

  12. Development of multiplex serological assay for the detection of human African trypanosomiasis.

    PubMed

    Nzou, Samson Muuo; Fujii, Yoshito; Miura, Masashi; Mwau, Matilu; Mwangi, Anne Wanjiru; Itoh, Makoto; Salam, Md Abdus; Hamano, Shinjiro; Hirayama, Kenji; Kaneko, Satoshi

    2016-04-01

    Human African trypanosomiasis (HAT) is a disease caused by Kinetoplastid infection. Serological tests are useful for epidemiological surveillance. The aim of this study was to develop a multiplex serological assay for HAT to assess the diagnostic value of selected HAT antigens for sero-epidemiological surveillance. We cloned loci encoding eight antigens from Trypanosoma brucei gambiense, expressed the genes in bacterial systems, and purified the resulting proteins. Antigens were subjected to Luminex multiplex assays using sera from HAT and VL patients to assess the antigens' immunodiagnostic potential. Among T. b. gambiense antigens, the 64-kDa and 65-kDa invariant surface glycoproteins (ISGs) and flagellar calcium binding protein (FCaBP) had high sensitivity for sera from T. b. gambiense patients, yielding AUC values of 0.871, 0.737 and 0.858 respectively in receiver operating characteristics (ROC) analysis. The ISG64, ISG65, and FCaBP antigens were partially cross-reactive to sera from Trypanosoma brucei rhodesiense patients. The GM6 antigen was cross-reactive to sera from T. b. rhodesiense patients as well as to sera from VL patients. Furthermore, heterogeneous antibody responses to each individual HAT antigen were observed. Testing for multiple HAT antigens in the same panel allowed specific and sensitive detection. Our results demonstrate the utility of applying multiplex assays for development and evaluation of HAT antigens for use in sero-epidemiological surveillance. PMID:26519611

  13. Localization of hepatitis B surface antigen in conventional paraffin sections of the liver. Comparison of immunofluorescence, immunoperoxidase, and orcein staining methods with regard to their specificity and reliability as antigen marker.

    PubMed Central

    Nayak, N. C.; Sachdeva, R.

    1975-01-01

    Hepatitis B antigen (HBAg) has been demonstrated in conventional formalin-fixed paraffin-embedded liver tissue by peroxidase and fluorescent immunostaining as well as by orcein. Complete locational and morphologic identity is seen between material stained by specific immunologic methods and by orcein. The antigen is restricted to the cytoplasm and is generally observed in the hepatocyte; it is present in three morphologic forms. Certain morphologic forms can even be identified in hematoxylin and eosin-stained tissue. Results of immunostaining procedures indicate that the antigen demonstrated in this study consists entirely of surface coat of hepatitis B virus (HBsAg). This seems to be the only component revealed by orcein staining. The latter is considered to be a good marker of the surface antigen and to have certain advantages over immunostaining. It is suggested that suitability of conventional paraffin sections for the detection of HBAg has wide and important implications. Images Figures 1-5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:55076

  14. Effects of T cell immunoglobulin and mucin domain-containing molecule-3 signaling molecule on human monocyte-derived dendritic cells with hepatitis B virus surface antigen stimulation in vitro.

    PubMed

    Yu, Zhenjun; Jiang, Ting; Zhu, Min; Pan, Kechuan; Yan, Fei; Zhu, Jiansheng

    2016-03-01

    The aim of the present study was to investigate the in vitro effects of hepatitis B virus surface antigen (HBsAg) on the immune function of human monocyte-derived dendritic cells (MD‑DCs), and the moderating role of T cell immunoglobulin and mucin domain‑containing molecule‑3 (Tim‑3) signaling molecule. The monocytes, obtained from healthy adult peripheral blood, were incubated with recombinant human granulocyte‑macrophage colony‑stimulating factor and interleukin (IL)‑4 to induce DCs. DC‑associated cell markers were detected using flow cytometry. MD‑DCs were treated with HBsAg (5 µg/ml) in vitro for 48 h and subsequently, cell markers, lymphocyte stimulatory capacity, signaling protein and downstream cytokines were assessed. In addition, a Tim‑3 monoclonal antibody was used to inhibit the Tim‑3 signaling pathway, and subsequently the immune responses of MD‑DCs to HBsAg stimulation were determined using the aforementioned method. The cell phenotype expressions of MD‑DCs were all significantly increased with cluster of differentiation (CD)11c at 70.09±0.57%, human leukocyte antigen‑DR at 79.83±2.12%, CD80 at 48.33±7.34% and CD86 at 44.21±5.35%. The treatment of MD‑DCs with HBsAg resulted in a CD80 and CD86 enhanced expression, enhanced lymphocyte stimulatory capacity, upregulated expression of Tim‑3 and nuclear factor‑κB (NF‑κB), as well as enhanced cytokine secretion of IL‑6, IL‑10 and interferon (IFN)‑γ. However, a reduced immune response of MD‑DCs in response to HBsAg stimulation was observed when the Tim‑3 signaling pathway was inhibited prior to stimulation. The expression of NF‑κB was decreased and the cytokine secretion level of IL‑6, IL‑10 and IFN‑γ were downregulated. The treatment with HBsAg in vitro resulted in an enhanced immune response of MD‑DCs, which may be positively regulated by the Tim-3 signaling molecule. PMID:26820685

  15. Response of health care workers with isolated antibody to hepatitis B core antigen to hepatitis B vaccine.

    PubMed

    Chiarakul, Supawadee; Eunumjitkul, Krissana; Vorapimol, Ar-Reerat; Kaewkungwal, Jaranit; Chimparlee, Nitinan; Poovorawan, Yong

    2011-07-01

    Isolated hepatitis B core antibody (antiHBc) without hepatitis B surface antigen (HBsAg) or hepatitis B surface antibody (antiHBs) is found during routine screening for hepatitis B virus (HBV) markers. Isolated antiHBc may indicate immunity against HBV or occult infection. To determine the immune response of health care workers (HCWs) with isolated antiHBc, HCWs were divided into two groups. A single dose of recombinant hepatitis B (HB) vaccine was administered to HCWs with isolated antiHBc (n = 36) and healthy HCWs (n = 20) seronegative for HBsAg, antiHBc and antiHBs. One month later, the subjects were tested for antiHBs. Twenty-one of 36 HCW (58.3%) in the antiHBc group had antiHBs, while only 1 of 20 HCW (5.0%) in the seronegative control group had a detectable antiHBs titer exceeding 10 mIU/ml. The antiHBs response in HCWs with antiHBc was significantly higher than in the seronegative group. The subjects' sera were tested for HBV DNA by nested PCR. Of those with antiHBc, 4 had detectable HBV DNA (occult HBV infection). None of these 4 responded to the vaccine. Therefore, the response elicited by a single dose of HB vaccine administered to patients with antiHBc may serve as an indicator of occult HBV infection. PMID:22299465

  16. Cationic Lipid-Formulated DNA Vaccine against Hepatitis B Virus: Immunogenicity of MIDGE-Th1 Vectors Encoding Small and Large Surface Antigen in Comparison to a Licensed Protein Vaccine

    PubMed Central

    Endmann, Anne; Klünder, Katharina; Kapp, Kerstin; Riede, Oliver; Oswald, Detlef; Talman, Eduard G.; Schroff, Matthias; Kleuss, Christiane; Ruiters, Marcel H. J.; Juhls, Christiane

    2014-01-01

    Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals. PMID:24992038

  17. Comparison of non-magnetic and magnetic beads in bead-based assays.

    PubMed

    Hansenová Maňásková, Silvie; van Belkum, Alex; Endtz, Hubert P; Bikker, Floris J; Veerman, Enno C I; van Wamel, Willem J B

    2016-09-01

    Multiplex bead-based flow cytometry is an attractive way for simultaneous, rapid and cost-effective analysis of multiple analytes in a single sample. Previously, we developed various bead-based assays using non-magnetic beads coated with Staphylococcus aureus and Streptococcus pneumoniae antigens for the detection of antibodies. Here, we compared the performance of the assay using non-magnetic beads with one based on the newly developed magnetic beads. We optimized the magnetic beads' coupling procedure and antibody detection assays for S. aureus and S. pneumoniae antigens and we measured IgG in human pooled serum against a series of S. aureus and S. pneumoniae-derived antigens in a singleplex and in a multiplex assay, respectively. For the multiplex assay, the comparison between magnetic and non-magnetic beads showed: i) in the majority of the cases (13 of the 17 tested S. pneumoniae antigens) significantly higher Median Fluorescence Intensity (MFI) values, ii) lower detection limits, iii) lower coefficient of variation (CV: 12% vs. 7% for non-magnetic vs. magnetic beads), so lower inter-assay variation and hence higher reproducibility. Magnetic bead coupling is cost effective, as we used 25% of the normal amount of antigen and only 50% of the beads in comparison to the non-magnetic beads. This optimized magnetic-based assay, which combines ease of use with an improved assay performance, allows detection of antibodies with a low titer that are potentially missed with the non-magnetic-based assay. PMID:27296810

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

    PubMed

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

    2016-07-01

    Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand. PMID:27101782

  19. Estimation of Recent and Long-Term Malaria Transmission in a Population by Antibody Testing to Multiple Plasmodium falciparum Antigens

    PubMed Central

    Ondigo, Bartholomew N.; Hodges, James S.; Ireland, Kathleen F.; Magak, Ng'wena G.; Lanar, David E.; Dutta, Sheetij; Narum, David L.; Park, Gregory S.; Ofulla, Ayub V.; John, Chandy C.

    2014-01-01

    Background. Tools that estimate recent and long-term malaria transmission in a population would be highly useful for malaria elimination programs. Methods. The prevalence of antibodies to 11 Plasmodium falciparum antigens was assessed by cytometric bead assay or enzyme-linked immunosorbent assay in 1000 people in a highland area of Kenya over 14 months, during a period of interrupted malaria transmission. Results. Antibodies differed by antigen in acquisition with age: rapid (>80% antibody positive by age 20 years, 5 antigens), moderate (>40% positive by age 20 years, 3 antigens), or slow (<40% positive by age 20 years, 3 antigens). Antibody seroreversion rates in the 14 months between samples decreased with age rapidly (7 antigens), slowly (3 antigens), or remained high at all ages (schizont extract). Estimated antibody half-lives in individuals >10 years of age were long (40 to >80 years) for 5 antigens, moderate (5–20 years) for 3 antigens, and short (<1 year) for 3 antigens. Conclusions. Antibodies to P. falciparum antigens in malaria-endemic areas vary by age, antigen, and time since last exposure to P. falciparum. Multiplex P. falciparum antibody testing could provide estimates of long-term and recent malaria transmission and potentially of a population's susceptibility to future clinical malaria. PMID:24737801

  20. Two-dimensional electrophoresis and characterization of antigens from Paracoccidioides brasiliensis.

    PubMed

    da Fonseca, C A; Jesuino, R S; Felipe, M S; Cunha, D A; Brito, W A; Soares, C M

    2001-06-01

    Paracoccidioides brasiliensis is a fungal pathogen of humans. To identify antigens from P. brasiliensis we fractionated a crude preparation of proteins from the fungus and detected the IgG reactive proteins by immunoblot assays of yeast cellular extracts with sera of patients with paracoccidioidomycosis (PCM). We identified and characterized six new antigens by amino acid sequencing and homology search analyses with other proteins deposited in a database. The newly characterized antigens were highly homologous to catalase, fructose-1,6-biphosphate aldolase (aldolase), glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and triosephosphate isomerase from several sources. The characterized antigens presented preferential synthesis in yeast cells, the host fungus phase. PMID:11418327

  1. Cancer testis antigen and immunotherapy

    PubMed Central

    Krishnadas, Deepa Kolaseri; Bai, Fanqi; Lucas, Kenneth G

    2013-01-01

    The identification of cancer testis (CT) antigens has been an important advance in determining potential targets for cancer immunotherapy. Multiple previous studies have shown that CT antigen vaccines, using both peptides and dendritic cell vaccines, can elicit clinical and immunologic responses in several different tumors. This review details the expression of melanoma antigen family A, 1 (MAGE-A1), melanoma antigen family A, 3 (MAGE-A3), and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) in various malignancies, and presents our current understanding of CT antigen based immunotherapy.

  2. A Computationally Designed Serological Assay for Porcine Epidemic Diarrhea Virus.

    PubMed

    Song, Yunfeng; Singh, Pankaj; Nelson, Eric; Ramamoorthy, Sheela

    2016-08-01

    The periodic emergence of new infectious agents and the genetic and antigenic evolution of existing agents necessitate the improvement of technology for the rapid development of diagnostic assays. The porcine epidemic diarrhea virus (PEDV) emerged in the United States in 2013, causing severe economic damage to the pork industry. The primary goal of this study was to develop methods to reduce the lead time for serological assay development. An approach involving the computational prediction of diagnostic targets, followed by a rapid synthesis of antigens, was adopted to achieve this objective. To avoid cross-reactivity with other closely related swine coronaviruses, the N protein sequences of PEDV were analyzed to identify sequences unique to PEDV. The potential antigenicity of the identified sequence was predicted computationally using the Jameson-Wolf method. A sequence with a high antigenic index was rapidly synthesized using an in vitro transcription and translation system to yield the diagnostic antigen. The computationally designed enzyme-linked immunosorbent assay (ELISA) was validated using 169 field sera, whose statuses were determined by a PEDV-specific immunofluorescence assay. Comparison of the computationally designed ELISA to a conventionally developed ELISA, using bacterially expressed N protein, and to the immunofluorescence assay showed a high degree of agreement among the three tests (mean kappa statistic, 0.842). The sensitivity and specificity, compared to the conventionally developed assay, were 90.62 and 95.18, respectively. Therefore, the described approach is useful in reducing the development time for serological assays in the face of an infectious disease outbreak. PMID:27225413

  3. Sandwich-ELISA detection of venom antigens in envenoming by Phoneutria nigriventer spider.

    PubMed

    Chávez-Olórtegui, C; Bohórquez, K; Alvarenga, L M; Kalapothakis, E; Campolina, D; Maria, W S; Diniz, C R

    2001-06-01

    Enzyme linked immunosorbent assays (ELISA) were developed to detect antigens from Phoneutria nigriventer spider venom. Horse anti-P. nigriventer immunoglobulins were prepared by immunoaffinity chromatography and used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to correctly discriminate between the circulating antigens in mice that were experimentally inoculated with P. nigriventer venom from those in mice inoculated with Lycosa sp. and Loxosceles intermedia spider venoms, Tityus serrulatus scorpion venom and Apis mellifera bee venom. Measurable absorbance signals were obtained with 0.8ng of venom per assay. The ELISA was used to follow the kinetic distribution of antigens in experimentally envenomed mice and to detect antigens in the sera of patients envenomed by P. nigriventer. PMID:11137553

  4. The antigenic structure of the influenza B virus hemagglutinin: operational and topological mapping with monoclonal antibodies.

    PubMed

    Berton, M T; Webster, R G

    1985-06-01

    We have probed the antigenic structure of the influenza B virus hemagglutinin (HA) with monoclonal antibodies specific for the HA of influenza B virus, B/Oregon/5/80. Seventeen laboratory-selected antigenic variants of this virus were analyzed by hemagglutination-inhibition (HI) assays or ELISA and an operational antigenic map was constructed. In addition, the monoclonal antibodies were tested in a competitive binding assay to construct a topological map of the antigenic sites. In contrast to the influenza A virus HA, only a single immunodominant antigenic site composed of several overlapping clusters of epitopes was defined by the HI-positive antibodies. Three variants could be distinguished from the parental virus with polyclonal antisera by HI and infectivity reduction assays suggesting that changes in this antigenic site may be sufficient to provide an epidemiological advantage to influenza B viruses in nature. In addition, two nonoverlapping epitopes of unknown biological significance were identified in the competitive binding analysis by two monoclonal antibodies with no HI activity and little or no neutralizing activity. We previously identified single amino acid substitutions in the HAs of the antigenic variants used in this study (M. T. Berton, C. W. Naeve, and R. G. Webster (1984), J. Virol. 52, 919-927). These changes occurred in regions of the molecule which, by amino acid sequence alignment, appeared to correspond to proposed antigenic sites A and B on the H3 HA of influenza A virus. Correlation with the antigenic map established in this report, however, demonstrates that the amino acid residues actually contribute to a single antigenic site on the influenza B virus HA and suggests significant differences in the antigenic structures of the influenza A and B virus HAs. PMID:2414911

  5. Serodiagnosis of American trypanosomosis by using nonpathogenic trypanosomatid antigen.

    PubMed Central

    Monteón, V M; Guzmán-Rojas, L; Negrete-García, C; Rosales-Encina, J L; Lopez, P A

    1997-01-01

    Crithidia luciliae, a nonpathogenic trypanosomatid, could provide a good alternative source of antigen for serodiagnosis of Chagas' disease. An enzyme-linked immunosorbent assay showed 100% sensitivity and 83% specificity when 91 human serum samples from Chagas' disease patients and 127 human serum samples from people suffering from toxoplasmosis (21 samples), leishmaniasis (32 samples), systemic rheumatic diseases (33 samples), and heart diseases (41 samples) were tested simultaneously with Trypanosoma cruzi and C. luciliae crude extracts. By Western blotting, an immunodominant band (30 kDa) was recognized by chagasic sera on the C. luciliae crude extract; specificity reached 97% with respect to this protein band. The carbohydrate moieties were not antigenic. PMID:9399545

  6. The detection of two antigenic groups among Renibacterium salmoninarum isolates.

    PubMed

    Bandín, I; Santos, Y; Magariños, B; Barja, J L; Toranzo, A E

    1992-07-01

    The analysis of the membrane proteins and their antigenic properties in a group of 14 geographically diverse strains of Renibacterium salmoninarum revealed the existence of antigenic diversity within this species. Eleven isolates, including the type strain ATCC 33209, shared a similar protein profile with a major component of 57 kDa whereas three strains showed a common pattern with a major protein of 30 kDa. The quantitative agglutination tests and Western blotting assays seem to indicate the existence of serological heterogeneity, with two distinct groups being detected. PMID:1521757

  7. A novel agglutination test for antigen-specific detection of platelet antibodies.

    PubMed

    Meyer, Oliver; Agaylan, Ashraf; Borchert, Hans-Hubert; Aslan, Tunay; Bombard, Stéphane; Kiesewetter, Holger; Salama, Abdulgabar

    2006-10-15

    A simple and rapid antigen-specific assay for the identification antibodies to platelets is lacking, yet. Red-dyed polystyrene microbeads were coated with monoclonal antibodies to various platelet glycoprotein complexes, and used for the detection of platelet autoantibodies and alloantibodies. The results were largely identical with those obtained by monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). The new test is reliable yet less complex and time-consuming than the currently available assays, and it can be implemented in any routine laboratory. PMID:16933262

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  9. Type VI secretion system sheaths as nanoparticles for antigen display

    PubMed Central

    Del Tordello, Elena; Danilchanka, Olga; McCluskey, Andrew J.; Mekalanos, John J.

    2016-01-01

    The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between cells by a mechanism analogous to phage tail contraction. T6SS sheaths are cytoplasmic tubular structures composed of stable VipA-VipB (named for ClpV-interacting protein A and B) heterodimers. Here, the structure of the VipA/B sheath was exploited to generate immunogenic multivalent particles for vaccine delivery. Sheaths composed of VipB and VipA fused to an antigen of interest were purified from Vibrio cholerae or Escherichia coli and used for immunization. Sheaths displaying heterologous antigens generated better immune responses against the antigen and different IgG subclasses compared with soluble antigen alone. Moreover, antigen-specific antibodies raised against sheaths presenting Neisseria meningitidis factor H binding protein (fHbp) antigen were functional in a serum bactericidal assay. Our results demonstrate that multivalent nanoparticles based on the T6SS sheath represent a versatile scaffold for vaccine applications. PMID:26929342

  10. A Novel Malaria Vaccine Candidate Antigen Expressed in Tetrahymena thermophila

    PubMed Central

    Eleni-Muus, Janna; Aldag, Ingo; Samuel, Kay; Creasey, Alison M.; Hartmann, Marcus W. W.; Cavanagh, David R.

    2014-01-01

    Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens. PMID:24489871

  11. Evaluation of the Procleix Ultrio Plus ID NAT assay for detection of HIV 1, HBV and HCV in blood donors

    PubMed Central

    Makroo, Raj Nath; Chowdhry, Mohit; Bhatia, Aakanksha; Antony, Minimol

    2015-01-01

    Introduction: The Procleix Ultrio Plusassay is a new-generation qualitative in vitro nucleic acid amplification test used to screen for human immunodeficiency virus type 1 (HIV-1) RNA, hepatitis C virus (HCV) RNA and hepatitis B virus (HBV) DNA in blood donors. This study was performed to compare the Procleix Ultrio assay with the new-generation Procleix Ultrio Plus Nucleic Acid Test (NAT) assays. Materials and Methods: Ten thousand three hundred and two donor samples were run in parallel for ID NAT using the Procleix Ultrio and the Procleix Ultrio Plus assay. Simultaneously, enzyme-linked immunosorbent assay testing was performed on an EVOLIS Walk away System for HIV, HCV, HBsAg and anti-HBc. Reactive samples were confirmed using polymerase chain reaction. Results: In the 10,302 samples tested during the study period, we identified 15 NAT yields, and all these revealed HBV DNA in the discriminatory assays. Eight of these were exclusive yields from the Ultrio Plus assay and the remaining seven cases were determined as HBV NAT yield, both by the Procleix Ultrio as well as the Ultrio Plus assays, i.e. “Combined” yields. No HCV or HIV 1 yields were detected during the study period by either of two assays. Conclusion: With an overall yield rate of 1 in 687 and an exclusive yield rate of 1 in 1287, the Procleix Ultrio Plus assay proved to be highly sensitive in detecting occult HBV infections. PMID:25722569

  12. Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes

    PubMed Central

    2012-01-01

    Background Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process. Results The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could

  13. T-cell recognition of a cross-reactive antigen(s) in erythrocyte stages of Plasmodium falciparum and Plasmodium yoelii: inhibition of parasitemia by this antigen(s).

    PubMed Central

    Lucas, B; Engels, A; Camus, D; Haque, A

    1993-01-01

    parasites displayed about a 50% lower level of parasitemia. These results demonstrated the existence of a cross-reactive antigen(s) between a murine and a human Plasmodium species, as determined from both in vivo and in vitro biological assays, and indicated the reactivity of mainly CD8+ T cells with this antigen. PMID:8104901

  14. Cutaneous lymphocyte antigen expression on human effector B cells depends on the site and on the nature of antigen encounter.

    PubMed

    Kantele, Anu; Savilahti, Erkki; Tiimonen, Heidi; Iikkanen, Katja; Autio, Soile; Kantele, Jussi M

    2003-12-01

    In contrast to T cells, information on skin-homing B cells expressing the cutaneous lymphocyte antigen (CLA) is sparse. CLA expression on human B cells was investigated among circulating immunoglobulin-secreting cells (ISC) and among antigen-specific antibody-secreting cells (ASC) elicited by parenteral, oral or rectal primary immunization, or by parenteral or oral secondary immunization with Salmonella typhi Ty21a. CLA expression was examined by combining cell sorting with an enzyme-linked immunospot assay. Among all ISC, the proportion of CLA(+) cells was 13-21%. Parenteral immunization induced antigen-specific ASC of which 13% were CLA(+), while oral and rectal immunizations were followed by only 1% of CLA(+) ASC (p<0.001). Oral re-immunization was followed by an up-regulation of CLA (34-48%) regardless of the route of priming. Parenteral re-immunization elicited ASC of which 9-14% were CLA(+). In conclusion, the expression of CLA on human effector B cells depends on the site of antigen encounter: intestinal stimulation elicits cells with no CLA, while parenteral encounter elicits significant numbers of CLA(+) cells. Even though primary antigen encounter in the intestine failed to stimulate CLA expression, up-regulation of CLA was found upon intestinal antigen re-encounter. These findings may be of relevance in the pathogenesis of some cutaneous disorders. PMID:14635035

  15. Suppression of furin by interferon-γ and the impact on hepatitis B virus antigen biosynthesis in human hepatocytes.

    PubMed

    Wu, Jia-Feng; Hsu, Hong-Yuan; Ni, Yen-Hsuan; Chen, Huey-Ling; Wu, Tzee-Chung; Chang, Mei-Hwei

    2012-07-01

    The roles of furin and intrahepatic cytokines in chronic heptatitis B virus (HBV) infection remain largely unknown. Here, we examined the relations between furin, IL-10, IL-12β, interferon (IFN)-γ, programed death (PD)-1, programed death ligand (PD-L)1, and the suppression of hepatitis B e antigen (HBeAg) and surface antigen (HBsAg) biosynthesis. Liver biopsies were performed on 20 chronically HBV-infected (15 HBeAg-positive and 5 HBeAg-negative) patients to assess liver inflammation/fibrosis, and mRNA levels of furin, IL-10, IL-12β, IFN-γ, PD-1, and PD-L1 were assessed by quantitative real-time PCR. IFN-γ mRNA abundance was associated with lower furin mRNA levels and higher PD-1 and PD-L1 mRNA levels in liver tissue from HBeAg-positive patients. IL-10 and IL-12β mRNA levels positively correlated with IFN-γ expression levels (P < 0.05). PD-L1 and furin mRNA levels were further assessed in IFN-γ-stimulated hepatoma cell lines with (HepG2.2.15 cells) and without (HepG2 and Huh7 cells) HBV replication. IFN-γ enhanced PD-L1 expression in hepatoma cells. In HepG2.2.15 cells, IFN-γ further suppressed furin and HBeAg expression. Furin inhibition and knockdown in HepG2.2.15 cells also down-regulated HBeAg and HBsAg biosynthesis. These data suggest that IFN-γ modulates the inflammatory response to avoid excessive hepatocyte damage through the enhancement of PD-1/PD-L1 expression, whereas furin suppression may contribute to a reduction in HBeAg/HBsAg biosynthesis. PMID:22634051

  16. Human immunodeficiency virus coinfection with hepatitis B virus leads to a decrease in extracellular and intracellular hepatitis B antigen.

    PubMed

    Pan, Wei; Wu, Zuoqiao; Wu, Shuwen; Guo, Deyin; Gong, Xiaoyan; Po, Tien

    2015-04-01

    Chronic hepatitis B virus (HBV) infection could cause severe liver disease including cirrhosis, hepatocellular carcinoma, and end-stage liver failure in HIV-positive individuals. The available data from clinical studies suggest that HIV infection modulates the HBV-specific T cell response. However, the virological and molecular aspects of HIV-HBV coinfection are currently poorly understood due to the lack of appropriate model systems. In this study, the effect of HIV infection on the life cycle of HBV was explored using an in vitro model system. The present data show that the extracellular and intracellular hepatitis B surface antigen (HBsAg) and e antigen (HBeAg) decrease significantly in HepG2 cells cotransfected with HIV NL4-3 and pHBV1.3 as compared to those cells transfected only with pHBV1.3. Moreover, a significant decrease in HBV DNA and mRNA expression was also observed in the cotransfected cells. HIV Rev protein, an RNA-bound regulatory protein, could significantly decrease the expression levels of extracellular and intracellular HBsAg and HBeAg by mediating the expression of HBV mRNA in cells cotransfected with plasmids containing HIV-1 Rev and pHBV1.3. Further experiments demonstrate that HIV Rev manipulated neither the promoters of HBV nor the nuclear export of HBV mRNA. These results from the in vitro model system might provide clues to further understand the rapid progression of liver disease in HIV-HBV-coinfected patients. PMID:25517882

  17. Lentiviral vector encoding ubiquitinated hepatitis B core antigen induces potent cellular immune responses and therapeutic immunity in HBV transgenic mice.

    PubMed

    Dai, Shenglan; Zhuo, Meng; Song, Linlin; Chen, Xiaohua; Yu, Yongsheng; Zang, Guoqing; Tang, Zhenghao

    2016-07-01

    Predominant T helper cell type 1 (Th1) immune responses accompanied by boosted HBV-specific cytotoxic T lymphocyte (CTL) activity are essential for the clearance of hepatitis B virus (HBV) in chronic hepatitis B (CHB) patients. Ubiquitin (Ub) serves as a signal for the target protein to be recognized and degraded through the ubiquitin-proteasome system (UPS). Ubiquitinated hepatitis B core antigen (Ub-HBcAg) has been proved to be efficiently degraded into the peptides, which can be presented by major histocompatibility complex (MHC) class I resulting in stimulating cell-mediated responses. In the present study, lentiviral vectors encoding Ub-HBcAg (LV-Ub-HBcAg) were designed and constructed as a therapeutic vaccine for immunotherapy. HBcAg-specific cellular immune responses and anti-viral effects induced by LV-Ub-HBcAg were evaluated in HBV transgenic mice. We demonstrated that immunization with LV-Ub-HBcAg promoted the secretion of cytokines interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), generated remarkably high percentages of IFN-γ-secreting CD8(+) T cells and CD4(+) T cells, and enhanced HBcAg-specific CTL activity in HBV transgenic mice. More importantly, vaccination with LV-Ub-HBcAg could efficiently decreased the levels of serum hepatitis B surface antigen (HBsAg), HBV DNA and the expression of HBsAg and HBcAg in liver tissues of HBV transgenic mice. In addition, LV-Ub-HBcAg could upregulate the expression of T cell-specific T-box transcription factor (T-bet) and downregulate the expression of GATA-binding protein 3 (GATA-3) in spleen T lymphocytes. The therapeutic vaccine LV-Ub-HBcAg could break immune tolerance, and induce potent HBcAg specific cellular immune responses and therapeutic effects in HBV transgenic mice. PMID:26874581

  18. Evaluation of the siemens HIV antigen-antibody immunoassay.

    PubMed

    Vallefuoco, Luca; Aden Abdi, Fatima; Sorrentino, Rosanna; Spalletti-Cernia, Daniela; Mazzarella, Claudia; Barbato, Sara; Perna, Enzo; Buffolano, Wilma; Di Nicuolo, Giuseppe; Portella, Giuseppe

    2014-01-01

    Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigen and antibodies are available on the international market and are currently used for blood donor screening and for HIV diagnosis. In this study we evaluated the performance of the novel automated fourth-generation ADVIA Centaur® HIV Ag/Ab Combo assay. The assay detected seroconversion at the same bleed or at least one bleed earlier in panels with respect to other assays and showed a detection efficacy equal to those of other assays in a low-titer panel. Samples obtained from blood donors (n = 2,778) or from HIV-positive patients (HIV-1 B subtype, n = 82; non-B subtype, n = 71) were also tested, showing a good correlation with other fourth-generation assays. We assessed the performance of 3 fourth-generation assays for detecting in utero transmitted anti-HIV antibodies and found a more specific detection efficiency with the ADVIA Centaur HIV Ag/Ab Combo assay compared to the other fourth-generation assays. PMID:24557036

  19. Human leucocyte antigens in tympanosclerosis.

    PubMed

    Dursun, G; Acar, A; Turgay, M; Calgüner, M

    1997-02-01

    This study was designed to evaluate the association between certain HLA antigens and tympanosclerosis. The serum concentrations of HLA antigens were measured by a microlymphocytotoxicity technique in patients with tympanosclerosis and compared with a healthy control group. The serum levels of HLA-B35 and -DR3 were significantly higher in the patients with tympanosclerosis. This result suggests that certain types of HLA antigens may play an important role as an indicator or mediator in the pathogenesis of tympanosclerosis. PMID:9088683

  20. Protein polymorphism of a human plasma apolipoprotein D antigenic epitope.

    PubMed

    Camato, R; Marcel, Y L; Milne, R W; Lussier-Cacan, S; Weech, P K

    1989-06-01

    Based on our previous observation that monoclonal antibody anti-apoD-4E11 reacted with several HDL proteins we studied them further with three questions in mind: i) is there common protein polymorphism in healthy individuals? ii) how many proteins are present and what are their characteristics? iii) are they all apolipoproteins and do they have the same lipoprotein distribution as apoD? Isolated, delipidated apoD was used as a standard for radioimmunometric assay of plasma with antibody 4E11. The antigen varied from 3 to 11 mumol-equivalents of apoD per liter of plasma (equivalent to 5-20 mg apoD/dl plasma) with means of 6.1 and 6.8 mumol/l in men and women, respectively. Two-dimensional electrophoresis of plasma found up to eight 4E11-antigenic-proteins of different Mr, each heterogeneous in pI. All plasmas tested contained apoD and an Mr 38,000 antigen, the latter being the most immunoreactive. Six proteins of Mr 70,000-94,000 were found, but the number varied between subjects. Eighty nine percent of the plasma antigen was associated with lipoproteins: 83% with HDL and VHDL, 5% with LDL and VLDL. Lipoproteins of all sizes, separated by polyacrylamide gradient gel electrophoresis, contained the antigen. ApoD was almost the only 4E11-antigen in LDL, and was in two states: the one free, the other an apoD-apoB mixed disulfide complex. The apparent proportions of higher Mr antigens increased with increasing lipoprotein density, and the proportion of apoD decreased reciprocally. None of these 4E11-antigenic-proteins cross-reacted with antiserum to retinol-binding protein. PMID:2477480

  1. Gender differences in chronic HBsAg carriers in Italy: Evidence for the independent role of male sex in severity of liver disease.

    PubMed

    Stroffolini, Tommaso; Esvan, Rozenn; Biliotti, Elisa; Sagnelli, Evangelista; Gaeta, Giovanni Battista; Almasio, Piero Luigi

    2015-11-01

    It has been shown that sexual hormones have an opposite effect on hepatic fibrosis progression and hepatocellular carcinoma development. Sex differences among 2,762 chronic HBsAg carriers consecutively referring Italian hospitals in 2001 and in 2007 have been evaluated, particularly focusing on the role of gender on severity of liver disease. The overall sex ratio (males/females) was 2.6. Females were more likely born abroad and new diagnosis cases; but less likely HIV coinfected. No sex difference was observed regarding coinfection with other hepatitis viruses. The sex ratio linearly increased with increasing severity of liver disease, being 1.3 in normal ALT, 2.8 in chronic hepatitis, 3.6 in liver cirrhosis, and 6.8 in hepatocellular carcinoma. Adjustment by multiple logistic regression analysis for the confounding effect of age, alcohol intake, HDV infection, HCV infection, and BMI shows that male gender is an independent predictor of the likelihood of more severe liver disease (O.R. 1.7; C.I. 95% = 1.3-2.1). HBV-DNA levels resulted not associated with the outcome of chronic HBV infection. Despite some potential risk factors associated with liver disease, such as HBV genotype or mutations, not having been controlled for due to lack of availability, the observed sex disparity in the outcome of chronic HBV infection may support biological observation that HBV infection could be considered a sex hormone-responsive virus. PMID:26037919

  2. Immune response elicited by an intranasally delivered HBsAg low-dose adsorbed to poly-ε-caprolactone based nanoparticles.

    PubMed

    Jesus, Sandra; Soares, Edna; Costa, João; Borchard, Gerrit; Borges, Olga

    2016-05-17

    Among new strategies to increase hepatitis B virus (HBV) vaccination, especially in developing countries, the development of self-administered vaccines is considered one of the most valuable. Nasal vaccination using polymeric nanoparticles (NPs) constitutes a valid approach to this issue. In detail, poly-ε-caprolactone (PCL)/chitosan NPs present advantages as a mucosal vaccine delivery system: the high resistance of PCL against degradation in biological fluids and the mucoadhesive and immunostimulatory properties of chitosan. In vitro studies revealed these NPs were retained in a mucus-secreting pulmonary epithelial cell line and were capable of entering into differentiated epithelial cells. The intranasal (IN) administration of 3 different doses of HBsAg (1.5μg, 5μg and 10μg) adsorbed on a fixed amount of PCL/chitosan NPs (1614μg) generated identical titers of serum anti-HBsAg IgG and anti-HBsAg sIgA in mice nasal secretions. Besides other factors, the NP surface characteristics, particularly, zeta potential differences among the administered formulations are believed to be implicated in the outcome of the immune response generated. PMID:26976502

  3. Immunochemistry of sea anemone toxins: structure-antigenicity relationships and toxin-receptor interactions probed by antibodies specific for one antigenic

    SciTech Connect

    Ayeb, M.E.; Bahraoui, E.M.; Granier, C.; Beress, L.; Rochat, H.

    1986-11-04

    Two antibody subpopulations directed against Anemonia sulcata toxin I or II have been purified by immunoaffinity chromatography. These antibodies are specific for a single antigenic region and were used in a structure-antigenicity relationship study using homologous toxins and chemically modified derivatives of A. sulcata toxin II. Asp-7 and/or Asp=9 and Gln-47 of toxin II were found to be implicated in the antigenic region recognized by the two antibody subpopulations. On the contrary, Arg-14, Lys-35, -36, and -46, and ..cap alpha..-NH/sub 2/ of the glycine residue of A. sulcata toxin II are not involved in the corresponding antigenic region. When assayed for interaction with the sodium channel, the antigenic region of toxin II, including Asp-9 and Gln-47, appeared fully accessible to its specific antibodies, suggesting that it is not involved in the binding of the toxin to its receptor.

  4. Novel antigen delivery systems.

    PubMed

    Trovato, Maria; De Berardinis, Piergiuseppe

    2015-08-12

    Vaccines represent the most relevant contribution of immunology to human health. However, despite the remarkable success achieved in the past years, many vaccines are still missing in order to fight important human pathologies and to prevent emerging and re-emerging diseases. For these pathogens the known strategies for making vaccines have been unsuccessful and thus, new avenues should be investigated to overcome the failure of clinical trials and other important issues including safety concerns related to live vaccines or viral vectors, the weak immunogenicity of subunit vaccines and side effects associated with the use of adjuvants. A major hurdle of developing successful and effective vaccines is to design antigen delivery systems in such a way that optimizes antigen presentation and induces broad protective immune responses. Recent advances in vector delivery technologies, immunology, vaccinology and system biology, have led to a deeper understanding of the molecular and cellular mechanisms by which vaccines should stimulate both arms of the adaptive immune responses, offering new strategies of vaccinations. This review is an update of current strategies with respect to live attenuated and inactivated vaccines, DNA vaccines, viral vectors, lipid-based carrier systems such as liposomes and virosomes as well as polymeric nanoparticle vaccines and virus-like particles. In addition, this article will describe our work on a versatile and immunogenic delivery system which we have studied in the past decade and which is derived from a non-pathogenic prokaryotic organism: the "E2 scaffold" of the pyruvate dehydrogenase complex from Geobacillus stearothermophilus. PMID:26279977

  5. Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay.

    PubMed

    Hunt, Ann R; Hall, Roy A; Kerst, Amy J; Nasci, Roger S; Savage, Harry M; Panella, Nicholas A; Gottfried, Kristy L; Burkhalter, Kristen L; Roehrig, John T

    2002-06-01

    An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay. PMID:12037058

  6. Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects

    PubMed Central

    Tognon, Mauro; Corallini, Alfredo; Manfrini, Marco; Taronna, Angelo; Butel, Janet S.; Pietrobon, Silvia; Trevisiol, Lorenzo; Bononi, Ilaria; Vaccher, Emanuela; Barbanti-Brodano, Giuseppe; Martini, Fernanda; Mazzoni, Elisa

    2016-01-01

    Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18–65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses. PMID:26731525

  7. Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects.

    PubMed

    Tognon, Mauro; Corallini, Alfredo; Manfrini, Marco; Taronna, Angelo; Butel, Janet S; Pietrobon, Silvia; Trevisiol, Lorenzo; Bononi, Ilaria; Vaccher, Emanuela; Barbanti-Brodano, Giuseppe; Martini, Fernanda; Mazzoni, Elisa

    2016-01-01

    Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18-65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses. PMID:26731525

  8. Antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells.

    PubMed Central

    Vapalahti, O; Lundkvist, A; Kallio-Kokko, H; Paukku, K; Julkunen, I; Lankinen, H; Vaheri, A

    1996-01-01

    Puumala virus (PUU) is a member of the genus Hantavirus in the family Bunyaviridae and the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. Sera of nephropathia epidemica patients react specifically with PUU nucleocapsid (N) protein. In order to safely provide large quantities of antigen for diagnostic purposes, PUU Sotkamo strain N protein was expressed by using the baculovirus system in Sf9 insect cells to up to 30 to 50% of the total cellular protein. The recombinant N protein (bac-PUU-N) was solubilized with 6 M urea, dialyzed, and purified by anion-exchange liquid chromatography. In an immunoglobulin M mu-capture assay purified and unpurified bac-PUU-N antigen showed identical results compared with the results of a similar assay based on native PUU antigen grown in Vero E6 cells. An immunoglobulin G monoclonal antibody-capture assay based on unpurified bac-PUU-N also showed results identical to those of an assay with native PUU-N antigen. Moreover, a panel of monoclonal antibodies reactive with eight different epitopes showed identical reactivity patterns with both natural and bac-PUU-N antigen, while two epitopes in PUU-N expressed as a fusion protein in Escherichia coli were not recognized. Puumala hantavirus N protein expressed by the baculovirus system offers a safe and inexpensive source of specific antigen for large-scale diagnostic and seroepidemiological purposes. PMID:8748286

  9. Antigenic differences between bovine viral diarrhea viruses and HoBi virus: Possible impacts on diagnosis and control

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Compare antigenic differences between HoBi virus and BVDV strains that might impact on diagnostics and control. Eighteen non-cytopathic isolates of pestiviruses including the 5 genotypic groups (BVDV1a-c, BVDV2, BDV) and HoBi virus, were tested using antigen capture enzyme-linked immunosorbent assay...

  10. Molecular characterization of a variant virus that caused de novo hepatitis B without elevation of hepatitis B surface antigen after chemotherapy with rituximab.

    PubMed

    Miyagawa, Masami; Minami, Masahito; Fujii, Kota; Sendo, Rei; Mori, Kojiro; Shimizu, Daisuke; Nakajima, Tomoaki; Yasui, Kohichiroh; Itoh, Yoshito; Taniwaki, Masafumi; Okanoue, Takeshi; Yoshikawa, Toshikazu

    2008-12-01

    Hepatitis B virus (HBV) reactivation in hepatitis B surface antigen (HBsAg)-negative patients following treatment with rituximab has been reported increasingly. The aim of this study was to investigate the molecular mechanisms underlying HBV reactivation in an HBsAg-negative patient. HBV was reactivated in a 75-year-old man following chemotherapy with rituximab, without elevation of HBsAg. The patient's full-length HBV genome was cloned and the entire sequence was determined. Transfection studies were performed in vitro using recombinant wild-type HBV (wild-type), the patient's HBV (patient), and two chimeric HBV constructs, in which the preS/S region of the patient and wild-type virus had been exchanged with one another. Secreted HBsAg and intra- and extra-cellular HBV DNA were measured. The number of amino acid substitutions in HBV from this patient was much higher than in previous reports of HBV mutants, such as occult HBV and vaccine escape HBV mutants. Levels of HBsAg and HBV DNA production in vitro were significantly lower in the patient compared to wild-type transfections. From analyses of the chimeric constructs, the altered preS/S region was responsible mainly for this impairment. These results show that highly mutated HBV can reactivate after chemotherapy with rituximab, despite an unusually large number of mutations, resulting in impaired viral replication in vitro. Severe immune suppression, probably caused by rituximab, may permit reactivation of highly mutated HBV. These findings have important clinical implications for the prevention and management of HBV reactivation and may explain partially the mechanism of recent, unusual cases of HBV reactivation. PMID:19040281

  11. Plasmids enriched with CpG motifs activate human peripheral blood mononuclear cells in vitro and enhance th-1 immune responses to hepatitis B surface antigen in mice.

    PubMed

    Chen, Zhihui; Cao, Jie; Liao, Xiaoling; Ke, Jinshan; Zhu, Shiying; Zhao, Ping; Qi, Zhongtian

    2011-06-01

    T helper-1 (Th-1)-type immune responses play an important role in viral clearance during infection with hepatitis B virus (HBV). Unmethylated CpG motifs present in bacterial DNA can activate toll-like receptor 9 (TLR9) signals and act as potent adjuvants to induce Th-1-type immune responses. Here, a mini-plasmid with 812 base pairs in length was constructed and used as a vector to prepare a series of plasmids containing 3-21 copies of D-type CpG motifs. In vitro, these CpG-enriched plasmids strongly stimulated proliferation of human peripheral blood mononuclear cells (PBMCs) and enhanced secretion of interferon-γ (IFN-γ) and interleukin-12 (IL-12). The responses of the PBMCs from healthy individuals to the plasmids were stronger than those obtained from HBV-infected individuals. Contrary to the strong Th-2-biased response induced by surface antigen of hepatitis B virus (HBsAg) plus alum adjuvant, immunization of BALB/c mice with HBsAg plus these plasmids induced a strong Th-1-biased response. The plasmids increased the titers of HBsAg-specific total immunoglobulin G (IgG) and IgG(2a). HBsAg-specific IL-2 and IFN-γ production and cytotoxic activity were also enhanced in the presence of the plasmids. The strength of the immune responses positively correlated with the number of CpG motifs in the plasmids. These results indicate that the use of CpG-enriched plasmids as an adjuvant to recombinant HBsAg could provide a promising and cost-effective approach for the development of efficacious therapeutic vaccines against HBV infection. PMID:21668361

  12. cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen.

    PubMed Central

    McVey, D; Ray, S; Gluzman, Y; Berger, L; Wildeman, A G; Marshak, D R; Tegtmeyer, P

    1993-01-01

    Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding. Images PMID:8394445

  13. Multiplex immunoassay for in vitro characterization of acellular pertussis antigens in combination vaccines.

    PubMed

    Agnolon, Valentina; Bruno, Cristina; Galletti, Bruno; Mori, Elena; Ugozzoli, Mildred; Pergola, Carlo; O'Hagan, Derek T; Baudner, Barbara C

    2016-02-17

    Vaccines characterization is required to ensure physical, chemical, and biological integrity of antigens and adjuvants. Current analytical methods mostly require complete antigen desorption from aluminum-based adjuvants and are not always suitable to distinguish individual antigens in multivalent formulations. Here, Luminex technology is proposed to improve the analytics of vaccine characterization. As proof of concept, TdaP (tetanus, diphtheria and acellular pertussis) combination, adjuvanted with aluminum hydroxide, was chosen as model formulation to quantify and determine the level of adsorption of acellular pertussis (aP) antigens onto adjuvant surface at the same time. The assay used specific antibodies bound to magnetic microspheres presenting unique digital signatures for each pertussis antigen, allowing the simultaneous recognition of respective antigens in the whole vaccine, avoiding laborious procedures for adjuvant separation. Accurate and reproducible quantification of aP antigens in TdaP vaccine has been achieved in the range 0.78-50 ng/mL, providing simultaneously information on antigen identity, quantity, and degree of adsorption to aluminum hydroxide. The current study could further be considered as a model to set up in vitro potency assays thus supporting the replacement of animal tests accordingly to the 3Rs concept. PMID:26784684

  14. Stool Test: H. Pylori Antigen

    MedlinePlus

    ... Things to Know About Zika & Pregnancy Stool Test: H. Pylori Antigen KidsHealth > For Parents > Stool Test: H. Pylori Antigen Print A A A Text Size ... en español Muestra de materia fecal: antígeno de H. pylori What It Is Helicobacter pylori ( H. pylori ) ...

  15. Novel antigen delivery systems

    PubMed Central

    Trovato, Maria; Berardinis, Piergiuseppe De

    2015-01-01

    Vaccines represent the most relevant contribution of immunology to human health. However, despite the remarkable success achieved in the past years, many vaccines are still missing in order to fight important human pathologies and to prevent emerging and re-emerging diseases. For these pathogens the known strategies for making vaccines have been unsuccessful and thus, new avenues should be investigated to overcome the failure of clinical trials and other important issues including safety concerns related to live vaccines or viral vectors, the weak immunogenicity of subunit vaccines and side effects associated with the use of adjuvants. A major hurdle of developing successful and effective vaccines is to design antigen delivery systems in such a way that optimizes antigen presentation and induces broad protective immune responses. Recent advances in vector delivery technologies, immunology, vaccinology and system biology, have led to a deeper understanding of the molecular and cellular mechanisms by which vaccines should stimulate both arms of the adaptive immune responses, offering new strategies of vaccinations. This review is an update of current strategies with respect to live attenuated and inactivated vaccines, DNA vaccines, viral vectors, lipid-based carrier systems such as liposomes and virosomes as well as polymeric nanoparticle vaccines and virus-like particles. In addition, this article will describe our work on a versatile and immunogenic delivery system which we have studied in the past decade and which is derived from a non-pathogenic prokaryotic organism: the “E2 scaffold” of the pyruvate dehydrogenase complex from Geobacillus stearothermophilus. PMID:26279977

  16. Radioimmunoassays of hidden viral antigens

    SciTech Connect

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

    1982-07-01

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

  17. Comparison of enzyme-linked immunosorbent assay with enzyme-linked fluorescence assay with automated readers for detection of rubella virus antibody and herpes simplex virus.

    PubMed

    Shekarchi, I C; Sever, J L; Nerurkar, L; Fuccillo, D

    1985-01-01

    The enzyme-linked immunosorbent assay (ELISA) was compared with the enzyme-linked fluorescence assay (ELFA) for the detection of rubella antibody and herpes simplex virus antigen. Test parameters, specimens, antigen or antibody, and conjugates for the two types of assays were identical except that p-nitrophenyl phosphate was used as the substrate for the ELISA and 4-methylumbelliferyl phosphate was used as the substrate for ELFA. Automated readers were used for both assays. Antibody titers and sensitivity of antigen detection were quite similar for ELISA and ELFA. ELFA for rubella antibody, however, could be conducted with less antigen or shorter substrate incubation time (5 min for ELFA versus 30 min for ELISA). For herpes simplex virus antigen detection, ELFA could also be read after a shorter substrate incubation time (15 min for ELFA versus 30 min for ELISA). Clear polystyrene microtiter plates routinely used for ELISA could be used for ELFA, but clear polyvinyl chloride plates had high background fluorescence. Black polystyrene and polyvinyl chloride plates gave lower background fluorescence than did clear plates. ELFA is of particular value as a substitute for ELISAs in which long substrate incubations are required or antigens of only low titer are available. PMID:2981902

  18. Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

    PubMed Central

    2012-01-01

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

  19. Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

    PubMed

    Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

    2015-02-21

    This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings. PMID:25474561

  20. Transplant immuno-diagnostics: crossmatch and antigen detection.

    PubMed

    South, Andrew M; Grimm, Paul C

    2016-06-01

    Identifying and monitoring donor-directed anti-human leukocyte antigen antibodies are a rapidly evolving area of solid organ transplantation. Donor-specific antibodies dictate pre-transplant donor choice and donor-recipient matching and underlie much acute and chronic allograft rejection and loss. The evolution of available technology has driven this progress. Early, labor-intensive, whole-cell assays based on complement-dependent cytotoxicity suffered from poor sensitivity and specificity, technical challenges and lack of precision. Sequential improvement in assay performance included anti-human immunoglobulin-enhanced, complement-dependent cytotoxicity techniques followed by cell-based flow cytometry. However, variable specificity and sensitivity inherent in cell-based testing continued to limit flow cytometry. The introduction of solid-phase assays led to a second revolution in histocompatibility testing with the use of purified antigens bound to artificial surfaces rather than whole cells. These techniques augmented sensitivity and specificity to detect even low-titer antibodies to previously undetected antigens. Identification of complement-activating antibodies is being introduced, but current technology is in the developmental stage. While the detection of alloantibodies has improved dramatically, our comprehension of their importance remains imperfect. Variability in methodology and a lack of standardization limits the clinical application of these tests. In spite of the hurdles that remain, antibody-mediated rejection has become a key target to improve graft survival. PMID:26139577

  1. Purification of Piscirickettsia salmonis and partial characterisation of antigens

    USGS Publications Warehouse

    Barnes, M.N.; Landolt, M.L.; Powell, D.B.; Winton, J.R.

    1998-01-01

    Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, an economically significant disease affecting the salmon aquaculture industry. As with other rickettsial pathogens, antigenic analysis of P. salmonis has been limited by the inherent difficulties of purifying an intracellular organism away from host cell material. In this report, we describe the use of diatrizoate meglumine and diatrizoate sodium (DMDS) density gradient centrifugation to purify P. salmonis grown in chinook salmon embryo (CHSE-214) cells. Plaque assay titers and total protein assays confirmed that viable P. salmonis was consistently concentrated in a visible band within the DMDS density gradient at a density of 1.15 to 1.16 g ml-1. Recovery of purified, viable organisms from DMDS density gradients varied from 0.6 to 3%. Preparations of uninfected CHSE-214 cells, CHSE-214 cells infected with P. salmonis, and gradient-purified P. salmonis were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis to assess the degree of purification and to identify P. salmonis-specific proteins. Although gradient-purified P. salmonis preparations were not completely free of host cell material, 8 bacterial proteins were identified. Polyclonal rabbit antiserum was used in an immunoblot of proteins from purified P. salmonis to identify 3 major and 5 minor antigens. The major antigens of 56, 30 and 20 kDa were potential candidates for experimental vaccines and development of novel diagnostic assays.

  2. Discovery of internalizing antibodies to tumor antigens from phage libraries

    PubMed Central

    Zhou, Yu; Marks, James D

    2014-01-01

    Phage antibody technology can be used to generate human antibodies to essentially any antigen. Many therapeutic target antigens are cell surface receptors, which can be challenging targets for antibody generation. In addition, for many therapeutic applications, one needs antibodies that not only bind the cell surface receptor but that also are internalized into the cell upon binding. This allows use of the antibody to deliver a range of payloads into the cell to achieve a therapeutic effect. In this chapter we describe how human phage antibody libraries can be selected directly on tumor cell lines to generate antibodies that bind cell surface receptors and which upon binding are rapidly internalized into the cell. Specific protocols show how to: 1) directly select cell binding and internalizing antibodies from human phage antibody libraries; 2) screen the phage antibodies in a high throughput flow cytometry assay for binding to the tumor cell line used for selection; 3) identify the antigen bound by the phage antibody using immunoprecipitation and mass spectrometry; and 4) direct cell binding and internalizing selections to a specific tumor antigen by sequential selection on a tumor cell line followed by selection on yeast displaying the target tumor antigen on the yeast surface. PMID:22208981

  3. Analyzing Antigen Recognition by Natural Killer T Cells

    PubMed Central

    Zeissig, Sebastian; Olszak, Torsten; Melum, Espen; Blumberg, Richard S.

    2013-01-01

    Natural Killer T (NKT) cells are a subset of T lymphocytes that recognize a wide variety of lipid antigens presented by CD1 molecules. NKT cells exhibit rapid activation after recognition of cognate antigens, secrete abundant amounts of T helper (Th) 1, Th2, and Th17 cytokines within hours of activation and shape the immune response through subsequent activation of dendritic, NK, T and B cells. NKT cells therefore play central roles in antimicrobial and anticancer immunity and in modulation of various autoimmune disorders. Consequently, recent research has focused on the discovery of microbial and self-antigens involved in NKT cell activation. In this chapter, we discuss different strategies for studying antigen recognition by NKT cells including CD1d tetramer-based approaches and in vitro assays characterizing NKT cell activation in response to lipid antigen presentation. While toll-like receptor (TLR) agonists and cytokines such as IL-12 are critical for NKT cell activation in vivo, particularly in the context of microbial infection, methods for detection of TLR- and cytokine-dependent NKT cell activation will not be discussed in this section. PMID:23329514

  4. Vaccination with a fusion DNA vaccine encoding hepatitis B surface antigen fused to the extracellular domain of CTLA4 enhances HBV-specific immune responses in mice: implication of its potential use as a therapeutic vaccine.

    PubMed

    Zhou, Cheng; Peng, Guoping; Jin, Xiaoli; Tang, Jie; Chen, Zhi

    2010-11-01

    Fusion of specific antigens to extracellular domain of cytotoxic-T-lymphocyte-associated antigen 4 (CTLA4) represents a promising approach to increase the immunogenicity of DNA vaccines. We evaluated this interesting approach for its enhancement on HBV-specific immune responses and its antiviral effects in HBV transgenic mice. A fusion plasmid encoding the extracellular domain of CTLA4 linked with HBsAg was constructed. Mice were immunized by this fusion plasmid. Vaccination with the CTLA4-fused DNA not only induced much higher level of anti-HBs antibody, but also increased HBsAg-specific CD8+ response as well as CTL response in BALB/c mice. Furthermore, both Th1 and Th2 responses were augmented. In HBV transgenic mice, the levels of circulating HBsAg and HBV DNA replication were down-regulated by induction of higher anti-HBs antibody and HBsAg-specific CD8+ response after vaccination with the fusion plasmid. Thus, the CTLA4-fused DNA vaccine led to breakdown of immune tolerance to viral infection in HBV transgenic mice, which might be used as a therapeutic vaccine in HBV infection. PMID:20692873

  5. PIKA as an adjuvant enhances specific humoral and cellular immune responses following the vaccination of mice with HBsAg plus PIKA.

    PubMed

    Shen, Erxia; Li, Li; Li, Lietao; Feng, Lianqiang; Lu, Lin; Yao, Ziliang; Lin, Haixiang; Wu, Changyou

    2007-04-01

    An adjuvant is usually used to enhance the immune response induced by vaccines. The choice of adjuvant or immune enhancer determines the effectiveness of the immune response. Currently, aluminium (Alum, a generic term for salts of aluminium) is the only FDA-approved adjuvant. Alum predominantly induces the differentiation of Th2 cells and thus mediates an antibody immune response. Therefore, there is an urgent need for new adjuvants that enhance not only humoral but also cellular immune responses. In the present study, we demonstrates that PIKA (a stabilized dsRNA) as an adjuvant directly induces the activation and the proliferation of both B and NK cells in vitro. Injection of PIKA into mice results in the production of cytokines in vivo. In addition, the study demonstrates that PIKA promotes the maturation of bone marrow-derived dendritic cells (BMDCs) including up-regulation of the co-stimulatory molecules CD80, CD86 and CD40, and the induction of cytokines such as IL-12p70, IL-12p40 and IL-6. Importantly, after immunization of mice with HBsAg plus PIKA, the presence of PIKA enhances the titers of HBsAg-specific IgG and HBsAg-specific IFN-gamma production. These results demonstrate that PIKA as an adjuvant can promote both humoral and cellular immune responses. These might have an implication in applying PIKA as an adjuvant to be used in the design and development of both therapeutic and preventive vaccines, and used in the clinical study. PMID:17484805

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

    EPA Science Inventory

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

  7. [Antigenic response against PPD and antigen 60 in tubercular patients: single antigen versus the combined test].

    PubMed

    Máttar, S; Broquetas, J M; Gea, J; Aran, X; el-Banna, N; Sauleda, J; Torres, J M

    1992-05-01

    We analyze serum samples from 70 patients with pulmonary tuberculosis and 50 healthy individuals. The antigenic activity (IgG) against protein purified antigen (PPD) and antigen 60 (A60) from M. tuberculosis. Thirteen patients were also HIV infected, and three patients had AIDS defined by the presence of disseminated tuberculosis. The test using antigen alone showed a 77% sensitivity and 74% specificity when PPD is used. When A60 was used, both values improved (81% sensitivity, 94% specificity). The use of a combined test (PPD and A60) improves the sensitivity (89%) but reduces the specificity (82%). The HIV infected patients showed similar responses to those of other patients. The combined use of different antigens might be useful for diagnosing tuberculosis. PMID:1390996

  8. Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach

    PubMed Central

    2014-01-01

    Background Novel vaccine candidates against Schistosoma japonicum are required, and antigens present in the vulnerable larval developmental stage are attractive targets. Post-genomic technologies are now available which can contribute to such antigen discovery. Methods A schistosome-specific protein microarray was probed using the local antibody response against migrating larvae. Antigens were assessed for their novelty and predicted larval expression and host-exposed features. One antigen was further characterised and its sequence and structure were analysed in silico. Real-time polymerase chain reaction was used to analyse transcript expression throughout development, and immunoblotting and enzyme-linked immunosorbent assays employed to determine antigen recognition by antibody samples. Results Several known and novel antigens were discovered, two of which showed up-regulated transcription in schistosomula. One novel antigen, termed S. japonicum Ly-6-like protein 1 (Sj-L6L-1), was further characterised and shown to share structural and sequence features with the Ly-6 protein family. It was found to be present in the worm tegument and expressed in both the larval and adult worms, but was found to be antigenic only in the lungs that the larvae migrate to and traverse. Conclusions This study represents a novel approach to vaccine antigen discovery and may contribute to schistosome vaccine development against this important group of human and veterinary pathogens. PMID:24964958