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Sample records for antigen hbsag assays

  1. Calibration of qualitative HBsAg assay results for quantitative HBsAg monitoring.

    PubMed

    Gunning, Hans; Adachi, Dena; Tang, Julian W

    2014-10-01

    Evidence is accumulating that quantitative hepatitis B surface antigen monitoring may be useful in managing patients with chronic HBV infection on certain treatment regimens. Based on these results with the Abbott Architect qualitative and quantitative HBsAg assays, it seems feasible to convert qualitative to quantitative HBsAg values for this purpose.

  2. A new HBsAg screening assay designed for sensitive detection of HBsAg subtypes and variants.

    PubMed

    van Roosmalen, M H; de Jong, J J; Haenen, W; Jacobs, T; Couwenberg, F; Ahlers-de Boer, G J C M; Hellings, J A

    2006-01-01

    The design of a new HBsAg screening assay, the Hepanostika HBsAg Ultra is based on the use of monoclonal antibodies raised against native wild-type HBsAg and reactive with HBsAg in which the common 'a'-determinant is modified by site-directed mutagenesis of four of the cysteine moieties. The design was checked using the same cysteine variants and samples from patients known to be infected with HBsAg variants. The results found were compared with other state-of-the-art commercial screening assays. The design of the Hepanostika HBsAg Ultra enabled detection of all variant HBsAg-positive samples in contrast to the other commercial assays. An additional 980 samples were tested to assess the specificity and sensitivity of the Hepanostika HBsAg Ultra. Screening of presumed negative serum and plasma samples resulted in a specificity of 100%. This makes the Hepanostika HBsAg Ultra the first screening assay with a design able to detect HBsAg variants with high sensitivity and specificity.

  3. Investigation of a Novel Hepatitis B Virus Surface Antigen (HBsAg) Escape Mutant Affecting Immunogenicity

    PubMed Central

    Hossain, Md. Golzar; Ueda, Keiji

    2017-01-01

    Mutation in the hepatitis B virus surface antigen (HBsAg) may affect the efficiency of diagnostic immunoassays or success of vaccinations using HBsAg. Thus, antigenicity and immunogenicity analyses of the mutated HBsAg are necessary to develop novel diagnostic tools and efficient vaccinations. Here, the in vitro antigenicity of three wild-type HBsAg open reading frames (ORFs) (adr4, W1S [subtype adr] and W3S [subtype adr]) isolated from clinically infected patients and nineteen synthesized single/double/multiple amino acid-substituted mutants were tested with commercial ELISA kits. Immunofluorescence staining of transfected cells and Western blot analysis confirmed that these ORFs were expressed at comparable levels in HEK-293 cells. W1S and adr4 were clearly detected, whereas W3S could not be detected. Using the same commercial immunoassay kit, we found that the single mutants, K120P and D123T, were marginally reactive, whereas W3S-aW1S and the double mutant, K120P/D123T, exhibited antigenicity roughly equivalent to the wild-type wako1S. On the other hand, the single mutants of W1S, P120K and T123D, significantly impaired the reactivity, while W1S-aW3S and the double mutant of W1S, P120K/T123D, resulted in a complete loss of antigenicity. In addition, ELISA revealed reduced HBs antigenicity of two mutants, W1S N146G and W1S Q129R/G145R. These commercial ELISA-based antigenic reactivities of HBsAg were also strongly correlated with the predicted Ai alterations of affected amino acids due to the specific mutation. In conclusion, this study showed for the first time that lysine (K120) and aspartate (D123) simultaneously affected HBsAg antigenicity, leading to diagnostic failure. These findings will improve diagnostic assays and vaccine development. PMID:28045894

  4. Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form.

    PubMed

    Smith, Mark L; Mason, Hugh S; Shuler, Michael L

    2002-12-30

    The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed.

  5. 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.

  6. Transgenic lettuce seedlings carrying hepatitis B virus antigen HBsAg.

    PubMed

    Marcondes, Jackson; Hansen, Ekkehard

    2008-12-01

    The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L.) using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety 'Vitória de Verão' genetically modified.

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

    PubMed

    Pondé, Robério Amorim de Almeida

    2011-02-01

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

  8. Cell-mediated immunity to hepatitis B virus antigens in mice: correlation of in vivo and in vitro assays.

    PubMed Central

    De Moerloose, P A; Frazer, I H; Sewell, W A; Collins, E J; Mackay, I R

    1986-01-01

    Cell mediated immunity (CMI) to hepatitis B viral antigens was studied in BALB/mice after immunization with purified hepatitis B surface antigen (HBsAg), or core antigen (HBcAg), with adjuvants. The two in vitro assays for cell-mediated immunity (CMI), utilizing lymph node cells, were release of interferon after exposure to antigen, and blast transformation of lymphocytes, and the in vivo assay was ear swelling at 24 h after local injection of antigen. Immunization with HBsAg or HBcAg with adjuvants induced antigen-specific cutaneous reactivity; if no adjuvants were given, immunization with HBcAg, but not HBsAg, induced cutaneous reactivity. CMI could be adoptively transferred by lymph node cells, but for only a limited period after immunization with HbsAg or HBcAg. The ability of lymph node cells from mice immunized with HBV antigens to transfer adoptively CMI correlated well with their production of interferon after challenge with antigen in vitro, but less well with blastogenesis after challenge with antigen in vitro, or with cutaneous reactivity to antigen in the donor mouse. Reliable antigen-specific lymphokine release assays, rather than blast transformation of lymphocytes or cutaneous reactivity after antigen challenge, are required to assess CMI to HBV antigens in the mouse and, by inference, in man. Images Fig. 1 PMID:3091300

  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. Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression

    PubMed Central

    Mirian, Mina; Taghizadeh, Razieh; Khanahmad, Hossein; Salehi, Mansour; Jahanian-Najafabadi, Ali; Sadeghi-aliabadi, Hojjat; Kouhpayeh, Shirin

    2016-01-01

    Hepatitis B virus (HBV) is considered as a global health concern and hepatitis B surface antigen (HBsAg) is the most immunogenic protein of HBV. The purpose of this study was to evaluate the expression of HBsAg on the cell surface of human embryonic kidney cell line (HEK293T). After transformation of expression vector pcDNA/HBsAg to E.coli TOP10F’, plasmid was extracted and digested with BglII. Afterwards, the linearized vector was transfected to cells and treated with hygromycin B for 5 weeks to expand the resulted clonies. The permanent expression of HBsAg followed by flow cytometry uptill now about one year. Genomic DNA was extracted from transfected cells and the existence of HBsAg gene was assessed by PCR. Real-time RT-PCR was utilized to measure the expression at the RNA level and flow cytometery was carried out to assess protein expression. Insertion of HBsAg cDNA in HEK293T genome was confirmed by PCR. The results of real-time RT-PCR illustrated that each cell expresses 2275 copies of mRNA molecule. Flow cytometry showed that compared with negative control cells, 99.9% of transfected cells express HBsAg on their surface. In conclusion, stable expression of hepatitis B surface antigen on the membrane of HEK293T provides an accurate post-translational modification, proper structure, and native folding in contrast with purified protein from prokaryotic expression systems. Therefore, these exposing HBsAg cells are practical in therapeutic, pharmaceutical, and biological sets of research. PMID:27920818

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    Lee, Jung Min; Ahn, Sang Hoon

    2011-01-21

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

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

    PubMed Central

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

    1995-01-01

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

  14. Role of quantitative hepatitis B surface antigen in predicting inactive carriers and HBsAg seroclearance in HBeAg-negative chronic hepatitis B patients

    PubMed Central

    Ungtrakul, Teerapat; Sriprayoon, Tassanee; Kusuman, Pattama; Chunnuan, Pitchayachuda; Soonklang, Kamonwan; Sornsamdang, Gaidganok; Auewarakul, Chirayu U.; Tanwandee, Tawesak

    2017-01-01

    Abstract To evaluate quantitative hepatitis B surface antigen (qHBsAg) as a diagnostic marker for inactive carriers (ICs) and hepatitis B surface antigen (HBsAg) seroclearance in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) patients. We retrospectively studied 300 HBeAg-negative CHB patients with initial serum hepatitis B virus (HBV) Deoxyribonucleic acid (DNA) levels <2000 IU/mL. Serum HBV DNA and alanine aminotransferase (ALT) levels were monitored every 6 months for 24 months. ICs were identified as having persistent HBV DNA levels <2000 IU/mL and normal ALT levels, whereas active carriers (ACs) were identified as having HBV DNA levels ≥2000 IU/mL, with or without elevated ALT levels. The serum qHBsAg level was defined at baseline and evaluated as a diagnostic predictor using a receiver-operating characteristic curve. The study group comprised 134 men and 166 women with a median age of 41.5 years. At baseline, 200 ICs displayed lower levels of qHBsAg (1492 IU/mL) compared with 100 ACs (2936 IU/mL) (P = 0.005). The qHBsAg level was independently associated with the IC state and HBsAg seroclearance. Baseline qHBsAg levels <1000 IU/mL and HBV DNA levels <2000 IU/mL, when detected simultaneously, allowed for identification of ICs with 41% sensitivity and 72% specificity. Fifteen patients (5%) displayed HBsAg seroclearance after 24 months. A qHBsAg cutoff value of <50 IU/mL provided 100% sensitivity and 92% specificity in predicting HBsAg seroclearance. The qHBsAg level at a single timepoint among HBeAg-negative CHB patients with low HBV DNA levels at baseline was not a predictive marker for ICs; however, it accurately predicted spontaneous HBsAg seroclearance at 24 months. PMID:28353619

  15. 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.

  16. 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.

  17. 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.

  18. 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 §...

  19. 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 §...

  20. 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 §...

  1. 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 §...

  2. 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 §...

  3. Antigen detection assay for identification of Penicillium marneffei infection.

    PubMed

    Chaiyaroj, Sansanee C; Chawengkirttikul, Runglawan; Sirisinha, Stitaya; Watkins, Pramuan; Srinoulprasert, Yuttana

    2003-01-01

    Two recently produced monoclonal antibodies were used to develop an antigen capture enzyme-linked immunosorbent assay (ELISA) for rapid diagnosis of Penicillium marneffei. The method was evaluated with 53 patients with culture-confirmed penicilliosis and 240 controls. The diagnostic sensitivity, specificity, and accuracy of the ELISA were 92.45, 97.5, and 96.59%, respectively.

  4. Comparison of four assays for the detection of cryptococcal antigen.

    PubMed

    Binnicker, M J; Jespersen, D J; Bestrom, J E; Rollins, L O

    2012-12-01

    We compared the performance of four assays for the detection of cryptococcal antigen in serum samples (n = 634) and cerebrospinal fluid (CSF) samples (n = 51). Compared to latex agglutination, the sensitivity and specificity of the Premier enzyme immunoassay (EIA), Alpha CrAg EIA, and CrAg lateral flow assay (LFA) were 55.6 and 100%, 100 and 99.7%, and 100 and 99.8%, respectively, from serum samples. There was 100% agreement among the four tests for CSF samples, with 18 samples testing positive by each of the assays.

  5. Cell-free antigens of Sporothrix brasiliensis: antigenic diversity and application in an immunoblot assay.

    PubMed

    Almeida-Paes, Rodrigo; Bailão, Alexandre Melo; Pizzini, Cláudia Vera; Reis, Rosani Santos; Soares, Célia Maria de Almeida; Peralta, José Mauro; Gutierrez-Galhardo, Maria Clara; Zancopé-Oliveira, Rosely Maria

    2012-11-01

    Sporotrichosis is a subcutaneous mycosis diagnosed by isolation of the fungus in culture. Serological tests for help in diagnosis in general do not use purified or recombinant antigens, because there is a paucity of described immunoreactive proteins, especially for the new described Sporothrix species, such as Sporothrix brasiliensis. This study aims to characterise antigens from S. brasiliensis and verify their application in serodiagnosis of sporotrichosis. An immunoblot assay allied with computer-based analysis was used to identify putative antigenic molecules in a cell-free extracts of both morphological phases of this fungus, and to delineate antigenic polymorphism among seven S. brasiliensis isolates and one S. schenckii Brazilian strain. The mycelial and yeast phase of the fungus originated 14 and 23 reactive bands, respectively, which were variable in intensity. An 85 kDa antigen, verified in the yeast phase of the fungus, was observed in all strains used and the immunodominant protein was identified. This protein, however, cross-react with serum samples from patients infected with other pathogens. The results show that the S. brasiliensis cell-free antigen extract is a single and inexpensive source of antigens, and can be applied on the sporotrichosis serodiagnosis.

  6. 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.

  7. Clinical features of HBsAg seroclearance in hepatitis B virus carriers in South Korea: A retrospective longitudinal study

    PubMed Central

    Park, Young Min; Lee, Seong Gyu

    2016-01-01

    AIM To investigate the characteristic features of hepatitis B surface antigen (HBsAg) seroclearance among Korean hepatitis B virus (HBV) carriers. METHODS Carriers with HBsAg seroclearance were selected by analyzing longitudinal data collected from 2003 to 2015. The period of time from enrollment to the negative conversion of HBsAg (HBsAg-NC) was compared by stratifying various factors, including age, sex, hepatitis B e antigen (HBeAg), HBV DNA, sequential changes in the signal-to-cutoff ratio of HBsAg (HBsAg-SCR), as measured by qualitative HBsAg assay, and chronic liver disease on ultrasonography (US-CLD). Quantification of HBV DNA and HBsAg (HBsAg-QNT) in the serum was performed by commercial assay. RESULTS Among the 1919 carriers, 90 (4.7%) exhibited HBsAg-NC at 6.2 ± 3.6 years after registration, with no differences observed among the different age groups. Among these carriers, the percentages of those with asymptomatic liver cirrhosis (LC) and hepatocellular carcinoma (HCC) at registration were 31% and 7.8%, respectively. The frequency of HBsAg-NC significantly differed according to the HBV DNA titer and US-CLD. HBeAg influenced HBsAg-NC in the 40-50 and 50-60 year age groups. HBsAg-SCR < 1000 was correlated with an HBsAg-QNT < 200 IU/mL. A gradual decrease in the HBsAg-SCR to < 1000 predicted HBsAg-NC. Six patients developed HCC after registration, including two before and four after HBsAg-NC. The rate at which the patients developed new HCC after HBsAg seroclearance was 4.8%. LC with excessive drinking and vertical infection were found to be risk factors for HCC in the HBsAg-NC group. CONCLUSION HCC surveillance should be continued after HBsAg seroclearance. An HBsAg-SCR < 1000 and its decrease in sequential testing are worth noting as predictive markers of HBsAg loss. PMID:27956808

  8. 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

  9. 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

  10. 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.

  11. Performance of the Cryptococcal Antigen Lateral Flow Assay in Non-HIV-Related Cryptococcosis.

    PubMed

    Jitmuang, Anupop; Panackal, Anil A; Williamson, Peter R; Bennett, John E; Dekker, John P; Zelazny, Adrian M

    2016-02-01

    The cryptococcal antigen lateral flow assay (CrAg LFA) was evaluated for the diagnosis of cryptococcosis in HIV-negative patients. The sensitivity was excellent, suggesting that this assay can replace conventional testing based on latex agglutination (LA). CrAg LFA and LA titers were correlated but were not directly comparable, with implications for conversion between assays.

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

    PubMed

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

    2015-03-01

    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.

  13. Preparation of Mycoplasma hyopneumoniae antigen for the enzyme-linked immunosorbent assay.

    PubMed Central

    Kazama, S; Yagihashi, T; Seto, K

    1989-01-01

    Methods of preparation of Mycoplasma hyopneumoniae antigens for the enzyme-linked immunosorbent assay to detect specific antibody, and properties of the antigens, are described. The reactivity and specificity of antigen prepared by Sephacryl S-300 column chromatography after treatment of M. hyopneumoniae cells with Tween 20 (S-300 antigen) were superior to those of antigen prepared by Sephadex G-25 column chromatography after treatment with Tween 20, or to lipid antigen. There were no differences among strains MI-3, J and VPP11 of M. hyopneumoniae. The S-300 antigen did not show cross-reactivity against porcine hyperimmune sera produced by M. hyorhinis, M. hyosynoviae, M. hyopharyngis, M. flocculare and Acholeplasma granularum. Antibody was first detected in sera of pigs inoculated intranasally with M. hyopneumoniae at two to four weeks after inoculation and seven to eight weeks after pigs were contact-exposed to the same mycoplasma. PMID:2523756

  14. 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

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

    PubMed Central

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

    2016-01-01

    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 from Borrelia 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 five B. burgdorferi antigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection of B. burgdorferi antigen-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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    DTIC Science & Technology

    2015-12-01

    homogenized insects . Two lots of SFFVA were purchased from VecTOR Test Systems. Viruses were purchased from the American Type Culture Collection...myia indicated that the titers of SFNV and TOSV can range from 103–104.5 and 103–104.7 PFU per insect , respectively, 5–7 days postinfection (Tesh and...validating the need for field- deployable insect pathogen detection assays. We also thank Brandy Russell (Arbovirus Disease Branch, Division of Vector

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

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

    PubMed

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

    2015-12-30

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

  1. 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.

  2. Hepatitis B viral antigenic structure: signature analysis by monoclonal radioimmunoassays.

    PubMed Central

    Wands, J R; Wong, M A; Shorey, J; Brown, R D; Marciniak, R A; Isselbacher, K J

    1984-01-01

    An approach has been developed for the analysis of hepatitis B viral (HBV) antigenic structure that creates numerical "signatures" of HBV strains. This technique employs high-affinity IgM and IgG monoclonal antibodies (anti-HBsAg) directed toward distinct and separate determinants on hepatitis B surface antigen (HBsAg). Such antibodies have been used to develop sensitive and specific radioimmuno-assays for measurement of HBsAg-associated determinants in serum. In performing "signature" analysis separate binding curves for each monoclonal anti-HBsAg are generated by measuring immunoreactivity in serial dilutions of HBsAg-positive serum. Since the HBsAg concentration in serum is unknown, the binding profiles of groups of samples from the same "classic" HBV subtype are aligned by an iterative maximum likelihood procedure to give the numerical signature of that HBV subtype. By using this approach, HBsAg shows far more antigenic heterogeneity than previously recognized by polyvalent anti-HBsAg antibodies. Indeed, there are subgroups within the classic HBsAg subtypes. In addition, the a domain (common to all known subtypes or strains of HBV) has been shown to be multideterminant. Thus, these studies have demonstrated heretofore unrecognized differences in HBV subtypes. This approach also has broader significance for the study of subtle or major antigenic changes among other viral agents since it is not necessary to know the concentration of virus or viral protein in complex protein mixtures. PMID:6585796

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

    NASA Astrophysics Data System (ADS)

    Kidwell, David A.

    1993-05-01

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

  4. Evaluation of Babesia bigemina 200 kDa recombinant antigen in enzyme-linked immunosorbent assay.

    PubMed

    Altangerel, Khukhuu; Alhassan, Andy; Iseki, Hiroshi; Sivakumar, Thillaiampalam; Boldbaatar, Damdinsuren; Yokoyama, Naoaki; Igarashi, Ikuo

    2009-07-01

    A truncated fragment of the gene encoding the 200-kDa protein (P200) of Babesia bigemina was cloned into a plasmid vector, pGEX-4 T-1 and expressed in Escherichia coli as a glutathione-S-transferase fused protein. An indirect enzyme-linked immunosorbent assay (ELISA) using the rp200/CT detected specific antibodies in cattle experimentally infected with B. bigemina. Furthermore, the antigen did not cross-react with antibodies to Babesia bovis, a closely related Babesia parasite indicating that rp200/CT is a specific antigen for the diagnosis of B. bigemina infection. Additionally, ELISA using p200/CT and polymerase chain reaction were conducted on serum and corresponding DNA samples obtained from field cattle to evaluate the diagnostic utility of the p200/CT antigen. Results from the current study suggest that p200/CT ELISA is a sensitive and specific method for improved serodiagnosis of B. bigemina infection.

  5. Estimation of low frequency antigen presenting cells with a novel RELISPOT assay

    PubMed Central

    Dzutsev, Amiran K.; Belyakov, Igor M.; Isakov, Dmitry V.; Gagnon, Susan J.; Margulies, David H.; Berzofsky, Jay A.

    2008-01-01

    Adequate presentation of self and foreign antigens is a key factor for efficient T-cell immunosurveillance against pathogens and tumors. Cells presenting foreign antigens usually comprise a rare population and are difficult to detect even at the peak of infection. Here we demonstrate a CD8+ T-cell-based approach that allows detection of specific antigen-presenting cells (APC) at a frequency of less than 0.0005%. When T cells are in excess, they form rosettes with rare APCs, which appear as single spots in an IFN-γ ELISPOT assay. Using this RELISPOT (Rosette ELISPOT) method we demonstrate the dynamic interplay between CD8 T cells and professional and non-professional APCs following virus challenge. PMID:18294650

  6. 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.

  7. Development of a serologic assay for cysticercosis, using an antigen isolated from Taenia spp cyst fluid.

    PubMed

    Hayunga, E G; Sumner, M P; Rhoads, M L; Murrell, K D; Isenstein, R S

    1991-03-01

    An ammonium sulfate-soluble fraction of Taenia hydatigena cyst fluid (ThFAS) was further evaluated for use in the immunodiagnosis of cysticercosis. Analysis of ThFAS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblot analysis confirmed earlier reports of a highly specific, low molecular weight antigen in this preparation; in contrast, other components of ThFAS were shown to react nonspecifically. Antibodies against the less than 12-kD diagnostic antigen were detected in sera from 10 cattle and 4 swine inoculated with metacestodes of T saginata and T solium, respectively, but not in animals inoculated with Fasciola hepatica, Trichinella spiralis, Brucella abortus, or Toxoplasma gondii, or in noninoculated controls. Isolation and immobilization of the less than 12-kD antigen on a hydrophobic transfer membrane resulted in development of an unambiguous dipstick assay capable of correctly identifying fully developed (10-week) experimentally induced infections in cattle and swine. In addition, the dipstick assay was highly specific for diagnosis of the disease in human beings, and offers the potential of distinguishing between human clinical cases of cysticercosis and taeniasis. A similar reactive antigen of diagnostic potential was also identified and isolated from T crassiceps and T taeniaeformis cyst fluids.

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

    PubMed

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

    2005-09-01

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

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

    PubMed

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

    1995-01-01

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

  10. Seroclearance of Hbsag in Chronic Hepatitis B Virus Patients on Lamivudine Therapy: A 10 Year Experience

    PubMed Central

    Sali, Shahnaz; Merza, Muayad A.; Saadat, Sina; Mustafa, Nazik H.; Queiky, Farzam; Yadegarynia, Davood

    2015-01-01

    Introduction: The aim of this study was to determine hepatitis B surface antigen (HBsAg) seroclearance rate among patients treated with lamivudine at a specialized tertiary care referral hospital in Tehran, Iran. Methods: All patients on lamivudine (biovudin®) therapy at a dose of 100 mg/day, who showed seroclearnace between March 2001 and September 2011 were recruited. The main evaluation parameters were duration of HBsAg seroclearance and duration of HBsAg seroconversion. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were evaluated using standard methods. HBsAg seroclearance was defined as two consecutive negative serums HBsAg at least 6 months apart, whereas HBsAg seroconversion was defined as the disappearance of serum HBsAg and the presence of anti-HBs for >6 months. Results: A total of 203 chronic HBV patients treated with lamivudine at a dose of 100 mg/day were included in the study. HBsAg seroclearance and seroconversion were observed in 11 patients after the initiation of the lamivudine therapy. Overall, in lamivudine responder patients, the mean time to HBsAg seroclearance was 26.90±10.93 months (range: 12-48 months). Furthermore, the responders showed seroconversion after a mean time of 26.90±11.08 months from the initiation of lamivudine therapy. When comparing the characteristics of those who have responded to lamivudine and those who have not responded, baseline HBV-DNA levels was significantly lower in responder than non responder patients (p<0.001). Meantime, there was no difference in age, sex, baseline ALT, AST and liver biopsy score between lamivudine responder and lamivudine non-responder patients. Conclusion: Despite introduction of tenofovir and entecavir as first line treatment for chronic HBV infection, lamivudine remains to be a low cost, safe and effective drug for HBsAg seroclearnace. PMID:26153167

  11. An immuno-enzymatic assay for herpes simplex virus tumour associated antigen in gynecological oncology.

    PubMed

    Magli, G; Scimone, C; Flaminio, G; D'Alessandro, G; Mascolo, A; Magli, R; Saladino, I

    1982-01-01

    The authors studied the HSV-TAA (Herpes Simplex Virus Tumor Associated Antigen) in patients affected by female genitale tract tumors, using the immunoenzymatic assay (ELISA). They found a positive frequence of 65% in sera of patients affected by uterine cervical carcinoma and of the 80% in sera of patients affected by vulvar carcinoma. The authors suggest that this enzymatic method has a real value and propose its use in the early diagnosis of the female genital tract neoplasms.

  12. Ultrasensitive p24 Antigen Assay for Diagnosis of Perinatal Human Immunodeficiency Virus Type 1 Infection▿

    PubMed Central

    Fiscus, Susan A.; Wiener, Jeffrey; Abrams, Elaine J.; Bulterys, Marc; Cachafeiro, Ada; Respess, Richard A.

    2007-01-01

    We evaluated an ultrasensitive p24 antigen enzyme immunosorbent assay on 802 plasma specimens from 582 infants and children of 0 to 180 days of age. Overall sensitivity and specificity were 91.7% and 98.5%, respectively. After exclusion of infants of less than 7 days of age, the sensitivity and specificity were 93.7% and 98.3%, respectively. PMID:17475763

  13. 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.

  14. HBsAg sT123N mutation induces stronger antibody responses to HBsAg and HBcAg and accelerates in vivo HBsAg clearance.

    PubMed

    Li, Songxia; Zhao, Kaitao; Liu, Shuhui; Wu, Chunchen; Yao, Yongxuan; Cao, Liang; Hu, Xue; Zhou, Yuan; Wang, Yun; Pei, Rongjuan; Lu, Mengji; Chen, Xinwen

    2015-12-02

    Immune escape mutants with mutations in the hepatitis B surface antigen (HBsAg) major hydrophilic region (MHR) often emerge in association with diagnostic failure or breakthrough of HBV infection in patients with anti-HBs antibodies. Some mutants harboring substitutions to Asn in HBsAg MHR may have an additional potential N-glycosylation site. We have previously showed that sT123N substitution could generate additional N-glycosylated forms of HBsAg. In the present study, 1.3-fold-overlength HBV genomes containing the sT123N substitution were digested from the pHBV1.3-sT123N construct and subcloned into the pAAV vector to generate pAAV1.3-sT123N for hydrodynamic injection (HI) in mice. Viral expression and replication were phenotypically characterized by transient transfection. The results demonstrated that sT123N substitution impaired virion secretion, resulting in intracellular retention of HBcAg. Using the HBV HI mouse model, we found that mice mounted significantly stronger antibody responses to HBsAg and HBcAg, which accelerated HBsAg clearance. Thus, additional N-glycosylation generated by amino acid substitutions in HBsAg MHR may significantly modulate specific host immune responses and influence HBV infection in vivo. Our results help further the understanding of the role of immune escape mutants with N-linked glycosylation in the biology of HBV infection.

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

    PubMed Central

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

    1991-01-01

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

  16. Demonstration of common antigens on cell surface of Clostridium chauvoei and C. septicum by indirect-immunofluorescence assay.

    PubMed

    Hamaoka, T; Terakado, N

    1994-04-01

    The common antigens between Clostridium chauvoei and C. septicum were examined by indirect-immunofluorescence assay (IFA). Antisera to formalized cells of C. chauvoei and C. septicum strains and to EDTA-soluble antigens of these strains were used. The antisera to formalized cells, which have reacted only with homologous antigens in agglutination tests, reacted not only with homologous antigens but also with heterologous antigens in IFA. The antisera to EDTA-soluble antigens, which have shown no reactivities in somatic agglutination tests, reached with both homologous and heterologous antigens in IFA. These results indicate that these species possess common antigens, which are undetected by agglutination tests, on the cell surface and a some of them are solubilized by EDTA-treatment.

  17. 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.

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

    SciTech Connect

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

    1984-10-01

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

  19. 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)

  20. 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.

  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. 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.

  3. [³H]serotonin release assay using antigen-stimulated rat peritoneal mast cells.

    PubMed

    Skaper, Stephen D; Facci, Laura

    2012-01-01

    The concentration of nerve growth factor (NGF) is elevated in a number of inflammatory and autoimmune states in conjunction with increased accumulation of mast cells. Mast cells, which are of hematopoietic lineage, and NGF appear to be involved in neuroimmune interactions and tissue inflammation. Mast cells themselves are capable of producing and responding to NGF. Here we describe a protocol for the isolation and culture of peritoneal-derived rat mast cells, together with a [(3)H]serotonin release assay which is useful in assessing the effects of antigens and neurotrophic factors on mast-cell activation.

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

    PubMed

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

    2013-10-01

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

  5. Point-of-care diagnosis and prognostication of cryptococcal meningitis with the cryptococcal antigen lateral flow assay on cerebrospinal fluid.

    PubMed

    Kabanda, Taseera; Siedner, Mark J; Klausner, Jeffrey D; Muzoora, Conrad; Boulware, David R

    2014-01-01

    The cryptococcal antigen (CRAG) lateral flow assay (LFA) had 100% sensitivity and specificity on cerebrospinal fluid samples. Pretreatment LFA titers correlated with quantitative cultures (R(2) = 0.7) and predicted 2- and 10-week mortality. The CRAG LFA is an accurate diagnostic assay for CSF and should be considered for point-of-care diagnosis of cryptococcal meningitis.

  6. Enzyme-linked immunosorbent assay for detection and quantitation of capsular antigen of Haemophilus influenzae type b.

    PubMed Central

    Crosson, F J; Winkelstein, J A; Moxon, E R

    1978-01-01

    An enzyme-linked immunosorbent assay was developed to detect the presence of the ribose-ribitol phosphate capsular antigen of Haemophilus influenzae type b in laboratory and clinical specimens. The assay is simple, sensitive, specific, and quantitative and should prove to be of value in the diagnosis and management of H. influenzae infections. PMID:310425

  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. Different Mechanisms May Exist for HBsAg Synthesis and Secretion During Various Phases of Chronic Hepatitis B Virus Infection

    PubMed Central

    Li, Yunsong; Cai, Qun; Xie, Qinxiu; Zhang, Yafei; Meng, Xiangling; Zhang, Zhenhua

    2017-01-01

    Background The aim of this study was to characterize the expression and secretion of hepatitis B surface-antigen (HBsAg) in the hepatocytes of hepatitis B virus (HBV)-infected patients at different phases of infection; as such, the association of intrahepatic HBsAg expression with virological markers and the histological characteristics were analyzed. Material/Methods 302 chronic HBV infection patients who had not received antiviral therapy were stratified by HBeAg status. The proportion of HBsAg-positive cells was used as an indicator for HBsAg expression level. Results In HBeAg-positive patients, there was a significant correlation between serum HBsAg and serum HBV DNA levels (r=0.569, p<0.001). Intrahepatic HBsAg expression and serum HBsAg level in HBeAg-positive patients were higher than those in HBeAg-negative patients (p=0.002 and p<0.001, respectively). A significant correlation between serum HBsAg level and intrahepatic HBsAg expression was found in HBeAg-negative patients (r=0.377, p<0.001), but not in HBeAg-positive patients (r=0.051, p=0.557). Very interestingly, the correlation between serum HBsAg level and HBsAg expression in hepatocytes gradually increased along with disease progression through the immune-tolerant, immune-clearance, inactive, and recovery phases of HBV infection (r=−0.184, 0.068, 0.492, and 0.575; and p=0,238, 0,722, 0.012, and 0.002, respectively). Conclusions Different mechanisms may be involved in HBsAg synthesis and secretion in different phases of chronic HBV infection. PMID:28321112

  9. 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.

  10. Immune modulation by cadmium and lead in the acute reporter antigen-popliteal lymph node assay.

    PubMed

    Carey, John B; Allshire, Ashley; van Pelt, Frank N

    2006-05-01

    Immune modulation by heavy metals may cause serious adverse health effects in humans, although the mechanisms involved are not well understood. Both cadmium and lead are important environmental and occupational toxins. Therefore, in the current study, the costimulatory/adjuvant effects and the T-cell-activating potential of these metals (i.e., CdCl2 and PbCl2), are examined. These immune-modulating properties are critical in the development of conditions such as allergy, hypersensitivity, and autoimmunity. Using the direct popliteal lymph node assay (PLNA) and reporter antigen-popliteal lymph node assay (RA-PLNA) both metals were examined individually for immunotoxicity. Mercury (i.e., HgCl2) was included for comparative purposes as its effects in the RA-PLNA are well documented. Seven days following a single footpad injection containing metal and/or RA (trinitrophenyl-ovalbumin [TNP-OVA] or TNP-Ficoll), BALB/c mice were sacrificed and the popliteal lymph nodes (PLNs) removed. PLN cellularity, TNP-specific antibody-secreting cells (ASCs), and lymphocyte subsets were assessed. All three metals strongly stimulated T- and B-cell proliferation and ASC production following coinjection with the RA TNP-OVA. In each case, ASC production was skewed towards the IgG1 isotype. In addition, all three metals induced IgG production to TNP-Ficoll (although relatively weakly in the case of Cd). These results show that each of these metals can provide adjuvant signals to promote lymphocyte proliferation and enhance adaptive immune responses to unrelated antigens. Skewing of immune responses towards T helper type 2 responses suggests that each of these metals can enhance allergic and hypersensitivity reactions to environmental antigens. Furthermore, the induction of IgG responses to TNP-Ficoll, a T-cell-independent antigen, indicates that each of these metals can activate neoantigen-specific T cells. T-cell activation by metals can lead to metal hypersensitivity and has been

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

    SciTech Connect

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

    1984-10-01

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

  12. Mutation in the S gene a determinant of the hepatitis B virus associated with concomitant HBsAg and anti-HBs in a population in Northeastern Brazil.

    PubMed

    de Campos Albuquerque, Ingrid; Sousa, Marinilde Teles; Santos, Max Diego Cruz; Nunes, Jomar Diogo Costa; Moraes, Maria Josélia Diniz; Gomes-Gouvêa, Michele Soares; Pinho, João Renato Rebelo; Carrilho, Flair José; Fonseca, Lena Maria Barros; de Sousa Paiva Ferreira, Adalgisa

    2017-03-01

    Mutations in the a determinant of S gene may develop co-existence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in the serum of infected hepatitis B virus (HBV) individuals. Mutations in this region may change the antigenicity of HBsAg, which in turn, lead to escape of neutralizing action of anti-HBs antibodies. This study identified individuals with concomitant HBsAg and anti-HBs serological markers in individuals of Maranhão, Northeastern Brazil. Samples from a population-based study were evaluated for HBsAg, anti-HBs, and anti-HBc, and those that tested positive for simultaneous HBsAg and anti-HBs were submitted to HBV DNA quantification and S gene characterization by Sanger sequencing. Mutations were investigated in the a determinant located in major hydrophilic region (MHR) of the S gene. Among 3,984 samples analyzed, 92 (2.3%) were positive for HBsAg and three had the atypical HBsAg and anti-HBs-positive profile (3.26%). The frequency of HBsAg and anti-HBs co-existence was similar to previous studies. Only one individual harbored mutation in the S gene a determinant associated with this profile. Little is known about this phenomenon; however, studies as ours may contribute for future enlightenment of this important issue. J. Med. Virol. 89:458-462, 2017. © 2016 Wiley Periodicals, Inc.

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

    PubMed

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

    2003-10-01

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

  14. Detection of non-jejuni and -coli Campylobacter Species from Stool Specimens with an Immunochromatographic Antigen Detection Assay

    PubMed Central

    Couturier, Brianne A.; Couturier, Marc Roger; Kalp, Kim J.

    2013-01-01

    The STAT! Campy immunochromatographic assay for Campylobacter antigen was compared to culture for 500 clinical stool specimens. Antigen was detected in six culture-negative, PCR-positive specimens. C. upsaliensis, a pathogenic species that is traditionally difficult to recover in routine stool cultures, was detected in two of these culture-negative specimens. This study provides evidence that antigen testing may cross-react with at least one additional non-jejuni and -coli Campylobacter species that may be missed by routine culture for campylobacteriosis. PMID:23554192

  15. 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

  16. 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

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

    PubMed

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

    2016-07-21

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

  18. Monitoring response to antiviral therapy for patients with chronic hepatitis C virus infection by a core-antigen assay.

    PubMed

    Rebucci, Chiara; Cerino, Antonella; Cividini, Agostino; Timo, Letizia; Furione, Milena; Mondelli, Mario U

    2003-08-01

    A recently released immunoassay detecting total serum hepatitis C virus (HCV) core antigen was used to prospectively monitor virological responses to antiviral treatment in patients with chronic HCV infection. Sustained responders cleared core protein from serum within the first month of therapy and maintained stably negative values for the entire duration of follow-up after treatment discontinuation. However, patients who relapsed or failed to respond showed transient negative values and could not be accurately discriminated either because of the intrinsic lower sensitivity of the core-antigen assay than those of molecular assays or because of differentially regulated secretion of immunoreactive core protein from infected hepatocytes.

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

    PubMed

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

    2008-11-05

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

  20. 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.

  1. Hepatitis B virus surface antigen as delivery vector can enhance Chlamydia trachomatis MOMP multi-epitope immune response in mice.

    PubMed

    Zhu, Shanli; Feng, Yan; Rao, Pinhuan; Xue, Xiangyang; Chen, Shao; Li, Wenshu; Zhu, Guanbao; Zhang, Lifang

    2014-05-01

    Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. There is currently no commercially available vaccine against C. trachomatis. Major outer membrane protein (MOMP) of C. trachomatis is considered to be an ideal candidate for prophylactic vaccine. We designed a MOMP multi-epitope containing T- and B-cell epitope-rich peptides and developed hepatitis B surface antigen (HBsAg) as antigen delivery vehicle. In order to study the immunogenicity and efficacy of the candidate vaccine in a murine model of chlamydial genital infection, we engineered a recombinant plasmid expressing HBsAg and MOMP multi-epitope genes. Results of reverse transcription polymerase chain reaction and immunofluorescence assay revealed successful expression of the recombinant HBsAg/MOMP multi-epitope gene at both the transcription and translation levels. Intramuscular administration in mice was able to elicit not only antibodies against Chlamydia and HBsAg but also cytotoxic T lymphocyte activity against Chlamydia. In addition, mice inoculated with the rHBsAg were highly resistant to C. trachomatis genital infection. The rHBsAg DNA with MOMP multi-epitope appended at the C terminus of the HBsAg stimulated a stronger immune response and protective response than that appended at the N terminus. Together, our results suggested that use of a recombinant HBsAg encoding the MOMP multi-epitope could be a powerful approach to developing a safe and immunogenic C. trachomatis vaccine.

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

    PubMed

    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 elimination of

  3. 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

  4. Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

    PubMed

    Almeida, Coral-Ann M; Roberts, Steven G; Laird, Rebecca; McKinnon, Elizabeth; Ahmed, Imran; Pfafferott, Katja; Turley, Joanne; Keane, Niamh M; Lucas, Andrew; Rushton, Ben; Chopra, Abha; Mallal, Simon; John, Mina

    2009-05-15

    The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, p<0.05). The precision results from the automated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

  5. Seroprevalence of hepatitis B surface antigen in pregnant women attending antenatal clinic in Honiara Solomon Islands, 2015

    PubMed Central

    Getahun, Aneley; Baekalia, Margaret; Panda, Nixon; Lee, Alice; Puiahi, Elliot; Khan, Sabiha; Tahani, Donald; Manongi, Doris

    2016-01-01

    AIM To determine the seroprevalence of hepatitis B surface antigen (HBsAg) among pregnant women attending antenatal clinic in Honiara, Solomon Islands. METHODS This descriptive cross-sectional study was carried out in seven area health centers in Honiara. From March to June 2015, identification of eligible pregnant women in each site was conducted using systematic random sampling technique. A total of 243 pregnant women who gave written informed consent were enrolled. Standardized tool was used to record demographics, obstetric history and serology results. HBsAg and hepatitis B e antigen (HBeAg) were tested using point-of-care rapid diagnostic test. All HBsAg positive samples were verified using enzyme-linked immunosorbent assay. RESULTS The mean age of participants was 26 ± 6 years. The overall hepatitis HBsAg prevalence was 13.8% with higher rate (22%) reported in women between 30-34 years of age. Majority of HBsAg positive participants were Melanesians (29 out for 33). None of the pregnant women in the 15-19 years and ≥ 40 years tested positive for HBsAg. There was no statistically significant difference in HBsAg prevalence by age, ethnicity, education and residential location. The overall HBeAg seroprevalence was 36.7%. Women between 20-24 years of age had the highest rate of 54.5%. Low level of knowledge about hepatitis B vaccination was reputed. Overall, 54.6% of participants were not aware of their hepatitis B vaccination status and only 65.2% of mothers reported their child had been vaccinated. CONCLUSION Hepatitis B is a disease of public health importance in Solomon Islands and emphasize the need for integrated preventative interventions for its control. PMID:28008343

  6. Sequence analysis of the HBV S protein in Chinese patients with coexisting HBsAg and anti-HBs antibodies.

    PubMed

    Ding, Feng; Yu, Hong-Gang; Li, Yan-Xia; Cui, Ning; Dai, Jin-Fen; Yu, Jie-Ping

    2015-12-01

    The coexistence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in patients with hepatitis B virus (HBV) infection has been discovered and explained for several decades, but debate still exists. This study was to explore the relationship between this special serological pattern and mutations in S gene region. Fifteen patients with coexisting HBsAg and anti-HBs were selected as the experimental group, and 27 patients with HBsAg positive only were selected as the control group. The S gene region was amplified and sequenced. No significant differences were observed between the two groups with regard to age, gender, alanine aminotransferase level, HBsAg titer, genotype, and HBV DNA level. The patients from the two groups were infected with HBV of the genotype B and C. Compared with the control group, the experimental group showed a higher variability in amino acid within the N-terminal region and the MHR, especially the "a" determinant. The most frequent change in patients from the experimental group was located at positions s126. The coexistence of HBsAg and anti-HBs might be associated with the increased amino acid mutations in the "a" determinant. Further studies should be performed to determine the clinical implication of this serological pattern, including the binding of anti-HBs to HBsAg, escape from immune system, and efficacy of antiviral therapy.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    SciTech Connect

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

    2013-09-13

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

  10. Evolution of full-length genomes of HBV quasispecies in sera of patients with a coexistence of HBsAg and anti-HBs antibodies.

    PubMed

    Zhou, Tai-Cheng; Li, Xiao; Li, Long; Li, Xiao-Fei; Zhang, Liang; Wei, Jia

    2017-04-06

    Although the evolutionary changes of viral quasispecies are correlated to the pathological status of a disease, little is known in the coexistence of hepatitis B surface antigen (HBsAg) and antibodies to these antigens (anti-HBs). To examine evolutionary changes in hepatitis B virus (HBV) and their relationship to the coexistence of HBsAg and anti-HBs antibodies, HBV genomes in patients with a coexistence of HBsAg and anti-HBs antibodies (experimental group) and HBsAg positive without anti-HBs (control group) were assessed. Our results showed that quasispecies diversity was significantly higher in the experimental group for large HBsAg (LHBsAg), middle HBsAg (MHBsAg), and HBsAg genes. LHBsAg harbored dN/dS values eight times higher in the experimental group; however, the mean dN/dS ratios in genes HbxAg, Pol and PreC/C of the experimental patients had an opposite trend. Phylogenetic trees in the experimental group were more complex than the control group. More positive selection sites, mutations and deletions were observed in the experimental group in specific regions. Furthermore, several amino acid variants in epitopes were potentially associated with the immune evasion. In conclusion, cumulative evolutionary changes in HBV genome that facilitate immune evasion provide insights into the genetic mechanism of a coexistence of HBsAg and anti-HBs antibodies.

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

    PubMed Central

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

    2015-01-01

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

  12. 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).

  13. Serodiagnosis of Chagas' disease by enzyme-linked immunosorbent assay using two synthetic peptides as antigens.

    PubMed Central

    Peralta, J M; Teixeira, M G; Shreffler, W G; Pereira, J B; Burns, J M; Sleath, P R; Reed, S G

    1994-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against Trypanosoma cruzi. Two synthetic T. cruzi peptides, TcD and PEP2, were used. The specificity and sensitivity of the peptide ELISA were determined with 260 serum samples from individuals living in an area in which Chagas' disease is endemic. ELISAs were performed with the peptides singly or in combination. The evaluation of these tests showed that 168 (93.8%) of 179 serum samples from T. cruzi-infected patients were positive when TcD peptide was used as antigen; 164 (91.6%) samples were positive with PEP2, and 178 (99.4%) samples were positive when the two peptides were combined. Thus, the sensitivity of the ELISA using the two peptides exceeded 99%. The specificity was evaluated by using a panel of 118 serum samples that included samples from 81 individuals living in an area of endemicity with negative serology for Chagas' disease and from 37 patients from areas in which T. cruzi was not endemic but with other pathologies, such as leishmaniasis, tuberculosis, and leprosy. Only two false-positive serum samples were found in this group of individuals, giving a test specificity of more than 98%. Because these peptides can be synthesized and are very stable at room temperature, the use of such reagents can improve the standardization and reproducibility of ELISAs for the serodiagnosis of T. cruzi infection. PMID:8027352

  14. Multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contrast.

    PubMed

    Boulware, David R; Rolfes, Melissa A; Rajasingham, Radha; von Hohenberg, Maximilian; Qin, Zhenpeng; Taseera, Kabanda; Schutz, Charlotte; Kwizera, Richard; Butler, Elissa K; Meintjes, Graeme; Muzoora, Conrad; Bischof, John C; Meya, David B

    2014-01-01

    Cryptococcal meningitis is common in sub-Saharan Africa. Given the need for data for a rapid, point-of-care cryptococcal antigen (CRAG) lateral flow immunochromatographic assay (LFA), we assessed diagnostic performance of cerebrospinal fluid (CSF) culture, CRAG latex agglutination, India ink microscopy, and CRAG LFA for 832 HIV-infected persons with suspected meningitis during 2006-2009 (n = 299) in Uganda and during 2010-2012 (n = 533) in Uganda and South Africa. CRAG LFA had the best performance (sensitivity 99.3%, specificity 99.1%). Culture sensitivity was dependent on CSF volume (82.4% for 10 μL, 94.2% for 100 μL). CRAG latex agglutination test sensitivity (97.0%-97.8%) and specificity (85.9%-100%) varied between manufacturers. India ink microscopy was 86% sensitive. Laser thermal contrast had 92% accuracy (R = 0.91, p<0.001) in quantifying CRAG titers from 1 LFA strip to within <1.5 dilutions of actual CRAG titers. CRAG LFA is a major advance for meningitis diagnostics in resource-limited settings.

  15. Evaluation of a commercially available enzyme-linked immunosorbent assay for Giardia lamblia antigen in stool.

    PubMed Central

    Addiss, D G; Mathews, H M; Stewart, J M; Wahlquist, S P; Williams, R M; Finton, R J; Spencer, H C; Juranek, D D

    1991-01-01

    The lack of a quick, simple, and inexpensive diagnostic test has limited the ability of public health officials to rapidly assess and control outbreaks of Giardia lamblia in child day-care centers. We evaluated the performance of a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of a G. lamblia-associated antigen in stool. Stool specimens were collected from the diapers of 426 children attending 20 day-care centers, fixed in 10% Formalin and polyvinyl alcohol, and examined by microscopy by Formalin concentration and trichrome staining techniques. Specimens were also tested visually and spectrophotometrically by ELISA. Of 99 tests positive by microscopy, 93 were visually positive by ELISA (sensitivity, 93.9%). Of 534 tests negative for G. lamblia by microscopy, 32 (6.0%) were ELISA positive. However, on the basis of examination of multiple specimens from the same child, none of these could be considered false-positive ELISAs; the specificity of the ELISA was therefore 100%. The sensitivity of both microscopy and ELISA improved as the number of specimens per child increased. An optical density value of greater than 0.040 was 98.0% sensitive and 100% specific for G. lamblia. This ELISA, which appeared to be more sensitive for G. lamblia than did microscopic examination of stool, should be useful as an epidemiologic tool, particularly in day-care settings, and may also have a role in confirming clinical diagnoses of giardiasis. PMID:1864930

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2013-01-01

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

  20. 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

  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. Gelatin particle indirect agglutination and enzyme-linked immunosorbent assay for diagnosis of strongyloidiasis using Strongyloides venezuelensis antigen.

    PubMed

    Huaman, Maria Cecilia; Sato, Yoshiya; Aguilar, Jose Luis; Terashima, Angelica; Guerra, Humberto; Gotuzzo, Eduardo; Kanbara, Hiroji

    2003-01-01

    Routine microscopical examination of stool specimens for diagnosis of strongyloidiasis is insensitive and serological methods using Strongyloides stercoralis antigen are at present not available for field studies. We evaluated 2 techniques, enzyme-linked immunosorbent assay (ELISA) and gelatin particle indirect agglutination (GPIA), using an antigen obtained from the rodent parasite, S. venezuelensis. Fifty-four Peruvian patients with different clinical forms of strongyloidiasis were studied: 12 asymptomatic, 31 symptomatic, and 11 hyperinfection cases. Our results demonstrate that both ELISA and GPIA using S. venezuelensis antigen are useful for diagnosis of strongyloidiasis, with sensitivities of 74.1% and 98.2%, respectively and a specificity of 100% for both techniques. We found that GPIA is a highly sensitive test for patients with suspected chronic infection and/or hyperinfection. In the hyperinfection cases, significantly lower concentrations of specific immunoglobulin antibodies and eosinophils (P < 0.001) were found compared with the asymptomatic and symptomatic cases.

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

    PubMed Central

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

    1993-01-01

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

  4. 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

  5. Development and validation of a rapid immunochromatographic assay for detection of Middle East respiratory syndrome coronavirus antigen in dromedary camels.

    PubMed

    Song, Daesub; Ha, Gunwoo; Serhan, Wissam; Eltahir, Yassir; Yusof, Mohammed; Hashem, Farouq; Elsayed, Elsaeid; Marzoug, Bahaaeldin; Abdelazim, Assem; Al Muhairi, Salama

    2015-04-01

    We present here a rapid immunochromatographic assay for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV) antigen in the nasal swabs of dromedary camels. The assay is based on the detection of MERS-CoV nucleocapsid protein in a short time frame using highly selective monoclonal antibodies at room temperature. The relative sensitivity and specificity of the assay were found to be 93.90% and 100%, respectively, compared to that of the UpE and open reading frame 1A (Orf1A) real-time reverse transcriptase PCR (RT-PCR). The results suggest that the assay developed here is a useful tool for the rapid diagnosis and epidemiological surveillance of MERS-CoV infection in dromedary camels.

  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. 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.

  8. [Nursing-home-acquired pneumococcal pneumonia--comparison of sputum cultures with Binax NOW Streptococcus pneumoniae urinary antigen assay].

    PubMed

    Rikimaru, Toru; Nishiyama, Mamoru; Yonemitsu, Junko; Nagabuchi, Masako; Shimada, Akiko; Koga, Takeharu; Aizawa, Hisamichi

    2008-11-01

    To clarify the clinical significance of Pneumococcal pneumonia in nursing-home-acquired pneumonia, we examined the positive disease rate of using sputum cultures and the Binax NOW Streptococcus pneumoniae urinary antigen assay in 154 nursing-home patients with pneumonia. These included 54 males and 100 females with a mean age of 86.2 years. Bacteriological findings for sputum culture in 130 patients showed Streptococcus pneumoniae to be cultured in 11 cases (8%). In 72 in whom the Streptococcus pneumoniae-urinary antigen test (Binax NOW) was done, the urinary-antigen-positive rate (26/72 ; 36%) was higher than the culture positive rate for S. pneumoniae. Both examinations were done in 64 patients, among whom 5 in whom S. pneumoniae was cultured also had positive results for the urinary antigen test. Almost half of those undergoing percutaneous endoscopic gastroscopy (PEG) tube nutrition had positive results for the urinary antigen test, but not all such patients had positive cultures for S. pneumoniae. Although the culture-positive rate for S. pneumoniae in sputum was low, we concluded that S. pneumoniae was frequently linked to nursing-home-acquired pneumonia, especially in "total-care" patients.

  9. 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.

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

    PubMed

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

    2008-05-01

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

  11. Detection of a low-molecular-weight antigen on melanoma cells by a human antiserum in leukocyte-dependent antibody assays.

    PubMed

    Hersey, P; Murray, E; Werkmeister, J; McCarthy, W H

    1979-10-01

    Biochemical characterization of serologically detected human melanoma antigens was undertaken for the development of immunodiagnostic assays in melanoma. An antiserum from a human melanoma patient, which detected melanoma antigens expressed on a large proportion of different melanoma cells, was used in leucocyte-dependent cytotoxic antibody (LDA) 51Cr-release assays to monitor the purification of melanoma antigens in urea/acetate extracts of lactoperoxidase 125I-labelled melanoma cell membranes. The separation procedures included affinity chromatography on Concanavalin A, gel filtration on porous polyacrylamide beads and preparative isoelectric focusing. The fractions were also monitored by polyacrylamide electrophoresis in sodium dodecyl sulphate and by measurement of beta 2 microglobulin and carcinoembryonic antigen content. The antigens detected by this antiserum appeared to be acidic (pI 3.5) low-mol.-wt glycoproteins of approximately 15,000 daltons which were resistant to heating at 56 degrees C and digestion with neuraminidase, but susceptible to repeated freeze-thawing and trypsin digestion. They did not appear to be related to HLA antigens, beta 2 microglobulin or known foetal antigens. The nature of the antigens detected in these studies is as yet unknown, but they appear similar to those described in the sera and urine of melanoma patients in previous reports. Thes combined results and the frequent expression of these antigens on melanoma cells from different patients suggest that assays to detect this antigen may provide a valuable immunodiagnostic aid in the management of melanoma.

  12. Status of HBsAg seroprevalence in 15 million rural couples in China: a cross-sectional study

    PubMed Central

    Zhang, Long; Wang, Yuan-Yuan; Huang, Yan-Jie; Wang, Qiao-Mei; Nelson, Kenrad E.; Wang, An-Qi; Shen, Hai-Ping; Liu, Xiao-Li; Zhang, Yi-Ping; Yan, Dong-Hai; Peng, Zuo-Qi; Zhang, Hong-Guang; Zhang, Ya; Zhao, Jun; Wang, Yan; Yang, Ying; He, Yuan; Xu, Ji-Hong; Liu, Du-Jia; Guo, Tong-Jun; Xin, Xiao-Na; Zhou, Hong; Ma, Xu

    2017-01-01

    A cross-sectional analysis of prevalence of hepatitis B virus infection (HBV) among rural couples was conducted between 2010 and 2014. Serologic HBV markers, including hepatitis B surface antigen (HBsAg) and e antigen (HBeAg), were tested. Primary outcome of interest comprised HBsAg positivity in couples (both positive: F+M+, only wife positive: F+M−, only husband positive: F−M+), and secondary outcome consisted of prevalence and risk factors of HBsAg positivity among husbands or wives. Of 14,816,300 couples included, 0.7% were F+M+; 6.3% were F−M+; 4.4% were F+M−, resulting in the overall seroprevalence of 11.4%. Individually, 6.1% were HBsAg positive with a higher rate seen in husbands (7.0%) than in wives (5.2%). Wife’s HBeAg(+)/HBsAg (+) (AOR = 2.61), HBeAg(−)/HBsAg (+) (AOR = 2.23), positivity of syphilis (AOR = 1.50), living in a high-risk region (AOR = 1.46) were significantly predictors of HBsAg positivity in husbands. Prevalence and predictors of HBsAg positivity in wives had similar results. Our data show a high burden and discordant pattern of HBV infection in rural couples, and partner’s double positivity of HBeAg and HBsAg was the most significant factor of HBV infection in couples. A comprehensive strategy that emphasizes vaccination and education is needed. PMID:28220812

  13. Fluorescent target array killing assay: a multiplex cytotoxic T-cell assay to measure detailed T-cell antigen specificity and avidity in vivo.

    PubMed

    Quah, Benjamin J C; Wijesundara, Danushka K; Ranasinghe, Charani; Parish, Christopher R

    2012-08-01

    Here we describe a multiplex, fluorescence-based, in vivo cytotoxic T-cell assay using the three vital dyes carboxyfluorescein diacetate succinimidyl ester, cell trace violet, and cell proliferation dye efluor 670. When used to label cells in combination, these dyes can discriminate >200 different target cell populations in the one animal due to each target population having a unique fluorescence signature based on fluorescence intensity and the different emission wavelengths of the dyes. This allows the simultaneous measurement of the in vivo killing of target cells pulsed with numerous peptides at different concentrations and the inclusion of many replicates. This fluorescent target array killing assay can be used to measure the fine antigen specificity and avidity of polyclonal cytotoxic T-cell responses in vivo, immunological parameters that were previously impossible to monitor.

  14. [Effect of conditions of monoclonal antibody adsorption on antigen-binding activity].

    PubMed

    Tarakanova, Iu N; Dmitriev, D A; Massino, Iu S; Smirnova, M B; Segal, O L; Fartushnaia, O V; Iakovleva, D A; Koliaskina, G I; Lavrov, V F; Dmitriev, A D

    2012-01-01

    The dependence of the antigen-binding activity of immobilized antibodies on pH of a saturating buffer has been investigated. We analyzed 28 monoclonal antibodies (MCAs) produced by various hybridomas to three virus antigens, i.e., the nuclear p23 protein of hepatitis C virus (C core protein p23), p24 protein of HIV 1, and the surface antigen of hepatitis B virus (HBsAg). Antibodies were adsorbed on the surfaces of immune plates in acidic (pH 2.8), neutral (pH 7.5), and alkaline (pH 9.5) buffers. The binding of labeled antigens, i.e., biotinylated or conjugated with horseradish peroxidase, with immobilized antigens was tested. It was shown that 10 out of 28 analyzed MCAs (36%) considerably better preserved their antigen-binding activity if their passive adsorption was carried out on the surface of polystyrene plates in an acidic buffer (pH 2.8). This approach allowed constructing a highly sensitive sandwich method for HBsAg assay with a minimal reliably determined antigen concentration of 0.013-0.017 ng/ml. The described approach may be recommended for the optimization of sandwich methods and solid-phase competitive methods.

  15. 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

  16. Detection of antibodies to hepatitis B core antigen using the Abbott ARCHITECT anti-HBc assay: analysis of borderline reactive sera.

    PubMed

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

    2008-12-01

    Routine use of the automated chemiluminescent microparticle immunoassay Abbott ARCHITECT anti-HBc for diagnosis of hepatitis B is limited in case of borderline reactive sera with low signal close to the cut-off index. In order to determine the significance of anti-HBc detection when borderline reactivity occurs using the ARCHITECT anti-HBc assay, a comparative study was designed. 3540 serum samples collected over a 2-month period in the hospital of Nice were examined for markers of HBV infection (HBsAg, anti-HBs and anti-HBc). One hundred seven samples with sufficient volume and with borderline reactivity by the ARCHITECT assay were tested by two other anti-HBc assays, a microparticle enzyme immunoassay (MEIA, AxSYM Core, Abbott Laboratories, IL, USA) and an enzyme linked fluorescent assay (ELFA, VIDAS Anti-HBc Total II, bioMérieux, Lyon, France). Only 46 samples were confirmed by the AxSYM and the VIDAS assays. Additional serological information linked to patient history showed that the remaining samples (61) were false positives (11), had low titer of anti-HBc antibodies (13), or were inconclusive (37). This comparative study highlighted the existence of a grey zone around the cut-off index. Confirmative results through a different immunoassay are needed to confirm the diagnosis of HBV on borderline reactive sera using the ARCHITECT anti-HBc assay.

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

    PubMed

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

    2006-12-01

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

  18. Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas' disease.

    PubMed Central

    Umezawa, E S; Nascimento, M S; Kesper, N; Coura, J R; Borges-Pereira, J; Junqueira, A C; Camargo, M E

    1996-01-01

    Immunoblotting with trypomastigote excreted-secreted antigens (TESA blot) of Trypanosoma cruzi was evaluated as a method for diagnosis of chronic and acute phases as well as congenital (in newborn children) Chagas' disease. Serum samples from acute-phase and congenital infections were considered to be positive when they reacted with ladder-like bands of 130- to 200-kDa antigens, recognized by immunoglobulin M (IgM) and IgG antibodies, while IgG from chronic-phase sera recognized a broad band antigen of 150 to 160 kDa. Nonchagasic sera were not reactive to these antigens. The study was carried out on 512 patients, 111 of whom were nonchagasic but included cases of leishmaniasis or other pathologies, and 401 chagasic patients. The latter group comprised 361 chronic cases, 36 acute cases, and 4 congenital cases in newborn children. Among the chronic cases, 256 were from areas in which T. cruzi is endemic but which differed widely in the pathogenic expression of T. cruzi infection and in parasitemia levels. These patients at the same time showed a broad range of low, medium, and high reactivity to conventional enzyme-linked immunosorbent assays and indirect immunofluorescence serotests for Chagas' disease. For these reasons they may better represent the universe of chagasic patients than would a sample of highly reactive sera obtained from chagasic patients in a single area endemic for T. cruzi. All acute and congenital cases showed positivity in the IgM and IgG TESA blots, while chronic cases were 100% positive for IgG antibodies. In nonchagasic sera, including 30 cases of visceral and muco-cutaneous leishmaniasis, the specificity index was 1.000, and no cross-reactions were observed. The TESA blot thus seems to be useful as a sensitive and specific diagnostic assay in cases of suspected acute or congenital T. cruzi infection and as a general confirmatory test for conventional Chagas' disease serology. PMID:8862574

  19. Japanese reference panel of blood specimens for evaluation of hepatitis C virus RNA and core antigen quantitative assays.

    PubMed

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

    2012-06-01

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

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

    PubMed Central

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

    1997-01-01

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

  1. A single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen

    PubMed Central

    Schild, G. C.; Wood, J. M.; Newman, R. W.

    1975-01-01

    Single-radial-diffusion techniques are proposed as possible alternatives to tests based on agglutination of erythrocytes for the assay of the haemagglutinin content of influenza vaccines. Two test procedures (microtest and macrotest) and the use of reference reagents to assay vaccines using these tests are described. The two tests are of similar reproducibility and accuracy but the macrotest is technically simpler to perform and results of assays may be obtained more rapidly. ImagesFig. 1aFig. 3a PMID:816480

  2. 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.

  3. 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

  4. An in vitro immune response model to determine tetanus toxoid antigen (vaccine) specific immunogenicity: Selection of sensitive assay criteria.

    PubMed

    Piersma, Sytse J; Leenaars, Marlies P P A M; Guzylack-Piriou, Laurence; Summerfield, Artur; Hendriksen, Coenraad F M; McCullough, Ken C

    2006-04-12

    Many vaccines employed in childhood vaccination programmes are produced by conventional techniques, resulting in complex biological mixtures for which batch-related quality control requires in vivo potency testing. Monitoring consistency via in vitro tests during the vaccine production has the capacity to replace certain of the in vivo methods. In this respect, determining vaccine antigen immunogenicity through functional immunological tests has high potential. Advances in immunology have made it possible to analyse this biological activity by in vitro means. The present study established such an in vitro test system for tetanus toxoid (TT). This measured vaccine immunogenicity through an antigen-specific secondary (recall) response in vitro, using a porcine model growing in value for its closeness to human immune response characteristics. Discrimination between the specific recall TT antigen and diphtheria toxoid (DT) was possible using both peripheral blood mononuclear cell cultures and monocyte-derived dendritic cells in co-culture with autologous specific lymphocytes. TT-specific activation was detected with highest discrimination capacity using proliferation assays, as well as IFN-gamma and TT-specific antibody ELISPOTS (measuring secreting T and B lymphocytes, respectively). These in vitro systems show a high potential for replacing animal experimentation to evaluate the immunogenicity of complex vaccines.

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

    PubMed

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

    2008-03-20

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

  6. Fluoroimmunoassay for antigen based on fluorescence quenching between quantum dots and gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Huang, Peng; Wang, Kan; Pandoli, Omar; Zhang, Xueqing; Gao, Feng; Shao, Jun; You, Xiaogang; He, Rong; Song, Hua; Cui, Daxiang

    2010-11-01

    A unique, sensitive, and highly specific fluoroimmunoassay system for antigen detection using gold and quantum dot nanoparticles has been developed. The assay is based on the fluorescence quenching of quantum dots caused by gold nanoparticles coated with antibody. To demonstrate its analytical capabilities, the CdTe quantum dots were coated with anti-HBsAg monoclonal antibodies (QDs-MAb1) and gold nanoparticles coated with another anti-HBsAg monoclonal antibodies (GNPs-MAb2) which specifically bound with HBsAg could sandwich the HBsAg captured by the immunoreactions. The sandwich-type immunocomplex was formed and the fluorescence intensity of quantum dots was measured. The results showed that the fluorescence intensity of quantum dots at 570 nm was negative linear proportional to the HBsAg concentration logarithm, and the limit of detection of the HBsAg was 0.928 ng/mL. This new system can be extended to detect target molecules with matched antibodies and has broad potential applications in immunoassay and disease diagnosis.

  7. The development of an enzyme-linked immunosorbent assay for measuring the potency of vaccines containing Clostridium chauvoei antigens.

    PubMed

    Crichton, R; Solomon, J; Barton, A M

    1990-01-01

    Current standards (British Pharmacopeia (Veterinary) 1985) for vaccines containing Clostridium chauvoei require a potency test based on a challenge assay in guinea-pigs. Animal welfare and cost considerations favour the development of alternatives. Most veterinary clostridial vaccines are multi component, requiring assays in rabbits to test the potency of components other than C. chauvoei. We describe the application of an ELISA to measure the response to C. chauvoei vaccines in rabbits. The antigen is a sonicated extract of C. chauvoei strain CH4, intended to include a mixture of cellular and soluble antigens. The rabbit response to more than 70 vaccines containing C. chauvoei has been assessed against a reference serum which has been assigned an arbitrary potency of 100 units ml-1. The antibody titres of rabbit sera have been compared with the results of guinea-pig challenge potency tests on the same vaccines. The pass level in the guinea-pig potency test is equivalent to a rabbit ELISA titre of 50 units ml-1.

  8. Nanoparticle-Based biobarcode amplification assay (BCA) for sensitive and early detection of human immunodeficiency type 1 capsid (p24) antigen.

    PubMed

    Tang, Shixing; Zhao, Jiangqin; Storhoff, James J; Norris, Philip J; Little, Richard F; Yarchoan, Robert; Stramer, Susan L; Patno, Tim; Domanus, Marc; Dhar, Arindam; Mirkin, Chad A; Hewlett, Indira K

    2007-10-01

    Nanotechnology-based techniques are being widely evaluated in medical testing and could provide a new generation of diagnostic assays due to their high degrees of sensitivity, high specificity, multiplexing capabilities, and ability to operate without enzymes. In this article, we have modified a nanoparticle-based biobarcode amplification (BCA) assay for early and sensitive detection of HIV-1 capsid (p24) antigen by using antip24 antibody-coated microplates to capture viral antigen (p24) and streptavidin-coated nanoparticle-based biobarcode DNAs for signal amplification, followed by detection using a chip-based scanometric method. The modified BCA assay exhibited a linear dose-dependent pattern within the detection range of 0.1 to 500 pg/ml and was approximately 150-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA). No false positive results were observed in 30 HIV-1-negative samples, while all 45 HIV-1 RNA positive samples were found HIV-1 p24 antigen positive by the BCA assay. In addition, the BCA assay detected HIV-1 infection 3 days earlier than ELISA in seroconversion samples. Preliminary evaluation based on testing a small number of samples indicates that the HIV-1 p24 antigen BCA may provide a new tool for sensitive and early detection of HIV-1 p24 antigen in settings where HIV-1 RNA testing is currently not routinely performed.

  9. 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.

  10. Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

    PubMed

    Merten, O W; Reiter, S; Scheirer, W; Katinger, H

    1983-01-01

    The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

  11. Linear antigenic regions of the structural proteins of human T-cell lymphotropic virus type I detected by enzyme-linked immunosorbent assays using synthetic peptides as antigens.

    PubMed Central

    Washitani, Y; Kuroda, N; Shiraki, H; Itoyama, Y; Sato, H; Ohshima, K; Kiyokawa, H; Maeda, Y

    1992-01-01

    We synthesized 46 sequential peptides 21 to 39 amino acids long over the structural protein of human T-cell leukemia virus type I (HTLV-I; the p19 and p24 gag protein and the gp46 and p20E env proteins) and tested their reactivities against antibodies in sera from HTLV-I healthy carriers and patients diagnosed as having human T-cell leukemia-lymphoma (ATLL) and myelopathy (HAM) by using an enzyme-linked immunosorbent assay. Of the 46 synthetic peptides, 18 peptides (2 corresponding to the p19 gag protein, 2 corresponding to the p24 gag protein, 8 corresponding to the gp46 env protein, and 6 corresponding to the p20E env protein) reacted with antibodies in the sera from HTLV-I healthy carriers. In particular, the peptides comprising amino acids 100 to 119 and 119 to 130 of the gag and 175 to 199, 213 to 236, 253 to 282, and 288 to 317 of the env proteins reacted with antibodies in sera from more than 30% of HTLV-I healthy carriers. These peptides also showed high reactivities to the antibodies in the sera from patients with ATLL and HAM. The results indicate that the predominant antigenic regions of the structural protein of HTLV-I were located at the C-terminal end of the p19 gag protein and the C-terminal half of the gp46 env protein, and the corresponding peptides proved to be useful antigens in detecting antibodies in the sera from individuals infected with HTLV-I. PMID:1537894

  12. Nationwide seroepidemiology of hepatitis B virus infection in South Korea in 2009 emphasizes the coexistence of HBsAg and anti-HBs.

    PubMed

    Lee, Byung Seok; Cho, Yong Kyun; Jeong, Sook-Hyang; Lee, Joon Hyeok; Lee, Don; Park, Neung Hwa; Ki, Moran

    2013-08-01

    Hepatitis B virus (HBV) infection is the major cause of chronic liver disease in Korea. This study investigated the seroprevalence of HBV infection with an emphasis on the coexistence of hepatitis B surface antigen (HBsAg) and antibody (anti-HBs). In all, 290,212 people undergoing health check-up examinations in 29 institutions during 2009 were recruited. The crude seroprevalences of HBsAg and anti-HBs was adjusted by age, sex, and geographic area using the 2009 estimated population of Korea. The adjusted seroprevalences of HBsAg and anti-HBs was 4.0% and 73.5%, respectively. Males showed higher HBsAg positivity and lower anti-HBs positivity than females (P<0.001). HBsAg positivity increased with age from 3.5% in people 20-29 years old to 4.8% in people 40-49 years old, followed by a decrease in people ≥ 50 years old. HBsAg positivity in Southern provinces (4.5%) including Jeju (5.9%), was significantly higher than that in Central provinces (3.6%; P<0.001). Interestingly, HBsAg and anti-HBs coexisted in 0.1% of the total subjects and in 2.9% of the HBsAg-positive group, showing distinct age distribution and higher alanine aminotransferase levels than those of the group positive for only HBsAg. In conclusion, the seroprevalence of HBsAg and anti-HBs in Korea varies significantly by age, sex and geographical location and coexisted in 2.9% of HBsAg-positive subjects. Continuous monitoring of seroepidemiology may facilitate the eventual eradication of HBV infection.

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

    PubMed Central

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

    2013-01-01

    Tumor marker endothelial 8 (TEM8) is a receptor for the Protective Antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared to normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z-prime value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complimentary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for anti-anthrax and anti-angiogenic diseases. PMID:23479355

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

    PubMed

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

    1989-01-01

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

  15. Sensitive detection and glycoprofiling of a prostate specific antigen (PSA) using impedimetric assays

    PubMed Central

    Pihíková, D; Belický, Š; Kasák, P; Bertok, T; Tkac, J

    2016-01-01

    The study presents proof-of-concept for development of an impedimetric biosensor for ultrasensitive glycoprofiling of PSA. The biosensor exhibits three unique characteristics: 1) analysis of PSA with LOD down to 4 aM; 2) analysis of a glycan part of PSA with LOD down to 4 aM level and 3) both assays (i.e. PSA quantification and PSA glycoprofiling) can be performed on the same interface due to label-free format of analysis. PMID:26647853

  16. 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.

  17. Development of Lateral Flow Assay Based on Size-Controlled Gold Nanoparticles for Detection of Hepatitis B Surface Antigen

    PubMed Central

    Kim, Dong Seok; Kim, Yong Tae; Hong, Seok Bok; Kim, Jinwoon; Heo, Nam Su; Lee, Moon-Keun; Lee, Seok Jae; Kim, Byeong Il; Kim, In Soo; Huh, Yun Suk; Choi, Bong Gill

    2016-01-01

    In this study, we developed lateral flow assay (LFA) biosensors for the detection of hepatitis B surface antigens using well-controlled gold nanoparticles (AuNPs). To enhance colorimetric signals, a seeded growth method was used for the preparation of size-controlled AuNPs with a narrow size distribution. Different sizes of AuNPs in the range of 342–137.8 nm were conjugated with antibodies and then optimized for the efficient detection of LFA biosensors. The conjugation stability was investigated by UV-vis spectroscopy of AuNP dispersion at various pH values and concentrations of antibody. Based on optimized conjugation conditions, the use of 42.7 ± 0.8 nm AuNPs exhibited superior performance for the detection of LFAs relative to other sizes of AuNPs. PMID:27999291

  18. 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%.

  19. Detection of Hepatitis B virus antigen from human blood: SERS immunoassay in a microfluidic system.

    PubMed

    Kamińska, Agnieszka; Witkowska, Evelin; Winkler, Katarzyna; Dzięcielewski, Igor; Weyher, Jan L; Waluk, Jacek

    2015-04-15

    A highly sensitive immunoassay utilizing surface-enhanced Raman scattering (SERS) has been developed with a new Raman reporter and a unique SERS-active substrate incorporated into a microfluidic device. An appropriately designed Raman reporter, basic fuchsin (FC), gives strong SERS enhancement and has the ability to bind both the antibody and gold nanostructures. The fuchsin-labeled immuno-Au nanoflowers can form a sandwich structure with the antigen and the antibody immobilized on the SERS-active substrate based on Au-Ag coated GaN. Our experimental results indicate that this SERS-active substrate with its strong surface-enhancement factor, high stability and reproducibility plays a crucial role in improving the efficiency of SERS immunoassay. This SERS assay was applied to the detection of Hepatitis B virus antigen (HBsAg) in human blood plasma. A calibration curve was obtained by plotting the intensity of SERS signal of FC band at 1178cm(-1) versus the concentration of antigen. The low detection limit for Hepatitis B virus antigen was estimated to be 0.01IU/mL. The average relative standard deviation (RSD) of this method is less than 10%. This SERS immunoassay gives exact results over a broad linear range, reflecting clinically relevant HBsAg concentrations. It also exhibits high biological specificity for the detection of Hepatitis B virus antigen.

  20. Azide and Tween-20 reduce binding to autoantibody epitopes of islet antigen-2; implications for assay performance and reproducibility.

    PubMed

    Williams, Alistair J K; Somerville, Michelle; Rokni, Saba; Bonifacio, Ezio; Yu, Liping; Eisenbarth, George; Akolkar, Beena; Steffes, Michael; Bingley, Polly J

    2009-12-31

    Autoantibodies to islet antigen 2 (IA-2A) are important markers for predicting diabetes in children and young adults. Harmonization of IA-2A assay measurement is essential if results from different laboratories are to be compared. We investigated whether sodium azide, a bacteriostatic agent added to some assays, could affect IA-2A binding and thereby contribute to differences in IA-2A measurement between laboratories. Addition of 0.1% azide to assay buffer was found to reduce median IA-2A binding of 18 selected sera from IA-2A positive patients with type 1 diabetes and their relatives by 41% (range, 78 to -33%, p<0.001). The effect on binding was epitope specific; median IA-2A binding by 14 sera with antibodies to the protein tyrosine phosphatase region of IA-2 was reduced by 48% (range, 11 to 78%, p<0.001), while binding by 4 sera with antibodies specific to only the juxtamembrane region of IA-2 showed no change (median increase 16% (range 6 to 33%, p=0.125). When the Tween-20 concentration was reduced from 1% to 0.15% the median reduction in IA-2A binding with azide by the 18 sera was only 10% (range, -12 to 41%, p<0.001). Tween-20 also exerted an independent effect, since median IA-2A binding increased by 23% (range 3% to 86%, p<0.001) when Tween-20 concentration was reduced from 1% to 0.15% in the absence of azide. We conclude that common assay reagents such as azide and Tween-20 can strongly influence IA-2A binding in an epitope-related manner, and their use may explain some of the differences between laboratories in IA-2A measurement.

  1. Improved primary immunodiagnosis of alveolar echinococcosis in humans by an enzyme-linked immunosorbent assay using the Em2plus antigen.

    PubMed Central

    Gottstein, B; Jacquier, P; Bresson-Hadni, S; Eckert, J

    1993-01-01

    Alveolar echinococcosis (AE) in humans is generally a fatal disease when not diagnosed early enough to provide curative treatment such as radical surgery. Immunodiagnosis for early detection of AE was improved by the isolation of an affinity-purified metacestode Em2 antigen and by the synthesis of recombinant Echinococcus multilocularis antigen II/3-10. Both antigens were individually assessed by enzyme-linked immunosorbent assay (ELISA) and demonstrated high specificities and diagnostic sensitivities, although both missed approximately 4 to 11% of diagnostic cases of AE. To provide an optimal serodiagnostic test, we investigated the two purified antigens by using a test employing a mixture of both purified antigens (designated Em2plus antigen) in one assay. For comparative purposes, crude E. multilocularis and Echinococcus granulosus metacestode antigens were investigated as well. The Em2plus ELISA proved to be the optimal diagnostic test with the highest diagnostic sensitivity, 97%, in serum samples from 140 patients with AE and an overall specificity of 99% for infections due to other Echinococcus and non-Echinococcus parasites. The new test combination (Em2plus ELISA) is suggested for the serodiagnosis of AE in patients and for seroepidemiological surveys. PMID:8432825

  2. 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.

  3. Sensitivities of antigen detection and PCR assays greatly increased compared to that of the standard culture method for screening for group B streptococcus carriage in pregnant women.

    PubMed

    Rallu, Fabien; Barriga, Peter; Scrivo, Carole; Martel-Laferrière, Valérie; Laferrière, Céline

    2006-03-01

    Group B streptococcus (GBS) is a major cause of serious infections in neonates. The 2002 revised guidelines of the Centers for Disease Control and Prevention (CDC) for the prevention of perinatal GBS disease recommend that all pregnant women be screened for GBS carriage at between 35 and 37 weeks of gestation and that intrapartum antibiotic prophylaxis be given to carriers. We studied the performances of four different GBS detection assays in the context of antenatal screening. Between May and August 2004, the 605 vaginorectal swab specimens received at our bacteriology laboratory for GBS antenatal detection were tested by the four assays. The standard culture method was done according to the CDC recommendations. The three experimental assays performed with the growth from the selective enrichment (LIM) broth (Todd-Hewitt broth with 15 mug/ml nalidixic acid and 10 mug/ml colistin) after overnight incubation were a GBS antigen detection assay (PathoDx) and two PCR assays (for cfb and scpB). The most accurate assay was the scpB PCR (sensitivity, 99.6%; specificity, 100%), followed by the cfb PCR (sensitivity, 75.3%; specificity, 100%), GBS antigen detection (sensitivity, 57.3%; specificity, 99.5%), and standard culture (sensitivity, 42.3%; specificity, 100%). The GBS antigen detection assay was found to be more sensitive than the standard culture method, and moreover, the assay has a low cost and is easy to perform in all obstetrical centers which have access to the most basic of diagnostic microbiology services. We believe that antigen detection on incubated LIM broth should replace the standard culture method for screening for GBS carriage at 35 to 37 weeks of gestation. The impact of the greater sensitivities of PCR assays on the diminution of neonatal GBS infections remains to be demonstrated.

  4. Histamine release-neutralization assay for sera of patients with atopic dermatitis and/or cholinergic urticaria is useful to screen type I hypersensitivity against sweat antigens.

    PubMed

    Shindo, Hajime; Ishii, Kaori; Yanase, Yuhki; Suzuki, Hidenori; Hide, Michihiro

    2012-10-01

    We previously reported that about 80 % of patients with atopic dermatitis and 60 % with cholinergic urticaria revealed type I allergy against sweat, by means of skin test against autologous sweat and/or histamine-release test for peripheral blood basophils with semi-purified sweat antigen. In this study, we developed an assay for sera to neutralize histamine-releasing activity of semi-purified sweat antigen. The semi-purified sweat antigen was pre-incubated with serially diluted sera for 30 min at 37 °C and was subjected to histamine-release activity. Histamine release-neutralization (HRN) activities were calculated by measuring the amount of histamine release from basophils in the presence or absence of semi-purified sweat antigen. Of 62 subjects, 39 showed positive histamine release (≥5 %) from their basophils in response to semi-purified sweat antigen, and sera of 34 out of 39 subjects (87.2 %) were also positive in HRN activity (≥10 %). The specificity of the HRN assay was 0.522. Moreover, HRN activities in sera were largely correlated with degrees of histamine release from peripheral blood basophils of the same donors in response to sweat antigen. To identify the substance that neutralizes histamine-release activity, we removed IgE and IgG from the sera of HRN (+) subjects by column chromatography. The HRN activities in 30 out of 42 sera were largely reduced by the removal of IgG. On the other hand, sera of four subjects lost HRN activity by the removal of IgE, suggesting that the majority of HRN (+) subjects have serum IgG against the sweat antigen as well as IgE bound to peripheral basophils. Thus, the HRN assay maybe useful for the screening of type I allergy against sweat antigen.

  5. 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

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

    2013-08-01

    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

  6. Highly sensitive detection of human cancer antigens by an immunogold-silver assay chip coupled with a polythiophene-based optical sensor.

    PubMed

    Pires, Nuno M M; Tao Dong

    2016-08-01

    This work presents a novel method for protein or cancer antigen detection in clinical samples by an immunogold-silver assay microfluidic biochip coupled with a polythiophene-based organic photodetector. The method has showed a detection limit below 1ng/mL and the low cost and high sensitivity of both organic photodetector and immunogold-silver assay make this method amenable for realization in a portable handheld probe tip biosensor.

  7. Mutations in surface and polymerase gene of chronic hepatitis B patients with coexisting HBsAg and anti-HBs

    PubMed Central

    Lu, Hai-Ying; Zeng, Zheng; Xu, Xiao-Yuan; Zhang, Nai-Lin; Yu, Min; Gong, Wei-Bo

    2006-01-01

    AIM: To investigate the clinical significance and presence of mutations in the surface (S) and overlapping polymerase gene of hepatitis B patients with coexisting HBsAg and anti-HBs. METHODS: Twenty-three patients with chronic hepatitis B were studied. Of the 23 patients, 11 were both positive for hepatitis B virus (HBV) surface antigen (HBsAg) and antibody to HBV surface antigen (anti-HBs), 12 were negative for anti-HBs while positive for HBsAg. DNA was extracted from 200 μL serum of the patients. Nucleotide of the surface and overlapping polymerase gene from HBV-infected patients was amplified by PCR, and the PCR products were sequenced. RESULTS: Forty-one mutations were found within the surface gene protein of HBV in 15 patients (10 with coexisting HBsAg and anti-HBs). Six (14.6%) out of 41 mutations were located at “α” determinant region in 5 patients (4 positive for HBsAg and anti-HBs). Eleven mutations (26.8%) occurred in the downstream or upstream of “α” determinant region. Lamivudine (LMV)-selected mutations were found in three patients who developed anti-HBs, which occurred in amino acid positions (196, 198, 199) of the surface protein and in YMDD motif (M204I/V) of the polymerase protein simultaneously. Presence of these mutations did not relate to changes in ALT and HBV DNA levels. CONCLUSION: Besides mutations in the “α” deter-minant region, mutations at downstream or upstream of the “α” determinant region may contribute to the development of anti-HBs. These mutations do not block the replicating competency of HBV in the presence of high titer of anti-HBs. PMID:16830379

  8. Understanding thread properties for red blood cell antigen assays: weak ABO blood typing.

    PubMed

    Nilghaz, Azadeh; Zhang, Liyuan; Li, Miaosi; Ballerini, David R; Shen, Wei

    2014-12-24

    "Thread-based microfluidics" research has so far focused on utilizing and manipulating the wicking properties of threads to form controllable microfluidic channels. In this study we aim to understand the separation properties of threads, which are important to their microfluidic detection applications for blood analysis. Confocal microscopy was utilized to investigate the effect of the microscale surface morphologies of fibers on the thread's separation efficiency of red blood cells. We demonstrated the remarkably different separation properties of threads made using silk and cotton fibers. Thread separation properties dominate the clarity of blood typing assays of the ABO groups and some of their weak subgroups (Ax and A3). The microfluidic thread-based analytical devices (μTADs) designed in this work were used to accurately type different blood samples, including 89 normal ABO and 6 weak A subgroups. By selecting thread with the right surface morphology, we were able to build μTADs capable of providing rapid and accurate typing of the weak blood groups with high clarity.

  9. An intravenous exposure mouse model for prediction of potential drug-sensitization using reporter antigens popliteal lymph node assay.

    PubMed

    Lin, Mingbao; Sun, Wei; Wang, Yingchao; Li, Xiang; Jin, Yecheng; Gong, Wan; Fan, Xiaohui; Wang, Yi

    2012-06-01

    Immune-mediated drug hypersensitivity is a particularly concerning health-safety issue among clinicians given its unpredictability and potentially life-threatening effects, especially with exposure to intravenous drugs. Therefore, the development of intravenous drug-exposure models for drug-hazard assessments has garnered increasing interest in recent years. In this study, we used reporter antigens popliteal lymph node assay to investigate the potential value of intravenous exposure to a selected variety of allergenic compounds, including ovalbumin (OVA), concanavalin A (ConA) and diclofenac. The trinitrophenyl (TNP)-specific antibody-forming cells were used to assess the systemic immune responses to a bystander antigen. Mice were subsequently sensitized by TNP-OVA, and then intravenous exposure to one of the selective compounds. As expected, all positive compounds induced significant popliteal lymph node (PLN) proliferation compared with the control. OVA significantly increased Cluster of Differentiation 4 receptors (CD⁴)⁺ interleukin-4 (IL-4)⁺ T-helper 2 (Th2) cells and, consequently, increased the ratios of IL-4/interferon-γ (IFN-γ) antibody-forming cells (AFCs) in PLNs, while bringing about a dose-dependent increase in immunoglobulin G1 (IgG1) AFCs; these findings indicate that a Th2 hypersensitivity response was induced. A Th2 response was also observed in diclofenac sodium-treated groups, and for ConA, a more mixed Th1/Th2 immune response appeared to be induced. In addition, there was no marked reaction with the negative compound. Together, it seems likely that the intravenous exposure model may be useful for drug-induced systemic hypersensitivity assessments.

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

    PubMed

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

    2003-11-01

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

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

    PubMed

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

    2015-06-01

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

  12. An easy and sensitive sandwich assay for detection of Mycobacterium tuberculosis Ag85B antigen using quantum dots and gold nanorods.

    PubMed

    Kim, Eun Ju; Kim, Eun Bee; Lee, Seung Woo; Cheon, Seon Ah; Kim, Hwa-Jung; Lee, Jaebeom; Lee, Mi-Kyung; Ko, Sungho; Park, Tae Jung

    2017-01-15

    Mycobacterium tuberculosis is a serious global infectious pathogen causing tuberculosis (TB). The development of an easy and sensitive method for the detection of M. tuberculosis is in urgent need due to complex and low specificity of the current assays. Herein, we present a novel method for M. tuberculosis detection based on a sandwich assay via antigen-antibody interaction using silica-coated quantum dots (SiQDs) and gold nanorods (AuNRs). A genetically engineered recombinant antibody (GBP-50B14 and SiBP-8B3) was bound to surfaces of AuNRs and SiQDs respectively, without any surface modification. The antigen-antibody interaction was revealed using M. tuberculosis-specific secretory antigen, Ag85B. Two biocomplexes showed a quenching effect in the presence of the target antigen through a sandwich assay. The assay response was in the range of 1×10(-3)-1×10(-10)μgmL(-1) (R=0.969) and the limit of detection for Ag85B was 13.0pgmL(-1). The Ag85B was selectively detected using three different proteins (CFP10, and BSA), and further specifically confirmed by the use of spiked samples. Compared with existing methods, a highly sensitive and selective method for Ag85B-expressing M. tuberculosis detection has been developed for better diagnosis of TB.

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

    PubMed Central

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

    1984-01-01

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

  14. Comparison of real-time PCR and antigen assays for detection of hepatitis E virus in blood donors.

    PubMed

    Vollmer, T; Knabbe, C; Dreier, J

    2014-06-01

    Hepatitis E virus (HEV) infection is recognized as an emerging and often undiagnosed disease in industrialized countries, with asymptomatic infections actually occurring in blood donors. Sensitive detection of HEV-RNA is crucial for diagnosis and monitoring of disease progression. We evaluated the analytical sensitivity and performance of three HEV RT-PCR assays (RealStar HEV reverse transcription-PCR [RT-PCR], hepatitis@ceeramTools, and ampliCube HEV RT-PCR) for screening of individuals for HEV infections (ID-nucleic acid amplification technology [ID-NAT]) and for blood donor pool screening (minipool-NAT [MP-NAT]). RNA was extracted using NucliSens easyMAG (ID-NAT) and a high-volume extraction protocol (4.8 ml, chemagic Viral 5K, MP-NAT). Three NAT assays were evaluated for ID-NAT but only two assays for MP-NAT due to inhibition of the ampliCube HEV RT-PCR kit using the corresponding RNA extract. Assays provided good analytical sensitivity, ranging from 37.8 to 180.1 IU/ml (ID-NAT) and from 4.7 to 91.2 IU/ml (MP-NAT). The applicability of HEV antigen (HEV-Ag) screening was compared to that of RT-PCR screening and detection of HEV-IgM antibodies using seroconversion panels of 10 HEV genotype 3-infected individuals. Four individuals revealed a positive HEV-Ag detection result, with corresponding viremias ranging from 1.92 E + 03 to 2.19 E + 05 IU/ml, while the progression of HEV-Ag followed that of HEV viremia. The other six individuals showed no presence of HEV-Ag although the corresponding viremias were also in the range of >1.0 E + 03. Anti-HEV-IgM antibodies were detectable in seven donors; one donor presented parallel positivities of HEV-Ag and anti-HEV IgM. The evaluated NAT methods present powerful tools providing sensitive HEV detection. Application of HEV-Ag or anti-HEV IgM screening is currently inferior for the early detection of HEV infection due to the decreased sensitivity compared to NAT methods.

  15. Biochemical and Biophysical Characterization of Maize-derived HBsAg for the Development of an Oral Vaccine

    PubMed Central

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

    2015-01-01

    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

  16. 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.

  17. 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.

  18. Evaluation of LIAISON® XL system for HBsAg, and anti-HCV and anti-HIV/Ag p24.

    PubMed

    De Paschale, Massimo; Manco, Maria Teresa; Belvisi, Luisa; Cagnin, Debora; Cerulli, Teresa; Paganini, Alessia; Arpino, Olivia; Cianflone, Annalisa; Agrappi, Carlo; Mirri, Paola; Clerici, Pierangelo

    2017-03-01

    The aim of this study was to compare the data obtained using the new LIAISON® XL chemiluminescence system to search for HBsAg, anti-HCV, and anti-HIV1-2/p24 Ag with those obtained using the VITROS system currently adopted by the Microbiology Unit of the Hospital of Legnano. Routine samples of patients who were referred by practitioners for the determination of HBsAg (1,000 samples) and/or anti-HCV (1,002 samples) and/or anti-HIV1-2 (995 samples) were simultaneously analyzed using both systems. The concordant positive and discordant samples were re-examined for confirmation by means of an HBsAg neutralization assay, anti-HCV immunoblot, or anti-HIV1-2 Western blot; HBV-DNA, or HCV-RNA or HIV-RNA was also sought in the discordant samples. Samples of patients known to be positive were tested (100 HBsAg positive, 100 anti-HCV positive, and 100 HIV 1-2 positive) as well throughout treatment, with viremia levels becoming undetectable after treatment. The HBsAg, anti-HCV, and anti-HIV1-2 concordance between the two systems in routine series was respectively 99.8%, 98.5% and 99.7%, and 100% for all markers in samples known positive. The various molecular biology and confirmatory tests of the discordant samples were all negative (except for one anti-HCV positive sample). Measure of Cohen's kappa coefficient for HBsAg, anti-HCV, and anti-HIV gave K values of respectively 0.992, 0.946, and 0.980. In conclusion, the performance of the LIAISON® XL system in the routine laboratory determination for all three markers was comparable with that of the VITROS system. J. Med. Virol. 89:489-496, 2017. © 2016 Wiley Periodicals, Inc.

  19. Hepatocellular carcinoma HBsAg positive in pregnancy.

    PubMed

    Gonçalves, C S; Pereira, F E; de Vargas, P R; Ferreira, L S

    1984-01-01

    The authors present a case of hepatocellular carcinoma diagnosed in a pregnant woman (four months pregnancy). The clinical evolution was complicated because of a severe hypoglicemia and the patient died 12 weeks after admission. The fetus died before a tentative of surgical delivery. The patient was HBsAg positive and five out of eight sons (inclusively the fetus), were HBsAg positive. There was not indication that the pregnancy had enhanced the tumor evolution.

  20. Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

    PubMed Central

    2013-01-01

    Background The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides. Methods Twenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay. Results The peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%. Conclusions The epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the

  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.

  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.

  3. Anti-HIV seropositivity was related to HBsAg seropositivity among injecting drug users in Taiwan.

    PubMed

    Hsieh, Meng-Hsuan; Hsieh, Ming-Yen; Huang, Chung-Feng; Yeh, Ming-Lun; Wang, Shu-Chi; Yang, Jeng-Fu; Chang, Ko; Lin, Wei-Ru; Lin, Chun-Yu; Chen, Tun-Chieh; Huang, Jee-Fu; Dai, Chia-Yen; Tsai, Jih-Jin; Chuang, Wan-Long; Yu, Ming-Lung

    2016-02-01

    In Taiwan, the number of new cases of human immunodeficiency virus (HIV) infection via drug injection has been increasing since 2003. Due to HIV and hepatitis B virus (HBV) having similar transmission routes, HBV and HIV infections among injecting drug users (IDUs) has become an important public health issue. The aim of this study was explore the prevalence of HBV infection among IDUs with and without HIV infection, and examine whether HIV infection is associated with HBV infection among IDUs in Southern Taiwan. We enrolled 566 IDUs, including 87 anti-HBV positive IDUs and 479 anti-HBV negative IDUs, and also analyzed the results of liver function tests, HBV DNA, anti-HIV, HIV RNA, and CD4 cell count. The results showed that the prevalence of HBV infection among IDUs was 15.4%. The prevalence of hepatitis B surface antigen (HBsAg) was higher among individuals born before 1985 (15.9% vs. 4.0%), but this was not significant. Anti-HIV seropositivity was related to HBsAg seropositivity [odds ratio (OR) = 2.47, 95% confidence interval = 1.26-4.82, p = 0.008). Anti-HCV and anti-HIV were risk factors for abnormal alanine aminotransferase (ALT; OR = 2.11, 95% confidence interval = 1.005-4.42, p = 0.048 and OR = 1.47, 95% confidence interval = 1.02-2.10, p = 0.04, respectively), and HBsAg was not a factor related to abnormal ALT. In conclusion, the prevalence of HBV infection was similar in the general population and in IDUs, and due to anti-HIV seropositivity being significantly related to HBsAg seropositivity, HBV infection among IDUs is still important. We suggest that for IDUs, HBsAg should be monitored closely.

  4. [Quantification of the hepatitis B surface antigen in the characterization and follow-up].

    PubMed

    Rodríguez, Manuel; González-Diéguez, María Luisa

    2014-07-01

    The recent availability of commercial techniques for the quantification of the hepatitis B surface antigen (HBsAg) has revived interest in this antigen. In recent years, the antigen's potential as a biomarker of the natural history of the disease and its response to antiviral treatment has been assessed. HBsAg serum values reflect the transcriptional activity of cccDNA; reading these values could therefore complement the reading of hepatitis B virus (HBV) DNA in the categorization of the various phases of chronic HBV infection. During its natural history, HBsAg values progressively decrease from the immune-tolerant phase to the inactive carrier phase. For patients who are HBeAg-negative, the combined reading of HBV DNA and HBsAg can be useful for differentiating inactive carriers from patients with chronic HBeAg-negative hepatitis, for stratifying the risk of developing hepatocarcinoma and for predicting HBsAg clearance.

  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. 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.

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

    PubMed

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

    1983-01-01

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

  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

    2015-06-10

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

  9. Use of the protective antigen of Erysipelothrix rhusiopathiae in the enzyme-linked immunosorbent assay and latex agglutination.

    PubMed

    Sato, H; Yamazaki, Y; Tsuchiya, K; Aoyama, T; Akaba, N; Suzuki, T; Yokoyama, A; Saito, H; Maehara, N

    1998-09-01

    To establish a safe and convenient serodiagnostic method for swine erysipelas, a purified protective protein antigen of Erysipelothrix rhusiopathiae, which included a large amount of protective protein (64 kDa protein), was used for enzyme-linked immunosorbent assay (ELISA) and the latex agglutination (LA) test. In the ELISA, the antisera to four different serovars (1a, 2, 5 and 20) of E. rhusiopathiae exhibit a positive reaction, while antisera to other species of bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Rhodococcus equi and Corynebacterium pseudotuberculosis) exhibit a negative reaction. In the LA test, the antisera to three different serovars (1a, 2 and 5) of E. rhusiopathiae reacted with P64-sensitized latex beads, while the antiserum to serovar 20 (2553 strain) did not. Moreover, the antisera to other species of bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Rhodococcus equi and Corynebacterium pseudotuberculosis) did not in this test. Comparing the results of the growth agglutination (GA), ELISA and LA tests of 284 swine sera, there was a high degree of correlation among the results. The detection of anti-E. rhusiopathiae antibodies in the GA, ELISA and LA tests were compared using sera from pigs immunized with P64, alkaline extract (AE) and live-cell vaccine (LV). In all three tests, anti-E. rhusiopathiae antibodies could be detected 1 week after immunization. The serum antibody titre as determined by the LA test increased moderately, as did that by the GA test, while that determined by ELISA increased rapidly. These results suggested that ELISA could be used to monitor changes in anti-E. rhusiopathiae antibody titre and the LA test could be used in the screening test for swine erysipelas.

  10. 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

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed Central

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

    1994-01-01

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

  13. New direct-acting antivirals for patients with chronic HCV infection: can we monitor treatment using an HCV core antigen assay?

    PubMed

    Alonso, R; Pérez-García, F; Ampuero, D; Reigadas, E; Bouza, E

    2017-03-01

    We evaluated the diagnostic usefulness of an HCV core antigen (HCV-Ag) assay in HCV-infected patients undergoing treatment with direct-acting antivirals. We analyzed 103 samples from 28 patients. Compared with RT-PCR, sensitivity was 96.2% and specificity was 100%. The correlation between techniques was excellent (Pearson coefficient: 0.871). HCV-Ag proved to be useful in patients with sustained viral response and in patients who experienced treatment failures.

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

    PubMed Central

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

    2003-01-01

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

  15. Kinetics of Hepatitis B Surface Antigen Level in Chronic Hepatitis B Patients who Achieved Hepatitis B Surface Antigen Loss during Pegylated Interferon Alpha-2a Treatment

    PubMed Central

    Li, Ming-Hui; Zhang, Lu; Qu, Xiao-Jing; Lu, Yao; Shen, Ge; Wu, Shu-Ling; Chang, Min; Liu, Ru-Yu; Hu, Lei-Ping; Li, Zhen-Zhen; Hua, Wen-Hao; Song, Shu-Jing; Xie, Yao

    2017-01-01

    Background: Hepatitis B surface antigen (HBsAg) loss/seroconversion is considered to be the ideal endpoint of antiviral therapy and the ultimate treatment goal in chronic hepatitis B (CHB). This study aimed to assess the patterns of HBsAg kinetics in CHB patients who achieved HBsAg loss during the treatment of pegylated interferon (PEG-IFN) α-2a. Methods: A total of 150 patients were enrolled, composing of 83 hepatitis B envelope antigen (HBeAg)-positive and 67 HBeAg-negative patients. Patients were treated with PEG-IFN α-2a180 μg/week until HBsAg loss/seroconversion was achieved, which occurred within 96 weeks. Serum hepatitis B virus deoxyribonucleic acid and serological indicators (HBsAg, anti-HBs, HBeAg, and anti-HBe) were determined before and every 3 months during PEG-IFN α-2a treatment. Biochemical markers and peripheral blood neutrophil and platelet counts were tested every 1–3 months. Results: Baseline HBsAg levels were 2.5 ± 1.3 log IU/ml, and decreased rapidly at 12 and 24 weeks by 48.3% and 88.3%, respectively. The mean time to HBsAg loss was 54.2 ± 30.4 weeks, though most patients needed extended treatment and 30.0% of HBsAg loss occurred during 72–96 weeks. Baseline HBsAg levels were significantly higher in HBeAg-positive patients (2.9 ± 1.1 log IU/ml) compared with HBeAg-negative patients (2.0 ± 1.3 log IU/ml; t = 4.733, P < 0.001), but the HBsAg kinetics were similar. Patients who achieved HBsAg loss within 48 weeks had significantly lower baseline HBsAg levels and had more rapid decline of HBsAg at 12 weeks compared to patients who needed extended treatment to achieve HBsAg loss. Conclusions: Patients with lower baseline HBsAg levels and more rapid decline during early treatment with PEG-IFN are more likely to achieve HBsAg loss during 96 weeks of treatment, and extended therapy longer than 48 weeks may be required to achieve HBsAg loss. PMID:28229987

  16. Detection of pulmonary and extrapulmonary tuberculosis patients with the 38-kilodalton antigen from Mycobacterium tuberculosis in a rapid membrane-based assay.

    PubMed Central

    Zhou, A T; Ma, W L; Zhang, P Y; Cole, R A

    1996-01-01

    A rapid membrane-based serologic assay using the 38-kDa antigen from Mycobacterium tuberculosis for the diagnosis of tuberculosis (TB) was evaluated with 201 patients with pulmonary TB, 67 patients with extrapulmonary TB, 79 Mycobacterium bovis BCG-vaccinated healthy controls, and 77 non-TB respiratory patients. The overall sensitivities, specificities, and positive and negative predictive values were, respectively, 92, 92, 84, and 96% for sputum-positive TB patients; 70, 92, 87, and 79% for sputum-negative TB patients; and 76, 92, 80, and 90% for extrapulmonary-TB patients. Only 2% (1 of 44) of the healthy control BCG-vaccinated subjects gave weak positive signals in the assay, indicating that this rapid serological assay is a valuable aid in clinical diagnosis for both pulmonary and extrapulmonary TB. PMID:8705680

  17. Hepatitis B surface antigen in blood donors. An epidemiologic study.

    PubMed

    Jayaprakash, P A; Shanmugam, J; Hariprasad, D

    1983-01-01

    Of 8085 volunteer donors attending the blood bank at SCTIMST screened for hepatitis B surface antigen (HBsAg) carrier state by counterimmunoelectrophoresis, 103 (1.27%) were HBsAg positive. The personal data of donors showed a higher rate of HBsAg among men than women and in the age group of 21 to 30 years than in the other age groups. A significantly higher rate was noted among donors belonging to the lower socioeconomic group (p less than 0.05).

  18. Highly-sensitive and specific enzyme-linked immunosorbent assays for GAD65 autoantibodies using a thioredoxin-GAD65 fusion antigen.

    PubMed

    Papouchado, M L; Valdez, S N; Ermácora, M R; Gañan, S; Poskus, E

    1997-09-24

    Autoantibodies against glutamic acid decarboxylase (GAD65) are present in the sera of most patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM). These antibodies appear years before the clinical symptoms, and they are considered to be early markers of the disease. To detect GAD65 autoantibodies (GADA), we developed new enzyme-linked immunosorbent assays (ELISA) with a fusion protein thioredoxin-GAD65 (Trx-GAD65) produced in E. coli as the antigen. These assays were compared with the reference radiobinding assay (RBA). Since most GADA are directed against native epitopes, and adsorption of GAD65 to plastic may cause disruption of its native conformation, the new assays rely on the following immobilization procedures: (a) capture ELISA (c-ELISA) with Trx-GAD65 (protocol A) or biotin-Trx-GAD (protocol B) indirectly immobilized by a non-adsorptive process; (b) ELISA with antigen-antibody preincubation in solution (p-ELISA) in which GADA were reacted first with Trx-GAD65 (protocol C) or biotin-Trx-GAD (protocol D) and the free antigen was determined by conventional ELISA. The results obtained with 42 newly diagnosed IDDM patients and 30 normal individuals were as follows: RBA had 79% sensitivity (percentage of IDDM patients detected) and 97% specificity (100% minus the percentage of false positives). c-ELISA showed low sensitivity (36 and 50%, respectively for protocols A and B), and high specificity (100 and 97%, respectively). p-ELISA were highly-sensitive (74 and 79%, respectively) and specific (97 and 93% for protocols C and D, respectively). Thus, protocols C and D had a performance similar to the reference method. The results reported here provide the basis for simple, highly-sensitive, specific, and widely-applicable tests for GADA that eliminate many of the drawbacks of the radioactive methods.

  19. Comparison of monolisa HCV Ag/Ab ULTRA with two anti-HCV assays for the detection of HCV infection in hospital setting.

    PubMed

    Yagci, Server; Padalko, Elizaveta

    2012-02-01

    In this study, we compared the performance of three serological assays (Monolisa HCV Ag/Ab ULTRA, Innotest HCV Ab IV enzyme immunoassay--EIA, and Ortho HCV 3.0 enzyme-linked immunosorbent assay--ELISA) for the detection of HCV infection. Ninety plasma samples were collected, representing 63 samples from groups at risk for acquiring HCV infection and 27 HCV RNA-positive samples. The results of Ortho HCV 3.0 ELISA, Innotest HCV Ab IV, and Monolisa HCV Ag/Ab ULTRA were fully concordant for 27 HCV RNA-positive samples. Ortho HCV 3.0 ELISA test and Innotest HCV Ab IV also gave the same results for risk groups, while three samples were found to be reactive by Monolisa HCV Ag/Ab ULTRA and were consequently found negative for HCV RNA. As two of the solely Monolisa HCV Ag/Ab ULTRA-positive samples were also hepatitis B s antigen (HBsAg)-positive, neutralization of HBsAg was performed but no arguments for the HBsAg interference were observed. In conclusion, the non-specific reactive signal was observed, in three samples using Monolisa HCV Ag/Ab ULTRA, to be negative by other serological assays, and observed to be negative in an HCV RNA assessment, a result that could not be attributed to the interference with HBsAg. In the context of diagnostic testing, no test for various HCV genotypes was observed to be superior to any other.

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

    SciTech Connect

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

    1982-01-01

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

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

    PubMed

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

    1982-01-01

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

  2. Antigen-specific secretion of IFNγ and CXCL10 in whole blood assay detects Mycobacterium leprae infection but does not discriminate asymptomatic infection from symptomatic leprosy.

    PubMed

    Hungria, Emerith Mayra; Freitas, Aline Araújo; Pontes, Maria Araci Andrade; Gonçalves, Heitor Sá; Sousa, Ana Lúcia Osório Maroccolo; Costa, Maurício Barcelos; Castilho, Mirian Lane Oliveira Rodrigues; Duthie, Malcolm S; Stefani, Mariane Martins Araújo

    2017-04-01

    To advance toward a whole blood assay (WBA)-based test capable of facilitating the diagnosis of paucibacillary (PB) leprosy, we evaluated a prototype in-tube WBA using combinations of Mycobacterium leprae antigens. Blood was collected from newly diagnosed untreated PB (n=38), multibacillary (MB) (n=30), healthy household contacts (HHC) of MB (n=27), and endemic controls (n=61) residing in Goiânia and Fortaleza, Brazil. Blood was incubated with M. leprae cell sonicate, recombinant proteins (46f+LID-1; ML0276+LID-1), or controls (phosphate-buffered saline, phytohemagglutinin, M. tuberculosis purified protein derivative). Antigen-specific IFNγ production was observed in 71-84% and 55% of PB and HHC, respectively. Antigen-specific CXCL10 levels were similarly assessed to determine if, unlike IFNγ, CXCL10 could differentiate PB from HHC with repeated exposure/asymptomatic M. leprae infection. The CXCL10 levels induced in response to M. leprae antigens could not, however, differentiate PB from HHC. Despite these limitations, the WBAs reported here still represent important tools for assessing M. leprae infection rates and evaluating the impact of control measures.

  3. 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

  4. 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.

  5. 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

  6. Cryptococcal phospholipase B antigen is not detected in serum of patients infected with Cryptococcus neoformans using a sandwich enzyme-linked immunosorbent assay.

    PubMed

    Wu, Qi Xuan; Chen, Sharon C A; Santangelo, Rosemary T; Martin, Patricia; Malik, Richard; Sorrell, Tania C

    2007-05-01

    Extracellular phospholipase B (PLB) is a virulence determinant of Cryptococcus neoformans and Cryptococcus gattii. In this study, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for PLB antigen with a detection limit of 3.9 ng mL(-1). PLB was detected in culture supernatants of C. neoformans and C. gattii. PLB, however, was not detected in sera of seven human patients and 10 feline patients with active cryptococcosis. Furthermore, none of five rats with extensive pulmonary C. gattii infection had a positive ELISA test result. In conclusion, cryptococcal PLB could not be detected in serum using a PLB antigen-based ELISA. Despite its sensitivity, this ELISA is of limited diagnostic value. Exploration of further extracellular molecules suitable for serodiagnosis of active cryptococcal infection is warranted.

  7. Development and Application of an ELISA Assay Using Excretion/Secretion Proteins from Epimastigote Forms of T. cruzi (ESEA Antigens) for the Diagnosis of Chagas Disease

    PubMed Central

    Berrizbeitia, Mariolga; Figueroa, Milagros; Ward, Brian J.; Rodríguez, Jessicca; Jorquera, Alicia; Figuera, Maria A.; Romero, Leomerys; Ndao, Momar

    2012-01-01

    An indirect enzyme-linked immunoabsorbent assay (ELISA) for Trypanosoma cruzi was developed using epimastigote secretion/excretion proteins (ESEA antigens) obtained from axenic culture supernatants. A panel of 120 serum samples from subjects with confirmed Chagas disease (n = 50), healthy controls (n = 50), and patients with other parasitic diseases (n = 20) was used to evaluate the new ESEA-based ELISA (ELISAESEA). This new test had excellent sensitivity (98%) and acceptable specificity (88%). Cross-reactivity was observed largely in sera from subjects with Leishmania and Ascaris infections. Using Western blotting and epimastigotes from two distinct T. cruzi isolates, several polypeptide bands with molecular masses ranging from 50 to 220 kDa were detected in pooled chagasic sera. However, the band pattern for each isolate was different. These data suggest that an inexpensive and technically simple ELISA based on ESEA antigens is a promising new tool for the diagnosis of Chagas disease. PMID:23049572

  8. [Dynamic expression profile of HBsAg according to hepatic parenchyma cells' volume at different liver fibrosis stages in the immune clearance phase].

    PubMed

    Wu, Zhe-bin; Cao, Hong; Liu, Ting; Wu, Ze-qian; Ke, Wei-min; Gao, Zhi-liang

    2012-10-01

    The aim of this study was to determine the dynamic expression profile of hepatitis B surface antigen (HBsAg) according to hepatic parenchyma cells' volume at different stages of liver fibrosis during the immune clearance phase. Eighty-nine patients with HBeAg-positive chronic hepatitis B (CHB) in the immune clearance stage were recruited for study. Each patient's serum HBsAg levels were detected by electrochemiluminescence. The serum HBsAg levels were apportioned according to hepatic parenchyma cells' volume at liver fibrosis stages 1, 2, 3, and 4 and compared by ANOVA. The unapportioned serum HBsAg levels (IU/mL) at liver fibrosis stages 1 (227.2+/-237.7), 2 (211.0+/-131.4), 3(300.1+/-144.6), and 4 (278.7+/-148.8) were not significantly different (all comparisons, P range: 0.061 to 0.759). However, when the serum HBsAg levels were apportioned by the same hepatic parenchyma cells' volume at liver fibrosis stages 1 (343.9+/-359.8), 2 (336.4+/-209.5), 3 (508.7+/-245.1), and 4 (525.2+/-274.8), the levels were significantly different (all comparisons, F = 3.045 and P = 0.033; stage 1 vs. 3, P = 0.041; stage 1 vs. 4, P = 0.046; stage 2 vs. 3, P = 0.028; stage 2 vs. 4, P = 0.034). During the immune clearance phase of chronic hepatitis B, increased HBsAg expression is associated with increased hepatic parenchyma cells' volume and progressive liver fibrosis stage.

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

    PubMed

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

    2015-06-01

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

  10. Evaluation of Point-of-Contact Circulating Cathodic Antigen Assays for the Detection of Schistosoma mansoni Infection in Low-, Moderate-, and High-Prevalence Schools in Western Kenya

    PubMed Central

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

    2015-01-01

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

  11. 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.

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

    PubMed

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

    1983-10-01

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

  13. Ultra-fast pg/ml anthrax toxin (protective antigen) detection assay based on microwave-accelerated metal-enhanced fluorescence.

    PubMed

    Dragan, Anatoliy I; Albrecht, Mark T; Pavlovic, Radmila; Keane-Myers, Andrea M; Geddes, Chris D

    2012-06-01

    Rapid presymptomatic diagnosis of Bacillus anthracis at early stages of infection plays a crucial role in prompt medical intervention to prevent rapid disease progression and accumulation of lethal levels of toxin. To detect low levels of the anthrax protective antigen (PA) exotoxin in biological fluids, we have developed a metal-enhanced fluorescence (MEF)-PA assay using a combination of the MEF effect and microwave-accelerated PA protein surface absorption. The assay is based on a modified version of our "rapid catch and signal" (RCS) technology previously designed for the ultra-fast and sensitive analysis of genomic DNA sequences. Technologically, the proposed MEF-PA assay uses standard 96-well plastic plates modified with silver island films (SiFs) grown within the wells. It is shown that the fluorescent probe, covalently attached to the secondary antibody, plays a crucial role of indicating complex formation (i.e., shows a strong MEF response to the recognition event). Microwave irradiation rapidly accelerates PA deposition onto the surface ("rapid catch"), significantly speeding up the MEF-PA assay and resulting in a total assay run time of less than 40 min with an analytical sensitivity of less than 1 pg/ml PA.

  14. 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

  15. 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

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

    PubMed

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

    2003-01-01

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

  17. 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

  18. Mutation in the S gene of hepatitis B virus and anti-HBs subtype-nonspecificity contributed to the co-existence of HBsAg and anti-HBs in patients with chronic hepatitis B virus infection.

    PubMed

    Fu, Xiaochun; Chen, Jing; Chen, Huijuan; Lin, Jinpiao; Xun, Zhen; Li, Shiqi; Liu, Can; Zeng, Yongbin; Chen, Tianbin; Yang, Bin; Ou, Qishui

    2017-02-15

    The mechanism for the co-existence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in chronic HBV infected patients remains controversial. This study aimed to explore the role of HBV S gene mutation and anti-HBs subtype-nonspecificity in patients with simultaneous HBsAg/anti-HBs positivity. Chronic HBV infections with (n = 145, group I) and without (n = 141, group II) anti-HBs were included. The S gene was amplified and sequenced. The neutralization experiment was used in group I patients' sera to determine the specificity of anti-HBs. Additionally, the HBV vaccinated persons' sera were used to estimate the neutralize capacity of anti-HBs against HBsAg in group I patients. Results showed that 2.63% (145/5513) chronic HBV infected patients had positive results for anti-HBs. HBsAg amino acid (aa) substitution rate in 35 patients of group I was significantly higher than that in 58 patients of group II (1.89% vs 0.95%, P < 0.05), especially within "a" determinant (4.05% vs 1.22%, P < 0.05). In group I patients, anti-HBs in (74.29%, 26/35) patients was not directed to the subtypes of the co-existing HBsAg. Besides, some HBsAg variations in group I patients, sG145R mutation, inserted mutations, and continuous aa mutations within the major hydrophilic region (MHR), decreased the neutralized capacity of anti-HBs from HBV vaccinated persons. In conclusion, both of HBsAg mutation and anti-HBs subtype-nonspecificity contributed to the co-existence of HBsAg and anti-HBs in chronic HBV infection. HBV vaccine recipients may still have a risk of HBV infection when exposure to patients with simultaneous HBsAg/anti-HBs positivity.

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

    PubMed

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

    2015-01-01

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

  20. [Comparison of the indirect immunofluorescence assay performance of Bartonella henselae antigens obtained by co-cultivation in Vero and HeLa cells].

    PubMed

    Ergin, Cağrı; Akkaya, Yüksel; Kiriş Satılmış, Ozgün; Yılmaz, Cansev

    2011-07-01

    The laboratory diagnosis of Bartonella henselae infection is mainly based on serological testing by indirect immunofluorescence assay (IFA). Cell line co-cultivation with B.henselae and agar derivated antigens are the two major procedures used for evaluation of anti-Bartonella antibodies. Vero and Hep-2 cell lines are the most commonly used media for co-cultivation both in-house and commercial diagnostic kits production. However, HeLa cells which are easily supplied and grown, also can easily be infected by B.henselae. The aim of this study was to compare the performances of antigens obtained by co-cultivation of B.henselae ATCC 49882 (Houston-1) in Vero and HeLa Cells in IFA serology. Out of 381 sera samples, 127 (33.3%) were found positive and 195 (51.2%) were found negative by IFA performed by both cell line co-cultivations. The total agreement between the methods were found as 84.5% (322/381), and Cohen kappa value was calculated as 0.68 (strong, coherent). As a result, He-La cells were found to be useful for the preparation of B.henselae antigens to be used in IFA for the serologic diagnosis of B.henselae infections. However different genotype strains and cross reactions with other infectious agents should be investigated by further studies before routine applications of HeLa cell co-cultivations procedure is established.

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

    PubMed

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

    2010-01-01

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

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

    PubMed Central

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

    1987-01-01

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

  3. Human fibroblast-derived molecules as antigens in enzyme-linked immunosorbent assay for coeliac disease-specific IgA.

    PubMed

    Marttinen, A; Sulkanen, S; Mäki, M

    1997-02-01

    We have recently shown that cultured human fibroblasts synthesize and secrete protein molecules that bind to IgA-class anti-reticulin and anti-endomysium antibodies but not to anti-gliadin antibodies in coeliac disease patient sera. In the present report, we describe a reproducible method for purification of these antigen molecules from fibroblast culture medium. Using reversed-phase chromatography as the final purification step, four different protein molecules reacting with coeliac disease patient sera IgA were obtained. In enzyme-linked immunosorbent assay (ELISA) for coeliac disease-specific IgA, a mixture of 0.5 microgram of the four reversed-phase-separated molecules was used as antigen. The optical density values in ELISA of the sera from newly diagnosed coeliac disease patients (n = 34) were 0.740-3.400 (mean 1.830) and in control patients (n = 66) 0.090-0.850 (mean 0.320). Using an arbitrary cut-off level of 0.700, the sensitivity of the present autoantibody test was 100%, specificity 91% and positive predictive value 85%. Our identified autoantigens may generate the production of the classical tissue antibodies, known as anti-reticulin and anti-endomysium antibodies, and may be used as antigen in an immunoassay for the antibodies.

  4. Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes.

    PubMed

    Woo, Wai-Ping; Doan, Tracy; Herd, Karen A; Netter, Hans-Jürgen; Tindle, Robert W

    2006-04-01

    Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.

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

    PubMed Central

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

    1997-01-01

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

  6. Detection of antibodies to hepatitis C virus (HCV) structural proteins in anti-HCV-positive sera by an enzyme-linked immunosorbent assay using synthetic peptides as antigens.

    PubMed Central

    Ishida, C; Matsumoto, K; Fukada, K; Matsushita, K; Shiraki, H; Maeda, Y

    1993-01-01

    We have defined 10 linear immunogenic regions encoded by the putative hepatitis C virus (HCV) structural proteins (core and envelope) by employing an enzyme-linked immunosorbent assay (ELISA) and by using 17 sequential synthetic peptides covering the N-terminal 330 amino acids of the structural polyproteins as antigens. These peptides correspond to amino acids 1 to 24, 21 to 44, 42 to 68, 64 to 91, and 100 to 120 of the putative core protein and amino acids 192 to 212, 223 to 238, 236 to 258, 250 to 266, and 307 to 330 of the putative envelope protein. In particular, the peptide covering amino acids 21 to 44 of the core protein was reactive with all but one (40 of 41) of the serum samples giving a positive signal in the passive hemagglutination assay (PHA) using the core and nonstructural proteins (NS 3/4) of the virus as antigens. We detected the HCV genome in 25 (61%) of 41 PHA-positive serum samples by the polymerase chain reaction (PCR) test. Of 25 PCR-positive serum samples, 17 serum samples had reactivity to the peptides derived from the envelope protein. On the other hand, only 1 of the 16 PCR-negative serum samples had reactivity to the peptides derived from the envelope protein. Interestingly, we often observed high serum alanine aminotransferase levels in PCR-positive individuals bearing antibodies to the envelope protein. PMID:7681849

  7. 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

  8. Noninfectious virus-like particle antigen for detection of swine vesicular disease virus antibodies in pigs by enzyme-linked immunosorbent assay.

    PubMed

    Ko, Young-Joon; Choi, Kang-Seuk; Nah, Jin-Ju; Paton, David J; Oem, Jae-Ku; Wilsden, Ginette; Kang, Shien-Young; Jo, Nam-In; Lee, Joo-Ho; Kim, Jae-Hong; Lee, Hee-Woo; Park, Jong-Myeong

    2005-08-01

    An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n=1,041). When tested using sera (n=186) collected periodically from pigs (n=19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.

  9. Expression of hepatitis B surface antigen in transgenic banana plants.

    PubMed

    Kumar, G B Sunil; Ganapathi, T R; Revathi, C J; Srinivas, L; Bapat, V A

    2005-10-01

    Embryogenic cells of bananan cv. Rasthali (AAB) have been transformed with the 's' gene of hepatitis B surface antigen (HBsAg) using Agrobacterium mediated transformation. Four different expression cassettes (pHBS, pHER, pEFEHBS and pEFEHER) were utilized to optimize the expression of HBsAg in banana. The transgenic nature of the plants and expression of the antigen was confirmed by PCR, Southern hybridization and reverse transcription (RT)-PCR. The expression levels of the antigen in the plants grown under in vitro conditions as well as the green house hardened plants were estimated by ELISA for all the four constructs. Maximum expression level of 38 ng/g F.W. of leaves was noted in plants transformed with pEFEHBS grown under in vitro conditions, whereas pHER transformed plants grown in the green house showed the maximum expression level of 19.92 ng/g F.W. of leaves. Higher monoclonal antibody binding of 67.87% of the antigen was observed when it was expressed with a C-terminal ER retention signal. The buoyant density in CsCl of HBsAg derived from transgenic banana leaves was determined and found to be 1.146 g/ml. HBsAg obtained from transgenic banana plants is similar to human serum derived one in buoyant density properties. The transgenic plants were grown up to maturity in the green house and the expression of HBsAg in the fruits was confirmed by RT-PCR. These transgenic plants were multiplied under in vitro using floral apex cultures. Attempts were also made to enhance the expression of HBsAg in the leaves of transgenic banana plants by wounding and/or treatment with plant growth regulators. This is the first report on the expression of HBsAg in transgenic banana fruits.

  10. Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

    PubMed Central

    Takehara, Shizuka; Takahashi, Masaharu

    2013-01-01

    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

  11. Comparison of a newly developed automated and quantitative hepatitis C virus (HCV) core antigen test with the HCV RNA assay for clinical usefulness in confirming anti-HCV results.

    PubMed

    Kesli, Recep; Polat, Hakki; Terzi, Yuksel; Kurtoglu, Muhammet Guzel; Uyar, Yavuz

    2011-12-01

    Hepatitis C virus (HCV) is a global health care problem. Diagnosis of HCV infection is mainly based on the detection of anti-HCV antibodies as a screening test with serum samples. Recombinant immunoblot assays are used as supplemental tests and for the final detection and quantification of HCV RNA in confirmatory tests. In this study, we aimed to compare the HCV core antigen test with the HCV RNA assay for confirming anti-HCV results to determine whether the HCV core antigen test may be used as an alternative confirmatory test to the HCV RNA test and to assess the diagnostic values of the total HCV core antigen test by determining the diagnostic specificity and sensitivity rates compared with the HCV RNA test. Sera from a total of 212 treatment-naive patients were analyzed for anti-HCV and HCV core antigen both with the Abbott Architect test and with the molecular HCV RNA assay consisting of a reverse transcription-PCR method as a confirmatory test. The diagnostic sensitivity, specificity, and positive and negative predictive values of the HCV core antigen assay compared to the HCV RNA test were 96.3%, 100%, 100%, and 89.7%, respectively. The levels of HCV core antigen showed a good correlation with those from the HCV RNA quantification (r = 0.907). In conclusion, the Architect HCV antigen assay is highly specific, sensitive, reliable, easy to perform, reproducible, cost-effective, and applicable as a screening, supplemental, and preconfirmatory test for anti-HCV assays used in laboratory procedures for the diagnosis of hepatitis C virus infection.

  12. 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.

  13. Assay Development and High-Throughput Screening for Inhibitors of Kaposi's Sarcoma-Associated Herpesvirus N-Terminal Latency-Associated Nuclear Antigen Binding to Nucleosomes.

    PubMed

    Beauchemin, Chantal; Moerke, Nathan J; Faloon, Patrick; Kaye, Kenneth M

    2014-07-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies, especially in immunocompromised hosts. KSHV latently infects tumor cells and persists as an extrachromosomal episome (plasmid). KSHV latency-associated nuclear antigen (LANA) mediates KSHV episome persistence. LANA binds specific KSHV sequence to replicate viral DNA. In addition, LANA tethers KSHV genomes to mitotic chromosomes to efficiently segregate episomes to daughter nuclei after mitosis. N-terminal LANA (N-LANA) binds histones H2A and H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high-throughput screening (HTS) of inhibitors of N-LANA binding to nucleosomes, here we develop, miniaturize, and validate a fluorescence polarization (FP) assay that detects fluorophore-labeled N-LANA peptide binding to nucleosomes. We also miniaturize a counterscreen to identify DNA intercalators that nonspecifically inhibit N-LANA binding to nucleosomes, and also develop an enzyme-linked immunosorbent assay to assess N-LANA binding to nucleosomes in the absence of fluorescence. HTS of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. No compounds survived all counterscreens, however. More complex small-molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A and H2B; these assays should prove useful for future screens.

  14. A rapid dipstick antigen capture assay for the diagnosis of falciparum malaria. WHO Informal Consultation on Recent Advances in Diagnostic Techniques and Vaccines for Malaria.

    PubMed Central

    1996-01-01

    Recent advances in the diagnosis of Plasmodium falciparum infections have made it possible to consider supplementing light microscopy with a standardized dipstick antigen capture assay based on the detection of a parasite-specific protein, which is secreted by the asexual blood stages and immature gametocytes but not by the other stages. Field trials indicate that this dipstick assay provides consistently reproducible results, with a threshold of detection of P. falciparum parasitaemia similar to that obtained by high quality routine malaria microscopy and a specificity and sensitivity of around 90% compared with standard thick blood film microscopy. The stability, reproducibility, and ease of use of the assay clearly indicate that it has potential for application in the management of malaria, particularly at the peripheral health care level, provided its accuracy can be assured and that it can be made affordable. Consideration should be given to its wider use where operational requirements and resources so justify, and where decisions are based on adequate evaluation of the existing health delivery systems. PMID:8653815

  15. Determination of Neutrophil Antigen HNA-3a and HNA-3b Genotype Frequencies in Six Racial Groups by High-Throughput 5’ Exonuclease Assay

    PubMed Central

    Bowens, Krista L.; Sullivan, Mia J.; Curtis, Brian R.

    2012-01-01

    BACKGROUND People with the human neutrophil antigen (HNA)-3b/3b type can make HNA-3a antibodies, which have been reported to cause immune neutropenia disorders, and are especially prone to cause severe cases of transfusion-related acute lung injury (TRALI). However, knowledge of HNA-3 allele frequencies outside Caucasian populations is limited. We developed a high-throughput genotyping assay and determined the HNA-3a/3b genotype frequencies in 6 different racial and ethnic groups. STUDY DESIGN AND METHODS Genotyping utilized Taqman 5’ exonuclease chemistry and real-time PCR. A total of 742 DNA samples from 6 different racial and ethnic groups were genotyped for HNA-3a and HNA-3b. RESULTS The genotyping assay showed 100% sensitivity and specificity compared to sequencing and phenotyping and had high throughput. A significant percentage of Caucasians (6.5%), Han Chinese (16%), and Asian Indians (6%) typed HNA-3b/3b, but only a small percentage of Hispanics (1%) and no African or Native Americans. CONCLUSIONS The HNA-3 genotyping assay had high sensitivity, specificity, and sample throughput. HNA-3b/b genotype results determined for 742 individuals representing 6 different racial and ethnic groups showed that there could be a significant risk of producing anti-HNA-3a in Chinese, as well as in Caucasian and Asian Indian blood donor populations, but a very low risk in Hispanic, African or Native American populations. PMID:22414054

  16. 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

  17. Effect of gamma irradiation on reactivity of rinderpest virus antigen with bovine immune serum in enzyme-linked immunosorbent assays and virus neutralization and indirect fluorescent-antibody tests.

    PubMed Central

    Saliki, J T; Berninger, M L; Torres, A; House, J A; Mebus, C A; Dubovi, E J

    1993-01-01

    Gamma irradiation effectively inactivated gradient-purified rinderpest virus. Irradiated antigen and sera remained functional in enzyme-linked immunosorbent assays, virus neutralization tests, and indirect fluorescent-antibody tests. Irradiation, however, led to a dose-dependent decrease in reactivity, particularly significant (P < 0.05) when both reagents were irradiated. To avoid false-positive reactions, only one reagent (serum or antigen) may be irradiated. PMID:8432831

  18. Enzyme-linked immunosorbent assay using recombinant SAG1 antigen to detect Toxoplasma gondii-specific immunoglobulin G antibodies in human sera and saliva.

    PubMed

    Chahed Bel-Ochi, Nouha; Bouratbine, Aïda; Mousli, Mohamed

    2013-04-01

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

  19. Sweet characterisation of prostate specific antigen using electrochemical lectin-based immunosensor assay and MALDI TOF/TOF analysis: Focus on sialic acid.

    PubMed

    Pihikova, Dominika; Pakanova, Zuzana; Nemcovic, Marek; Barath, Peter; Belicky, Stefan; Bertok, Tomas; Kasak, Peter; Mucha, Jan; Tkac, Jan

    2016-12-01

    The construction of a sensitive electrochemical lectin-based immunosensor for detection of a prostate specific antigen (PSA) is shown here. Three lectins with different carbohydrate specificities were used in this study to glycoprofile PSA, which is the most common biomarker for prostate cancer (PCa) diagnosis. The biosensor showed presence of α-L-fucose and α-(2,6)-linked terminal sialic acid within PSA´s glycan with high abundance, while only traces of α-(2,3)-linked terminal sialic acid were found. MALDI TOF/TOF mass spectrometry was applied to validate results obtained by the biosensor with a focus on determination of a type of sialic acid linkage by two methods. The first direct comparison of electrochemical immunosensor assay employing lectins for PSA glycoprofiling with mass spectrometric techniques is provided here and both methods show significant agreement. Thus, electrochemical lectin-based immunosensor has potential to be applied for prostate cancer diagnosis.

  20. Purification of a ribonuclease from potato tubers and its use as an antigen in the immunochemical assay of this protein following tuber damage.

    PubMed

    Pitt, D

    1971-12-01

    A method for the purification of a ribonuclease from potato tubers is described. The preparation was free from deoxyribonuclease and phosphodiesterase activities and possessed only slight phosphomonoesterase activity. Specific antibodies against the ribonuclease preparation were raised in rabbits. Two precipitin arcs were observed on Ouchterlony plates and three by the use of immunoelectrophoresis suggesting that the preparation contained three antigens. Development of one of the arcs on the diffusion plates could be prevented by prior absorption of the RNase preparation with an antiserum specific for phosphomonoesterase from potato tubers. Two of the arcs developing upon immunoelectrophoresis, one of which had low electrophoretic mobility and the other which migrated to the anode, corresponded in position to that of ribonuclease fractionated by agar gel electrophoresis. The remaining arc corresponded to the position of that arising when the RNase antigen was cross-reacted with specific antibodies against phosphomonoesterase from potato tubers. It was concluded that the anti-acid RNase antiserum may be useful for the immunochemical assay of RNase protein when used in conjunction with an anti-phosphomonoesterase antiserum and it was used for this purpose with homogenates derived from damaged and undamaged tuber tissue cv. Majestic. The observations suggested that RNase protein did not parallel the increase in ribonuclease activity following tissue damage and it was concluded that the enhanced RNase activity following mechanical damage may be due to activation of the pre-formed enzyme.

  1. Seroprevalence of Cryptosporidium parvum infection of dairy cows in three northern provinces of Thailand determined by enzyme-linked immunosorbent assay using recombinant antigen CpP23.

    PubMed

    Inpankaew, T; Jittapalapong, S; Phasuk, J; Pinyopanuwut, N; Chimnoi, W; Kengradomkit, C; Sunanta, C; Zhang, G; Aboge, G O; Nishikawa, Y; Igarashi, I; Xuan, X

    2009-06-01

    Cryptosporidium parvum is the most frequent parasitic agent that causes diarrhoea in AIDS patients in Thailand. Cryptosporidiosis outbreaks in humans may be attributed to contamination of their drinking water from infected dairy pastures. A 23-kDa glycoprotein of C. parvum (CpP23) is a sporozoite surface protein that is geographically conserved among C. parvum isolates. This glycoprotein is a potentially useful candidate antigen for the diagnosis of cryptosporidiosis by enzyme-linked immunosorbent assay. Therefore, we investigated the seroprevalence of C. parvum infection in dairy cows in northern Thailand using an ELISA based on recombinant CpP23 antigen. Sera were randomly collected from 642 dairy cows of 42 small-holder farmers, which had the top three highest number of the dairy cows' population in Northern Thailand, that included Chiang Mai, Chiang Rai and Lumpang provinces. The overall seroprevalence of the infection was 4.4%, and the seropositive rates for the three provinces were 3.3% in Chiang Mai, 5.1% in Chiang Rai and 3% in Lumpang. These results suggest that cattle could play a role in zoonotic cryptosporidiosis in Thailand.

  2. 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

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

    PubMed

    Vidal, Jose E; Boulware, David R

    2015-09-01

    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.

  4. 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.

  5. Manufacturer's recall of rapid assay kits based on false positive Cryptosporidium antigen tests--Wisconsin, 2001-2002.

    PubMed

    2002-03-08

    The Wisconsin Division of Public Health and the Wisconsin State Laboratory of Hygiene (WSLH) reported that a recent cluster of cryptosporidiosis cases in a three-county area in southeastern Wisconsin was the result of false-positive tests. During December 1, 2001-February 1, 2002, approximately 30 cases of cryptosporidiosis were diagnosed at a laboratory in southeastern Wisconsin using the Becton, Dickinson, and Company (Franklin Lakes, New Jersey) ColorPAC Cryptosporidium/Giardia rapid assay (lot number 219370, expiration date 2002-06-05). Seventeen stool specimens, which were collected from 11 patients and tested positive by the rapid assay, were re-evaluated at WSLH. Six of these stool specimens were in EcoFix (Meridian Bioscience Inc., Cincinnati, Ohio), eight were in Cary-Blair transport media, and three were formalin fixed. All 17 specimens tested negative for Cryptosporidium at WSLH using the hot safranin stain and MeriFluor (Meridian Bioscience Inc., Cincinnati, Ohio) Cryptosporidium/Giardia direct fluorescent antibody kit with concentrated specimens.

  6. Mapping of Schistosoma mansoni in the Nile Delta, Egypt: Assessment of the prevalence by the circulating cathodic antigen urine assay.

    PubMed

    Haggag, Ayat A; Rabiee, Amal; Abd Elaziz, Khaled M; Gabrielli, Albis F; Abdel Hay, Rehab; Ramzy, Reda M R

    2017-03-01

    In line with WHO recommendations on elimination of schistosomiasis, accurate identification of all areas of residual transmission is a key step to design and implement measures aimed at interrupting transmission in low-endemic settings. To this purpose, we assessed the prevalence of active S. mansoni infection in five pilot governorates in the Nile Delta of Egypt by examining schoolchildren (6-15 years) using the Urine-Circulating Cathodic Antigen (Urine-CCA) cassette test; we also carried out the standard Kato-Katz (KK) thick smear, the monitoring and evaluation tool employed by Egypt's national schistosomiasis control programme. Prevalence rates determined by the Urine-CCA test for all governorates were higher than those determined by KK (p<0.01). Of 35 districts surveyed in the five governorates, S. mansoni infection was detected in 19 districts (54.3%) using KK, and in 31 districts (88.6%) by Urine-CCA (χ2=9.94; P=0.0016). S. mansoni infections were detected by Urine-CCA, but not by KK in 12 districts (34.3%), and infection was not detected by either of the two diagnostic methods in four districts in Qalyubia governorate. Males and higher age-groups have significantly higher Urine-CCA prevalence rates. Based on the findings of the current S. mansoni mapping exercise, authorities of the Ministry of Health and Population (MoHP) adopted a new elimination strategy by readjusting thresholds for mass treatment with praziquantel and targeting all transmission areas. MoHP is now planning to remap in all other endemic governorates using Urine-CCA with the aim of identifying all areas of transmission where the elimination strategy should be applied.

  7. Flow cytometry-based TCR-ligand Koff -rate assay for fast avidity screening of even very small antigen-specific T cell populations ex vivo.

    PubMed

    Nauerth, Magdalena; Stemberger, Christian; Mohr, Fabian; Weißbrich, Bianca; Schiemann, Matthias; Germeroth, Lothar; Busch, Dirk H

    2016-09-01

    High epitope-specific sensitivity of CD8(+) T cells is required for optimal immune protection against intracellular pathogens as well as certain malignancies. The quality of antigen recognition of CD8(+) T cells is usually described as "avidity" to its cognate peptide MHCI complex. T cell avidity is mainly dependent on the structural qualities of the T cell receptor (TCR), as convincingly demonstrated by recombinant TCR re-expression experiments. Based on reversible MHCI multimer staining and koff -rate measurements of monomeric peptide MHCI complexes, we recently established a microscopic assay for determining the structural avidity of individual CD8(+) T cells. Here we demonstrate that this assay can be adapted for rapid flow-cytometric avidity screening of epitope-specific T cell populations. Furthermore, we show that-in combination with conventional nonreversible MHCI multimer staining-even very small epitope-specific CD8(+) T cell populations can be analyzed directly ex vivo without the need for previous TCR cloning or T cell sorting. This simplified approach provides highly accurate mean TCR-ligand koff -rate values for poly- or oligoclonal T cell populations and is ideally suited for high-throughput applications in basic research as well as clinical settings. © 2016 International Society for Advancement of Cytometry.

  8. Serodiagnosis of sporotrichosis infection in cats by enzyme-linked immunosorbent assay using a specific antigen, SsCBF, and crude exoantigens.

    PubMed

    Fernandes, Geisa Ferreira; Lopes-Bezerra, Leila Maria; Bernardes-Engemann, Andréa Reis; Schubach, Tânia Maria Pacheco; Dias, Maria Adelaide Galvão; Pereira, Sandro Antonio; de Camargo, Zoilo Pires

    2011-01-27

    The main objective of this study is to standardize an ELISA for the diagnosis of feline sporotrichosis. Sporothrix schenckii is the etiological agent of human and animal sporotrichosis. Cats may act as reservoirs for S. schenckii and can transmit the infection to humans by a bite or scratch. There are few methods for the serological diagnosis of fungal diseases in animals. In this paper, an ELISA test for the diagnosis of cat sporotrichosis is proposed, which detects S. schenckii-specific antibodies in feline sera. Two different kinds of antigens were used: "SsCBF", a specific molecule from S. schenckii that consists of a Con A-binding fraction derived from a peptido-rhamnomannan component of the cell wall, and a S. schenckii crude exoantigen preparation. The ELISA was developed, optimized, and evaluated using sera from 30 cats with proven sporotrichosis (by culture isolation); 22 sera from healthy feral cats from a zoonosis center were used as negative controls. SsCBF showed 90% sensitivity and 96% specificity in ELISA; while crude exoantigens demonstrated 96% sensitivity and 98% specificity. The ELISA assay described here would be a valuable screening tool for the detection of specific S. schenckii antibodies in cats with sporotrichosis. The assay is inexpensive, quick to perform, easy to interpret, and permits the diagnosis of feline sporotrichosis.

  9. Detection of antibodies specific for sheeppox and goatpox viruses using recombinant capripoxvirus antigens in an indirect enzyme-linked immunosorbent assay.

    PubMed

    Bowden, Timothy R; Coupar, Barbara E; Babiuk, Shawn L; White, John R; Boyd, Victoria; Duch, Christine J; Shiell, Brian J; Ueda, Norihito; Parkyn, Geoff R; Copps, John S; Boyle, David B

    2009-10-01

    Viruses in the genus Capripoxvirus, family Poxviridae, cause sheeppox, goatpox and lumpy skin disease, which are the most serious poxvirus diseases of production animals. Despite the considerable threat that these viruses pose to livestock production and global trade in sheep, goats, cattle and their products, convenient and effective serodiagnostic tools are not readily available. To develop a more effective antibody detection capability, selected open reading frames from capripoxvirus DNA were amplified and expressed in Escherichia coli as His-tagged fusion proteins. By screening 42 candidate antigens, two sheeppox virus virion core proteins that were expressed efficiently, purified readily using affinity chromatography and reactive against capripoxvirus immune sera in an indirect enzyme-linked immunosorbent assay (ELISA) were identified. The ELISA performed favourably when sera from sheep and goats infected experimentally with virulent capripoxvirus isolates were tested, with sensitivity and diagnostic specificity ranging between 95 and 97%, but it was unable to detect antibodies reliably in vaccinated sheep or goats. Furthermore, no cross-reactivity with antibodies against orf virus was detected. This assay offers the prospect of a convenient and standardised ELISA-based serodiagnostic test, with no requirement for infectious reagents, that is well suited to high-throughput capripoxvirus surveillance on a flock or herd basis.

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

    PubMed

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

    2009-08-01

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

  11. 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

  12. 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.

  13. 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

  14. Evaluation of circulating cathodic antigen (CCA) urine-cassette assay as a survey tool for Schistosoma mansoni in different transmission settings within Bugiri District, Uganda.

    PubMed

    Adriko, M; Standley, C J; Tinkitina, B; Tukahebwa, E M; Fenwick, A; Fleming, F M; Sousa-Figueiredo, J C; Stothard, J R; Kabatereine, N B

    2014-08-01

    Diagnosis of schistosomiasis at the point-of-care (POC) is a growing topic in neglected tropical disease research. There is a need for diagnostic tests which are affordable, sensitive, specific, user-friendly, rapid, equipment-free and delivered to those who need it, and POC is an important tool for disease mapping and guiding mass deworming. The aim of present study was to evaluate the relative diagnostic performance of two urine-circulating cathodic antigen (CCA) cassette assays, one commercially available and the other in experimental production, against results obtained using the standard Kato-Katz faecal smear method (six thick smears from three consecutive days), as a 'gold-standard', for Schistosoma mansoni infection in different transmission settings in Uganda. Our study was conducted among 500 school children randomly selected across 5 schools within Bugiri district, adjacent to Lake Victoria in Uganda. Considering results from the 469 pupils who provided three stool samples for the six Kato-Katz smears, 293 (76%) children had no infection, 109 (23%) were in the light intensity category, while 42 (9%) and 25 (5%) were in the moderate and heavy intensity categories respectively. Following performance analysis of CCA tests in terms of sensitivity, specificity, negative and positive predictive values, overall performance of the commercially available CCA test was more informative than single Kato-Katz faecal smear microscopy, the current operational field standard for disease mapping. The current CCA assay is therefore a satisfactory method for surveillance of S. mansoni in an area where disease endemicity is declining due to control interventions. With the recent resolution on schistosomiasis elimination by the 65th World Health Assembly, the urine POC CCA test is an attractive tool to augment and perhaps replace the Kato-Katz sampling within ongoing control programmes.

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

    PubMed

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

    2013-04-01

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

  16. Utility of Schistosoma bovis Adult Worm Antigens for Diagnosis of Human Schistosomiasis by Enzyme-Linked Immunosorbent Assay and Electroimmunotransfer Blot Techniques

    PubMed Central

    Pardo, J.; Carranza, C.; Turrientes, M. C.; Arellano, J. L. Pérez; Vélez, R. López; Ramajo, V.; Muro, A.

    2004-01-01

    Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive. PMID:15539523

  17. Utility of Schistosoma bovis adult worm antigens for diagnosis of human schistosomiasis by enzyme-linked immunosorbent assay and electroimmunotransfer blot techniques.

    PubMed

    Pardo, J; Carranza, C; Turrientes, M C; Pérez Arellano, J L; López Vélez, R; Ramajo, V; Muro, A

    2004-11-01

    Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive.

  18. Development of EMA-2 recombinant antigen based enzyme-linked immunosorbent assay for seroprevalence studies of Theileria equi infection in Indian equine population.

    PubMed

    Kumar, Sanjay; Kumar, Rajender; Gupta, Ashok K; Yadav, Suresh C; Goyal, Sachin K; Khurana, Sandip K; Singh, Raj K

    2013-11-15

    Equine piroplasmosis is a tick-transmitted protozoan disease caused by Theileria equi and/or Babesia caballi. In the present study, we expressed a 53kDa protein from the truncated EMA-2 gene of T. equi (Indian strain) and developed EMA-2ELISA using this expressed protein. This ELISA is able to detect T. equi-specific antibodies in experimentally infected animals as early as 9 days post-infection. The assay developed was validated with the OIE recommended competitive ELISA (cELISA) on 120 serum samples and significant agreement (kappa=0.93) was observed between results of both the ELISAs which indicates suitability of EMA-2ELISA for use in sero-diagnosis. Diagnostic sensitivity and specificity of EMA-2ELISA - as compared with cELISA - were 0.97 and 0.96, respectively. Analysis of 5651 equine serum samples - collected during 2007-2012 from 12 states of India representing eight agro-climatic zones - by EMA-2ELISA revealed 32.65% seroprevalence of T. equi in India. In conclusion, the EMA-2ELISA developed using the T. equi EMA-2 recombinant protein as antigen for detecting T. equi-specific antibodies has good diagnostic potential for sero-epidemiological surveys.

  19. 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

  20. 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

  1. 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.

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

    PubMed

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

    2015-02-01

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

  3. A recombinant cysteine proteinase from Leishmania (Leishmania) chagasi as an antigen for delayed-type hypersensitivity assays and serodiagnosis of canine visceral leishmaniasis.

    PubMed

    Pinheiro, Paulo Henrique da Costa; Pinheiro, Adriana Nunes; Ferreira, Josie Haydée Lima; Costa, Francisco Assis Lima; Katz, Simone; Barbiéri, Clara Lúcia

    2009-05-26

    A recombinant protein, rLdccys1, produced by expression of the gene encoding a 30kDa cysteine proteinase from Leishmania (Leishmania) chagasi, was used to detect specific antibodies in serum by enzyme-linked immunosorbent assays and to test for reactivity in delayed-type hypersensitivity (DTH) responses of dogs from an endemic region of visceral leishmaniasis (VL), Teresina, Piauí State, Brazil. Amastigote or promastigote extracts were also assayed for comparison. The sensitivity for detection of specific antibodies to L. (L.) chagasi using rLdccys1, lysates from L. (L.) chagasi promastigotes and amastigotes was 96%, 68%, and 69%, respectively. No cross-reactivity between rLdccys1 and Chagas disease was observed, and little reactivity was found with sera from dogs with babesiosis and ehrlichiosis. Among 106 sera from symptomatic dogs and 22 from non-infected controls, no false negatives and only two false positive sera were found for rLdccys1. In contrast, amastigote lysates yielded 11 false positives and 13 false negatives, whereas the corresponding numbers for promastigote lysates were 17 and 16. DTH responses were determined after intradermal injection of rLdccys1 or amastigote extract and the induration area was measured at 24, 48 and 72h after injection. All asymptomatic dogs showed a positive intradermal response to rLdccys1 (>10mm) which peaked at 48h, whereas no significant reactivity to the recombinant antigen was found in the symptomatic group. Histological analysis of the intradermal induration showed a predominance of necrotic and hemorrhagic areas in sections from asymptomatic dogs injected with L. (L.) chagasi amastigote extract, whereas a typical granulomatous reaction mediated by mononuclear cells was observed in sections from asymptomatic animals injected with rLdccys1. Grouping data from ELISA and DTH assays with rLdccys1 and L. (L.) chagasi amastigote extracts showed that humoral and cellular responses were inversely correlated during the

  4. Qualitative and quantitative analyses of Epstein-Barr virus early antigen diffuse component by western blotting enzyme-linked immunosorbent assay with a monoclonal antibody.

    PubMed Central

    Lin, J C; Choi, E I; Pagano, J S

    1985-01-01

    We report the use of monoclonal antibody against the early antigen diffuse component (anti-EA-D) of Epstein-Barr virus (EBV) to analyze, both qualitatively and quantitatively, the expression of EA-D in various human lymphoblastoid cell lines activated by chemical inducers. The kinetics of synthesis of EA-D in P3HR-1, B95-8, and Ramos/AW cells were similar in that they all reached the peak of synthesis on day 5 after induction. Surprisingly, no expression of EA-D was found in induced BJAB/GC, an EBV-genome-containing cell line. EBV-negative cell lines, BJAB and Ramos, were negative for EA-D. Raji cells had no detectable EA-D but responded rapidly to induction, reaching a peak on day 3. Superinfection of Raji cells also resulted in marked induction of EA-D, which reached a plateau between 8 to 12 h postinfection. Western blotting coupled with the enzyme-linked immunosorbent assay was employed to identify polypeptides representing EA-D. A family of four polypeptides with molecular weights of 46,000 (46K protein), 49,000, 52,000, and 55,000 were identified to be reactive with monoclonal anti-EA-D antiserum. The pattern of EA-D polypeptides expressed in each cell line was different. Of particular interest was the expression of a large quantity of 46K protein both in induced Raji and P3HR-1 cells, but not in superinfected Raji cells. A 49K doublet was expressed in activated p3HR-1, B95-8, and Ramos/AW cells and in superinfected Raji cells. In addition, two distinct 52K and 55K polypeptides were expressed in induced Ramos/AW and superinfected Raji cells. However, none of these EA-D polypeptides was detectable in BJAB/GC, BJAB, Ramos, and mock-infected Raji cells. To approximate relative concentrations of EA-D in cell extracts, we employed the enzyme-linked immunosorbent assay and immunoblot dot methods by using one of the purified EA-D components to construct a standard curve. Depending upon the cell lines, it was estimated that ca. 1 to 3% (determined by the enzyme

  5. 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

  6. Production of hepatitis B surface antigen in transgenic plants for oral immunization.

    PubMed

    Richter, L J; Thanavala, Y; Arntzen, C J; Mason, H S

    2000-11-01

    Here we present data showing oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) in preclinical animal trials. Mice fed transgenic HBsAg potato tubers showed a primary immune response (increases in HBsAg-specific serum antibody) that could be greatly boosted by intraperitoneal delivery of a single subimmunogenic dose of commercial HBsAg vaccine, indicating that plants expressing HBsAg in edible tissues may be a new means for oral hepatitis B immunization. However, attainment of such a goal will require higher HBsAg expression than was observed for the potatoes used in this study. We conducted a systematic analysis of factors influencing the accumulation of HBsAg in transgenic potato, including 5' and 3' flanking elements and protein targeting within plant cells. The most striking improvements resulted from (1) alternative polyadenylation signals, and (2) fusion proteins containing targeting signals designed to enhance integration or retention of HBsAg in the endoplasmic reticulum (ER) of plant cells.

  7. 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

  8. Hepatitis B surface antigen in blood donors: further observations.

    PubMed

    Szmuness, W; Hirsch, R L; Prince, A M; Levine, R W; Harley, E J; Ikram, H

    1975-02-01

    A survey of 128,000 volunteer blood donors from the Greater New York metropolitan area revealed that first-time male donors were positive for hepatitis B surface antigen (HBsAg) 2.5 times more frequently than were first-time female donors; Negroes and Mongols were positive four to 20 times more frequently than Caucasians. The ratio of ad to ay seemed to be higher in non-Caucasian antigen carriers than in Caucasian carriers. Among both Caucasians and non-Caucasians the rate of positivity declined after the age of 50. An excess prevalence of HBsAg was observed in donors with the lowest level of education and in those with the highest level. HBsAg was detectable nine times less frequently among repeat donors than among first-time donors (0.2 vs.1.90 per 1,000). Detection of HBsAg was unrelated to ABO-Rh blood groups. Several mechanisms for these wide variations of antigen detection are possible.

  9. Overview of expression of hepatitis B surface antigen in transgenic plants.

    PubMed

    Guan, Zheng-jun; Guo, Bin; Huo, Yan-lin; Guan, Zheng-ping; Wei, Ya-hui

    2010-10-28

    Hepatitis B virus (HBV), a pathogen for chronic liver infection, afflicts more than 350 million people world-wide. The effective way to control the virus is to take HBV vaccine. Hepatitis B surface antigen (HBsAg) is an effective protective antigen suitable for vaccine development. At present, "edible" vaccine based on transgenic plants is one of the most promising directions in novel types of vaccines. HBsAg production from transgenic plants has been carried out, and the transgenic plant expression systems have developed from model plants (such as tobacco, potato and tomato) to other various plant platforms. Crude or purified extracts of transformed plants have been found to conduct immunological responses and clinical trials for hepatitis B, which gave the researches of plant-based HBsAg production a big boost. The aim of this review was to summarize the recent data about plant-based HBsAg development including molecular biology of HBsAg gene, selection of expression vector, the expression of HBsAg gene in plants, as well as corresponding immunological responses in animal models or human.

  10. 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.

  11. 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.

  12. Mechanism of inactivation of hepatitis B surface antigen by N alpha-cocoyl-L-arginine ethyl ester.

    PubMed Central

    Sugimoto, Y; Toyoshima, S

    1981-01-01

    The mechanism of N alpha-cocoyl-L-arginine ethyl ester (CAE) in the inactivation of hepatitis B surface antigen (HBsAg) was investigated. The CAE increased the density of HBsAg, and particles of the antigen were destroyed in amorphous clusters, suggesting that CAE influences the lipid components of HBsAg. The lipid components such as cholesterol and phospholipid were mostly removed from the antigen by the treatment with CAE. N alpha-Lauroyl-L-[U-14C] arginine ethyl ester (LAE), a principal component of CAE, became tightly bound to HBsAg in place of the lipid components. The binding amounts of LAE in the HBsAg-LAE complex reached 3.04 +/- 0.44 microgram/mg of protein. The formation of the complex was not influenced by the presence of CAE-related compounds such as L-arginine, L-arginine ethyl ester, and N alpha-cocoyl-L-arginine. Treatment with mercaptoethanolurea, guanidine hydrochloride, and some detergents failed to resolve appreciably the labeled LAE from the labeled complex. All attempts to reactivate the CAE-treated HBsAg and to restore it morphologically from the denatured aggregates were unsuccessful. These results indicate that CAE tightly binds to HBsAg, followed by formation of stable aggregates of the denatured HBsAg-CAE complex. Images PMID:7283411

  13. Early hepatitis B surface antigen decline predicts treatment response to entecavir in patients with chronic hepatitis B

    PubMed Central

    Peng, Cheng-Yuan; Lai, Hsueh-Chou; Su, Wen-Pang; Lin, Chia-Hsin; Chuang, Po-Heng; Chen, Sheng-Hung; Chen, Ching-Hsiang

    2017-01-01

    Early declines in serum hepatitis B surface (HBsAg) levels, their optimal cutoffs, and association with therapeutic endpoints in chronic hepatitis B (CHB) patients receiving entecavir treatment remain unclear. We prospectively enrolled 529 patients (195 hepatitis B e antigen [HBeAg]-positive and 334 HBeAg-negative) with a median treatment duration of 49.2 months. Median HBsAg levels declined significantly in both groups at Month 3, but only at Months 6–12 in the HBeAg-negative group. Both groups exhibited a significant HBsAg decline with each successive year of treatment. An HBsAg decline of ≥75% from baseline, assessed at Months 3 and 12 of treatment in the HBeAg-positive and -negative patients, respectively, independently predicted a virological response and HBeAg seroconversion in the HBeAg-positive patients, an HBsAg level of <100 IU/mL in the HBeAg-negative patients, and HBsAg loss in all the patients during treatment. HBsAg levels of <3,000 IU/mL at baseline combined with an HBsAg decline of ≥75% from baseline provided a predictive algorithm for HBsAg loss (positive and negative predictive values: 70% and 100%, respectively) during 5 years of treatment. The proposed cutoffs for defining an HBsAg decline may assist clinicians in early assessments of treatment responses in genotype B-infected or C-infected CHB patients receiving entecavir therapy. PMID:28220833

  14. 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

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

    PubMed

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

    1993-07-01

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

  16. A Combinatory Antibody–Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries

    PubMed Central

    Jansson, Bo; Stuhr-Hansen, Nicolai; Kovács, András; Welinder, Charlotte

    2016-01-01

    We have developed a combinatory antibody–antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a “spot-on-spot” print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation. PMID:28002485

  17. 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...

  18. 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 ...

  19. Antibodies to liver membrane antigens in chronic active hepatitis (CAH). II. Specificity for autoimmune CAH.

    PubMed Central

    Frazer, I H; Kronborg, I J; Mackay, I R

    1983-01-01

    An immunoradiometric assay for IgG class autoantibody to liver membrane antigens, based on serum binding to glutaraldehyde treated monkey hepatocytes, was used to examine sera from patients with chronic active hepatitis (CAH) and other acute and chronic liver diseases. All sera from normals and patients showed binding, up to a titre of 1/2,048. For comparison of assays, results were normalized by selecting two reference sera, one with a high degree of binding, and one from a healthy subject with a low degree of binding: at a dilution of 1/2,048, these sera were given binding values of 100% and 0%. The values for the binding of unknown sera at the same dilution were calculated from these two reference values. For 26 patients with autoimmune CAH, the mean (+/- s.d.) percentage binding value (70 +/- 33%) was significantly higher than the mean value for 26 healthy subjects (10 +/- 15%), and high binding values were significantly associated with biochemically active hepatitis. The mean percentage binding value was moderately increased for eight patients with HBsAg associated CAH (42 +/- 12%), 13 patients with alcoholic hepatitis with cirrhosis (37 +/- 25%) and 45 patients with acute viral hepatitis A (40 +/- 27%) or B (52 +/- 37%). At a cut-off binding value of 65%, the assay as a single diagnostic procedure was shown to have a 70% sensitivity and a 95% specificity for the diagnosis of autoimmune CAH. Better understanding of the pathogenetic significance of antibodies to liver membrane antigens in CAH and other liver diseases will depend upon biochemical analysis of the presumably multiple antigenic determinants on the hepatocyte membrane. PMID:6616969

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

    PubMed

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

    2015-12-01

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

  1. 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

  2. Agreement between assays of cell-mediated immunity utilizing Mycobacterium bovis-specific antigens for the diagnosis of tuberculosis in African buffaloes (Syncerus caffer).

    PubMed

    Goosen, Wynand J; Miller, Michele A; Chegou, Novel N; Cooper, David; Warren, Robin M; van Helden, Paul D; Parsons, Sven D C

    2014-07-15

    We assessed the use of Mycobacterium bovis-specific peptides for the diagnosis of tuberculosis in African buffaloes (Syncerus caffer) by evaluating the agreement between the single intradermal comparative tuberculin test (SICTT), the Bovigam(®) EC (BEC) assay, the Bovigam(®) HP (BHP) assay and two assays utilizing the QuantiFERON(®) TB-Gold (in tube) system employing 20 h (mQFT20 assay) and 30 h (mQFT30 assay) whole blood incubation periods. Of 84 buffaloes, 45% were SICTT-positive, 48% were BEC-positive, 50% were BHP-positive, 37% were mQFT20-positive and 43% were mQFT30-positive. Agreement between the BEC and BHP Bovigam(®) assays was high (κ=0.86, 95% CI 0.75-0.97) and these detected the most test-positive animals suggesting that they were the most sensitive assays. Interferon-gamma release was significantly greater in buffaloes that were test-positive for all tests than in animals with discordant but positive Bovigam(®) results. Agreement between the mQFT assays was equally high (κ=0.88, 95% CI 0.77-0.98); however, all buffaloes with discordant mQFT results (n=6) were mQFT30-positive/mQFT20-negative, including three confirmed M. bovis-infected animals, suggesting that the mQFT30 assay is the more sensitive of the two. Agreements between the two Bovigam(®) and two mQFT assays were moderate, suggesting that in its current format the mQFT assay is less sensitive than either the BEC or the BHP assays.

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

    PubMed

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

    2012-02-01

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

  4. Highly sensitive electrochemical stripping detection of hepatitis B surface antigen based on copper-enhanced gold nanoparticle tags and magnetic nanoparticles.

    PubMed

    Shen, Guangyu; Zhang, Yun

    2010-07-26

    On the basis of copper-enhanced gold nanoparticle tags as an amplification approach, we introduced, in this paper, magnetic nanoparticles for further improving performance of electrochemical immunoassay by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. Due to the use of antibody-immobilized magnetic nanoparticles, the immunoreaction between antibody and antigen takes place in a homogeneous bulk solution phase. Compared with traditional solid interface reaction, the proposed strategy can provide some advantages such as easy of separation, shorter analytical time, wider linear range, and lower detection limit. It was also successfully applied to HBsAg determination in a linear range of 0.1-1500 ng mL(-1) with a detection limit of 87 pg mL(-1). The proposed analytical strategy holds good selectivity, sensitivity and repeatability and also great promise for the extended application in the fields of clinical diagnosis, bio-affinity assay and environmental monitoring.

  5. 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

  6. Hepatitis B Surface Antigen Reduction by Switching from Long-term Nucleoside/nucleotide Analog Administration to Pegylated Interferon.

    PubMed

    Tamaki, Nobuharu; Kurosaki, Masayuki; Kusakabe, Atsunori; Orito, Etsuro; Joko, Kouji; Kojima, Yuji; Kimura, Hiroyuki; Uchida, Yasushi; Hasebe, Chitomi; Asahina, Yasuhiro; Izumi, Namiki

    2017-02-15

    Hepatitis B surface antigen (HBsAg) reduction during nucleoside/nucleotide analog (NA) therapy is slow and an alternative strategy for patients receiving ongoing NA to facilitate HBsAg reduction is required. We investigated whether switching to pegylated interferon (PEG-IFN) after long-term NA administration enhances HBsAg reduction. Forty-nine patients who switched from long-term NA to 48 weeks of PEG-IFN alfa-2a were studied. The mean duration of previous NA was 48 months (sequential group). A total of 147 patients who continued NA and matched for baseline characteristics were analyzed for comparison (NA continuation group). The treatment response was defined as HBsAg reduction ≥1.0 logIU/ml at the end of PEG-IFN. HBsAg reduction at week 48 was 0.81 ± 1.1 logIU/ml in the sequential group, which was significantly higher than that in the NA continuation group (0.11 ± 0.3 logIU/ml, p <0.001). The treatment response was achieved in 29% and 2% of the sequential group and NA continuation group (p <0.001), and the odds ratio of sequential therapy for treatment response was 19 compared with NA continuation (p <0.001). In patients positive for hepatitis B e antigen (HBeAg), HBeAg seroconversion was higher in the sequential group (44% vs. 8%, p <0.001). In HBeAg negative patients, only patients in the sequential group achieved HBsAg loss. No patient needed to resume NA administration because of HBV DNA increase accompanied by alanine aminotransferase flares. In summary, sequential therapy with PEG-IFN after long-term NA enhances the reduction of HBsAg and may represent a treatment option to promote HBsAg loss. This article is protected by copyright. All rights reserved.

  7. 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.

  8. [Partial purification of HBsAg by the progressive elimination of serum proteins].

    PubMed

    Babeş, V T; Anton, G

    1988-01-01

    Partial elimination of proteins from HBsAg-positive sera was realised by raising the temperature and simultaneously lowering the pH. Products with a convenient level of purity (2.2 mg of proteins per ml) were obtained as well as a good recovery rate of the HBsAgs (8-24%).

  9. Quantitation of IgE and carcinoembryonic antigen (CEA) by optical beam deflection (OBD) measurement of dot-immunobinding assay patterns visualized by an ELISA technique.

    PubMed

    Matsuzawa, S; Kimura, H; Tu, C Y; Kitamori, T; Sawada, T

    1993-05-05

    Dot-immunobinding assays of IgE and CEA were performed by a conventional dot-ELISA technique with diaminobenzidine staining, and the quantitative results were compared by densitometry and a new, spectroscopic, optical beam deflection (OBD) method using the same membrane. It was possible with the OBD method to detect quantities of these substances at least ten times smaller than with densitometry. Better intra-assay reproducibility for IgE and CEA measurements was obtained by the OBD method. The measurable ranges of the OBD method was broader than that of densitometry, because dark bands caused OBD in proportion to their color densities. When the dot-immunobinding assay with OBD measurement for CEA was also compared with a microtube ELISA using biotin-avidin conjugates, the sensitivities and reproducibilities of the two methods were found to be similar, with a correlation coefficient of 0.991.

  10. Quantification of hepatitis B surface antigen: a new concept for the management of chronic hepatitis B.

    PubMed

    Moucari, Rami; Marcellin, Patrick

    2011-01-01

    HBsAg is a very important clinical test that might not only indicate active hepatitis B virus (HBV) infection but might also be used to predict clinical and treatment outcome. Clearance of HBsAg in patients with chronic HBV infection is associated with a much better clinical outcome, although surveillance for early detection of hepatocellular carcinoma (HCC) should continue. HBV DNA quantification is currently used for selecting candidates for therapy, monitoring response to therapy and detecting the emergence of drug resistance. Assays for HBsAg quantification are less expensive than HBV DNA and fully automated with a high throughput capacity. HBsAg titering may be a useful tool to manage patients with chronic HBV, to more clearly define which patients may, and more importantly, may not, benefit from treatment. Baseline and on-treatment HBsAg quantification may help to refine future treatment algorithms for both immune-modulator therapy and nucleos(t)ide analogues. Both HBV markers provide complementary information on the status of HBV infection. However, the relevance of serum HBsAg levels and its use as a reliable replacement for both covalently closed circular DNA and HBV DNA remain unclear.

  11. 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

  12. Severe sensitivity loss in an influenza A molecular assay due to antigenic drift variants during the 2014/15 influenza season.

    PubMed

    Overmeire, Yarah; Vanlaere, Elke; Hombrouck, Anneleen; De Beenhouwer, Hans; Simons, Guus; Brink, Antoinette; Van den Abeele, Anne-Marie; Verfaillie, Charlotte; Van Acker, Jos

    2016-05-01

    The 2014-2015 influenza season in Belgium was dominated by the circulation of 2 influenza A(H3N2) subgroups: 3C.2a and 3C.3b. Analysis of 166 nasopharyngeal aspirates, collected in patients with respiratory illness at the start of the epidemic season, showed a decreased sensitivity for the detection of influenza A(H3N2)/3C.2a using a commercially available multiplex assay. Gene sequencing of the matrix protein showed a point mutation (C163T) leading to a mismatch with the assay probes.

  13. Different functions and associations of HLA-DR and HLA-DQ(DC) antigens shown by serological, cellular and DNA assays.

    PubMed

    Navarrete, C; Jaraquemada, D; Hui, K; Awad, J; Okoye, R; Festenstein, H

    1985-03-01

    Two consanguineous Caucasoid HTCs, DHI and FPA, were investigated, the latter having an unusual HLA-DR/DQ(DC) association. Both these HTCs typed clearly as HLA-DRw11. However, while DHI typed as DRw11/DQw3(DC4) as expected, FPA typed as DRw11/DQw1(DC1) instead. Although extremely rare in Caucasoids, DRw11/DQw1 is a common pattern of association in Nigerian Negroids. Southern blots of DNA extracted from EBV cell line derived from FPA, hybridized with HLA-DC alpha and HLA-DC beta probes, confirmed this unusual DRw11/DQw1(DC1) association. In addition the DC alpha probe showed a unique additional restriction fragment length polymorphism (8 kb) attributable to the DX gene in the FPA DNA. When DHI and FPA were used as stimulators in MLC, the patterns to typing responses obtained were not completely concordant although they overlap to some extent. For this reason FPA has been locally designated Dw'F5', distinct from Dw5. Furthermore, the HLA-DQ antigens of the responder cells were not necessarily the same as those of the HTCS to which they gave typing responses (FPA and DHI). Functional studies using these two HTCs showed that the DQ(DC) antigens probably have no direct lymphocyte activating properties but rather have a regulatory role in controlling responses to allodeterminants in MLC.

  14. 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

  15. Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: myosin-V assay.

    PubMed

    Calábria, Luciana Karen; Teixeira, Renata Roland; Coelho Gonçalves, Sybelli Magda; Passos Lima, Andreia Barcelos; Santos, Ana Alice Diniz Dos; Martins, Antônio Roberto; Espindola, Foued Salmen

    2010-01-01

    The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 ºC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.

  16. Occurrence of hepatitis and hepatitis B surface antigen in adult patients with acute leukemia.

    PubMed Central

    Cowan, D. H.; Kouroupis, G. M.; Leers, W. D.

    1975-01-01

    Fifty-eight adult patients with acute leukemia were screened at the onset of the disease for hepatitis B antigen (HBSAg) in the serum, and during the course of the disease for the development of hepatitis B. One patient had a positive test for HBSAg by the radioimmunoassay technique only at the time leukemia was diagnosed; this patient had received transfusions some years before. In six patients icteric hepatitis B developed; five recovered completely and one died of leukemia during the course of hepatitis. All patients in whom hepatitis developed had received transfusions as a part of supportive therapy for leukemia. The hepatitis risk for patients who received transfusions of blood found to be negative for HBSAg by counterimmunoelectrophoresis was 0.26 percent per unit of blood administered. PMID:1054615

  17. Sequence heterogeneity in the equi merozoite antigen gene (ema-1) of Theileria equi and development of an ema-1-specific TaqMan MGB assay for the detection of T. equi.

    PubMed

    Bhoora, Raksha; Quan, Melvyn; Matjila, Paul T; Zweygarth, Erich; Guthrie, Alan J; Collins, Nicola E

    2010-08-27

    detected in more samples and at lower C(q) values when the new assay was used. Phylogenetic analyses of the 18S rRNA gene sequences and ema-1 amino acid sequences from the same samples showed inconsistencies between the clades, indicating that the T. equi 18S rRNA genetic groups previously identified in South Africa may not represent distinct T. equi lineages. It is possible that the different T. equi ema-1 genotypes could be related to antigenic variability and pathogenicity and may be associated with clinical differences in equine piroplasmosis cases, but this remains to be elucidated.

  18. Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells.

    PubMed

    Chen, H C; Chou, C K; Lee, S D; Wang, J C; Yeh, S F

    1995-05-01

    We have examined the antiviral activity of the crude extract prepared from the root of Saussurea lappa Clarks, a Chinese medicinal herb which is widely used for many illnesses including cancer. Two active components, costunolide and dehydrocostus lactone, were identified which show strong suppressive effect on the expression of the hepatitis B surface antigen (HBsAg) in human hepatoma Hep3B cells, but have little effect on the viability of the cells. Both costunolide and dehydrocostus lactone suppress the HBsAg production by Hep3B cells in a dose-dependent manner with IC50s of 1.0 and 2.0 microM, respectively. Northern blotting analysis shows that the suppression of HBsAg gene expression by both costunolide and dehydrocostus lactone were mainly at the mRNA level. Furthermore, the suppressive effect of costunolide and dehydrocostus lactone on HBsAg and hepatitis B e antigen (HBeAg), a marker for hepatitis B viral genome replication in human liver cells, was also observed in another human hepatoma cell line HepA2 which was derived from HepG2 cells by transfecting a tandemly repeat hepatitis B virus (HBV) DNA. Similarly, the mRNA of HBsAg in HepA2 cells was also suppressed by these two compounds. Our findings suggest that costunolide and dehydrocostus lactone may have potential to develop as specific anti-HBV drugs in the future.

  19. Morphological and physiological characteristics of transgenic cherry tomato mutant with HBsAg gene.

    PubMed

    Guan, Z J; Guo, B; Huo, Y L; Hao, H Y; Wei, Y H

    2011-08-01

    HBsAg gene was previously introduced into cherry tomato (Lycopersicum esculentum var. cerasiforme) by Agrobacterium-mediated transformation. To investigate the side effect of HBsAg gene in cherry tomato, we analyzed morphological and physiological characteristics of the transgenic mutant N244. The process was performed under field conditions. The results suggested that the mutant N244 exhibited morphological, cytological and physiological variation. First of all, compared with the wild plants NK, N244 had fleshy and dark green leaves, the fewer notches of leaf edge, more adventitious roots and barren seeds. Moreover, the chromosome of N244 were found to be triploid (n = 36) by flow cytometric analysis. Furthermore, N244 has obvious physiological alterations, as compared to NK. It was speculated that transformation of the genes probably led to ploidy variation, and further caused phenotype and physiological changes of plants. Our study will reveal side effects of the mutants, and promote cultivation of transgenic plants in the field.

  20. Development of an enzyme-linked immunoelectrotransfer blot (EITB) assay using two baculovirus expressed recombinant antigens for diagnosis of Taenia solium taeniasis.

    PubMed

    Levine, Min Z; Lewis, Melissa M; Rodriquez, Silvia; Jimenez, Juan A; Khan, Azra; Lin, Sehching; Garcia, Hector H; Gonzales, Armando E; Gilman, Robert H; Tsang, Victor C W

    2007-04-01

    Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

  1. Maturation of recombinant hepatitis B virus surface antigen particles.

    PubMed

    Zhao, Qinjian; Wang, Yang; Freed, Daniel; Fu, Tong-Ming; Gimenez, Juan A; Sitrin, Robert D; Washabaugh, Michael W

    2006-01-01

    The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with associated lipids can self-assemble into 22-nm immunogenic spherical particles, which are used in licensed Hepatitis B vaccines. Little is known about the structural evolvement or maturation upon assembly beyond an elevated level of disulfide formation. In this paper, we further characterized the maturation of HBsAg particles with respect to their degree of cross-linking, morphological changes, and changes in conformational flexibility. The lipid-containing rHBsAg particles undergo KSCN- and heat-induced maturation by formation of additional intra- and inter-molecular disulfide bonds. Direct measurements with atomic force microscopy (AFM) revealed morphological changes upon maturation through KSCN-induced and heat-/storage-incurred oxidative refolding. Particle uniformity and regularity was greatly improved, and protrusions formed by the protein subunits were more prominent on the surface of the mature particles. Decreased conformational flexibility in the mature rHBsAg particles was demonstrated by millisecond-scale unfolding kinetics in the presence of an environment-sensitive conformation probe. Both the accessible hydrophobic cavities under native conditions and the changeable hydrophobic cavities upon denaturant-induced unfolding showed substantial decrease upon maturation of the rHBsAg particles. These changes in the structural properties may be critical for the antigenicity and immuno-genicity of this widely-used vaccine component.

  2. 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.

  3. Combined assays for serum carcinoembryonic antigen and microRNA-17-3p offer improved diagnostic potential for stage I/II colon cancer

    PubMed Central

    ZHU, JINHAI; DONG, HUIMING; ZHANG, QIONG; ZHANG, SHANGWU

    2015-01-01

    Colorectal cancer is among the leading causes of cancer-related mortality, one of the main reasons for which is the lack of an effective screening method for early-stage disease. The levels of carcinoembryonic antigen (CEA) and microRNA (miR)-17-3p in the serum of 70 patients with stage I/II colon cancer and 70 healthy volunteers were determined, and the diagnostic value of CEA plus miR-17-3p detection for colon cancer was assessed. The levels of CEA were measured by a radioimmunoassay method, and those of miR-17-3p using the reverse transcription-quantitative polymerase chain reaction method. miR-16 was used as the endogenous control, as it displayed high stability, high abundance and low variability in the analyzed serum samples. The receiver operating characteristic (ROC) curve analysis indicated the potential diagnostic value of the two markers and the area under the ROC curve (AUC) for CEA and miR-17-3p was 0.719 (95% CI: 0.658–0.843) and 0.807 (95% CI: 0.748–0.906), respectively. At a threshold of 9.6 ng/ml for CEA, the optimal sensitivity and specificity were 74.6 and 84.3%, respectively, in discriminating colon cancer patients from healthy controls. At a threshold of 2.98 for miR-17-3p, the sensitivity and the specificity were 83.6 and 72.9%, respectively. A combined ROC analysis using CEA and miR-17-3p revealed an AUC of 0.929 (95% CI: 0.834–0.978) with a sensitivity of 96.4% and a specificity of 95.7% in discriminating colon cancer patients from healthy controls. In conclusion, both CEA and miR-17-3p were highly expressed in the serum of our series of colon cancer patients. CEA plus miR-17-3p detection significantly increased the sensitivity and specificity in discriminating stage I/II colon cancer patients from healthy controls. Therefore, combined detection of serum CEA and miR-17-3p levels may have the potential to become a new laboratory method for the early clinical diagnosis of colon cancer. PMID:26807240

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

    PubMed

    Kong, Q; Richter, L; Yang, Y F; Arntzen, C J; Mason, H S; Thanavala, Y

    2001-09-25

    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.

  5. Evaluation of spore wall protein 1 as an alternative antigen for the diagnosis of Encephalitozoon cuniculi infection of farmed foxes using an enzyme-linked immunosorbent assay.

    PubMed

    Meng, Xiangchun; Zheng, Jun; Gao, Yang; Zhang, Yanlong; Jia, Honglin

    2014-07-14

    The sequence encoding SWP1 was cloned from the genome of Encephalitozoon cuniculi. Recombinant SWP1 (rSWP1) was expressed in Escherichia coli and used to detect E. cuniculi infections in farmed foxes and dogs with an indirect enzyme-linked immunosorbent assay (ELISA) in the present study. The sera of foxes infected with E. cuniculi could be distinguished from the sera of foxes infected with Toxoplasma gondii, Neospora caninum, and Cryptosporidium parvum using the ELISA. In total, 198 fox samples collected in Liaoning were used to determine the prevalence of antibodies against this disease. The results showed that 16.7% of the fox serum samples were positive according to the ELISA using rSWP1, which agreed with the ELISA results based on recombinant PTP2 (rPTP2). The sensitivity and specificity of the ELISA based on rSWP1 suggest that this could be an alternative method for the diagnosis of E. cuniculi infections in foxes. In addition, 298 dog samples collected in Beijing, Shanghai, and Hunan were also detected in this study, of which six dog samples (2%) were positive according to the ELISA using rSWP1. To the best of our knowledge, this is the first study to demonstrate the serological prevalence of E. cuniculi infections in dogs and foxes in China.

  6. Two-dimensional paper network format that enables simple multistep assays for use in low-resource settings in the context of malaria antigen detection.

    PubMed

    Fu, Elain; Liang, Tinny; Spicar-Mihalic, Paolo; Houghtaling, Jared; Ramachandran, Sujatha; Yager, Paul

    2012-05-15

    The lateral flow test has become the standard bioassay format in low-resource settings because it is rapid, easy to use, and low in cost, uses reagents stored in dry form, and is equipment-free. However, lateral flow tests are often limited to a single chemical delivery step and not capable of the multistep processing characteristic of high performance laboratory-based assays. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions; this allows incorporation of multistep chemical processing, while still retaining the advantages of conventional lateral flow tests. Here, we demonstrate this format for an easy-to-use, signal-amplified sandwich format immunoassay for the malaria protein PfHRP2. The card contains reagents stored in dry form such that the user need only add sample and water. The multiple flows in the device are activated in a single user step of folding the card closed; the configuration of the paper network automatically delivers the appropriate volumes of (i) sample plus antibody conjugated to a gold particle label, (ii) a rinse buffer, and (iii) a signal amplification reagent to the capture region. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.

  7. 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.

  8. 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

  9. Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System.

    PubMed

    Gorton, R L; White, P L; Bagkeris, E; Cotterall, D; Desai, R; McHugh, T; Kibbler, C C

    2015-07-01

    The galactomannan enzyme immunoassay (GM-EIA) is widely utilized for the diagnosis of invasive aspergillosis (IA). There is inconsistent reproducibility of results between centers when the assay is processed manually. Automation of EIAs can reduce variation. This study investigated the semiautomation of the GM-EIA on the DS2 (Dynex) platform in the following three stages: (i) DS2 GM-EIA method validation with experimental samples, (ii) DS2 retesting of case-defined clinical samples, and (iii) a 12-month audit of DS2 GM-EIA performance. In stage i, Bland-Altman analysis demonstrated a reduced variance between optical density index (ODI) values for samples processed on two DS2 platforms (mean difference, -0.02; limits of agreement [LOA], -0.19 to 0.14) compared with the variance between samples processed manually and on a DS2 platform (mean difference, 0.02; LOA, -0.25 to 0.3). In stage ii, 100% (14/14 samples) qualitative agreement was observed for serum samples from patients with IA, with no significant change in the ODI values when samples were processed on the DS2 platform. A significant decrease in ODI values was observed for control serum samples on the DS2 platform (difference, 0.01; P = 0.042). In stage iii, a significant reduction in the frequency of equivocal results, from 5.56% (136/2,443 samples) to 1.56% (15/961 samples), was observed after DS2 automation (difference, 4.0%; 95% confidence interval [CI], 2.7 to 5.2%; P < 0.01), with an equivalent increase in negative results. This study demonstrates that GM-EIA automation may reduce intersite variability. Automation does not have an impact on the repeatability of truly positive results but contributes to a reduction in false-positive (equivocal) GM-EIA results, reducing the need to retest a significant proportion of samples.

  10. Measurement of IgG, IgA and IgE antibodies to Dermatophagoides pteronyssinus by antigen-binding assay, using a partially purified fraction of mite extract (F4P1).

    PubMed Central

    Chapman, M D; Platts-Mills, T A

    1978-01-01

    An extract of Dermatophagoides pteronyssinus culture has been fractionated by chromatography on Sephadex G-100 and Pevikon block electrophoresis to obtain a partially purified allergen (F4P1). This preparation has a molecular weight of between 15--25,000 Dalton, migrates slowly on electrophoresis, and is colourless in solution. The skin-test reactivity of F1P1 was comparable to that of crude D. pteronyssinus extract. F4P1 was radio-labelled with 125I and used in an antigen-binding radioimmunoassay to measure IgG, IgA and IgE antibody (ab) to D. pteronyssinus. IgG, ab was detected in serum from 32/34 (94%) mite-allergic persons, and from 10/31 (30%) nonallergic persons. IgA ab and IgE ab were found in sera from 22/34 (65%) and 37/34 (79%) allergic persons respectively. Neither IgA nor IgE ab could be detected in sera from non-allergic persons. An excellent correlation was found between radioallergo-sorbent technique (RAST), using crude D. pteronyssinus extract and IgE-binding activity (BA) for F4P1, (r=0.94, P less than 0.001). The antigen-binding assay for IgE BA was as sensitive as RAST, but less sensitive than PK testing. There was a very good quantitative correlation between IgG BA and IgE BA (r = 0.84, P less than 0.001). IgG BA was shown to rise in the serum of three patients treated with injections of D. pteronyssinus extract. PMID:750116

  11. Anti-apical-membrane-antigen-1 antibody is more effective than anti-42-kilodalton-merozoite-surface-protein-1 antibody in inhibiting plasmodium falciparum growth, as determined by the in vitro growth inhibition assay.

    PubMed

    Miura, Kazutoyo; Zhou, Hong; Diouf, Ababacar; Moretz, Samuel E; Fay, Michael P; Miller, Louis H; Martin, Laura B; Pierce, Mark A; Ellis, Ruth D; Mullen, Gregory E D; Long, Carole A

    2009-07-01

    Apical membrane antigen 1 (AMA1) and the 42-kDa merozoite surface protein 1 (MSP1(42)) are leading malaria vaccine candidates. Several preclinical and clinical trials have been conducted, and an in vitro parasite growth inhibition assay has been used to evaluate the biological activities of the resulting antibodies. In a U.S. phase 1 trial with AMA1-C1/Alhydrogel plus CPG 7909, the vaccination elicited anti-AMA1 immunoglobulin G (IgG) which showed up to 96% inhibition. However, antibodies induced by MSP1(42)-C1/Alhydrogel plus CPG 7909 vaccine showed less than 32% inhibition in vitro. To determine whether anti-MSP1(42) IgG had less growth-inhibitory activity than anti-AMA1 IgG in vitro, the amounts of IgG that produced 50% inhibition of parasite growth (Ab(50)) were compared for rabbit and human antibodies. The Ab(50)s of rabbit and human anti-MSP1(42) IgGs were significantly higher (0.21 and 0.62 mg/ml, respectively) than those of anti-AMA1 IgGs (0.07 and 0.10 mg/ml, respectively) against 3D7 parasites. Ab(50) data against FVO parasites also demonstrated significant differences. We further investigated the Ab(50)s of mouse and monkey anti-AMA1 IgGs and showed that there were significant differences between the species (mouse, 0.28 mg/ml, and monkey, 0.14 mg/ml, against 3D7 parasites). Although it is unknown whether growth-inhibitory activity in vitro reflects protective immunity in vivo, this study showed that the Ab(50) varies with both antigen and species. Our data provide a benchmark for antibody levels for future AMA1- or MSP1(42)-based vaccine development efforts in preclinical and clinical trials.

  12. Hepatitis B Surface Antigen Loss and Hepatocellular Carcinoma Development in Patients With Dual Hepatitis B and C Infection.

    PubMed

    Yang, Wan-Ting; Wu, Li-Wei; Tseng, Tai-Chung; Chen, Chi-Ling; Yang, Hung-Chih; Su, Tung-Hung; Wang, Chia-Chi; Kuo, Stephanie Fang-Tzu; Liu, Chen-Hua; Chen, Pei-Jer; Chen, Ding-Shinn; Liu, Chun-Jen; Kao, Jia-Horng

    2016-03-01

    Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are 2 major causes of chronic viral hepatitis. It is still unclear how HCV coinfection affects HBV replication and clinical outcomes in HBV/HCV coinfected patients.We conducted a longitudinal study, which enrolled 111 patients with HBV/HCV coinfection and 111 propensity score-matched controls with HBV monoinfection. Both groups had comparable baseline age, sex, fibrosis stage, levels of HBV DNA, and HBV surface antigen (HBsAg). The HCV coinfection and other host/viral factors were correlated with various outcomes, including HBsAg loss and cirrhosis/hepatocellular carcinoma (HCC) development.After a 10-year follow-up, we found that HCV coinfection itself was not associated with HBsAg loss. However, coinfected patients with alanine aminotransferase (ALT) level >80 U/L had a higher chance of HBsAg loss than those with ALT level ≤80 U/L [hazard ratio (95% confidence interval): 4.41 (1.75-11.15)] or matched controls with HBV monoinfection [hazard ratio (95% confidence interval): 3.40 (1.54-7.50)]. Besides, both HCV coinfection and higher ALT levels were associated with higher HCC risks and the HCC risks remained even after HBsAg loss in HBV/HCV con-infected patient.HCV coinfection is not associated with HBsAg loss. A higher ALT level is a major determinant of HBsAg loss in patients with HBV/HCV coinfection. Both HCV coinfection and a higher ALT level were independent risk factors of HCC.

  13. Hepatocellular carcinoma. A study of 50 autopsy cases with detection of hepatitis B surface antigen in fixed tissues.

    PubMed

    Perez-Barrios, A; Colina-Ruizdelgado, F; Gallego, I; Martinez-Tello, F J

    1983-03-01

    Fifty patients who died of hepatocellular carcinoma (HCC) were autopsied at the Ciudad Sanitaria "1 degree de Octubre" and the Hospital de la Cruz Roja (Madrid) from 1974 to 1980. Formalin fixed paraffin-embedded autopsy tissue of liver and tumor from the 50 HCC and liver tissue from 50 liver cirrhosis (LC) and from 50 autopsy of non cirrhotic control cases were examined for the presence of cytoplasmic hepatitis B surface antigen (HBsAg). The study was carried out using orcein staining, immunoperoxidase technique (IP) and indirect immunofluorescence (IF). In livers with HCC the HBsAg was detected in the cytoplasm of the hepatocytes in 10 cases (20%) with the orcein staining and in 11 (22%) with the IP and IF techniques. In one case (2%) HBsAg was found in the cytoplasm of tumor cells with the three methods--In four cases (8%) of LC and 2 (4%) control cases cytoplasmic positive cells were found. In 41 patients with HCC HBsAg was studied in the serum by radio-immunoassay (RIA) (13 cases) and immunodiffussion (28 cases). 5 patients (12,1%) were positive and 36 (72%) were negative. In the 5 serum positive HBsAg HCC the staining methods for cytoplasmic HBsAg were positive (100%). In 36 serum negative HBsAg HCC the staining method were positive in 2 cases. The results let us to conclude that HBV is a probable important etiologic factor of HCC in our milieu. 54% of the patients with HCC had a previous history of alcohol abuse; however, histologic features compatible with an alcoholic etiology were found in only 5 cases. Nevertheless we consider that the described histopathologic findings do not exclude excess alcohol consumption as a possible etiologic factor for HCC in our series.

  14. [Comparison of antibody responses to hepatitis B surface antigen among four recipient groups of hepatitis B vaccines that have been approved in Japan: evaluation using passive hemagglutination assay and chemiluminescent immunoassay].

    PubMed

    Ogata, Norio

    2009-10-01

    In hepatitis B virus (HBV) infection-preventing programs, serum or plasma levels of antibody to hepatitis B surface antigen (anti-HBs) are important to determine whether individuals are protective or not. We compared anti-HBs responses using passive hemagglutination assay (Mycell) and chemiluminescent immunoassay (Architect) among four recipient groups of HB vaccines, Meinyu, HBY, Bimmugen and Heptavax II, that have been approved in Japan. Overall, in a total of 1875 vaccinees Mycell results showed recipient groups of Meinyu and HBY acquired higher anti-HBs levels than those of Bimmugen and Heptavax II. Comparison of anti-HBs responses by both Mycell and Architect in recipient groups of Meinyu (n=150), HBY (n=218), Bimmugen (n=260), and Heptavax II (n=47) demonstrated the order of vaccinees' responses, such as geometric mean titers, ratios of acquiring high antibody levels (Mycell titers over 1024, Architect measurements over 1000 mIU/mL), and ratios of having unsuccessful antibody responses (Mycell titers under 8, Architect measurements under 10 mIU/mL), were somewhat different between the two assays. Comparison of Architect measurements at given Mycell titers revealed Bimmugen-recipients showed significantly lower values than HBY- or Heptavax II-recipients. Around critical protective levels, 5 of 22 Bimmugen-recipients with Mycell titers 16 or 32 showed Architect measurements under 10 mIU/mL, while 8 of 11 Heptavax II-recipients with Mycell titers below 8 demonstrated Architect measurements over 10 mIU/mL. Thus, discrepancies in anti-HBs evaluation between Mycell and Architect seemed to partly depend on administered vaccines. These results indicate anti-HBs concentration should be evaluated carefully so that we could completely prevent HBV infection.

  15. Effects of 1,2,4,6-tetra-O-galloyl-β-D-glucose from P. emblica on HBsAg and HBeAg secretion in HepG2.2.15 cell culture.

    PubMed

    Xiang, Yang-Fei; Ju, Huai-Qiang; Li, Shen; Zhang, Ying-Jun; Yang, Chong-Ren; Wang, Yi-Fei

    2010-10-01

    A polyphenolic compound, 1,2,4,6-tetra-O-galloyl-β-D-glucose (1246TGG), was isolated from the traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae) and assayed for its potential as an anti-hepatitis B virus (HBV) agent. The cytotoxicity of 1246TGG on HepG2.2.15 as well as HepG2 cells was determined by observing cytopathic effects, and the effects of 1246TGG on secretion of HBsAg and HBeAg in HepG2.2.15 cells were assayed by enzyme immunoassay. Results indicates that treatment with 1246TGG (6.25 μg/mL, 3.13 μg/mL), reduced both HBsAg and HBeAg levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. This study provides a basis for further investigation of the anti-HBV activity and possible mechanism of action of 1246TGG.

  16. Surface antigen-negative hepatitis B virus infection in Dutch blood donors.

    PubMed

    Lieshout-Krikke, R W; Molenaar-de Backer, M W A; van Swieten, P; Zaaijer, H L

    2014-01-01

    Hepatitis B virus (HBV) surface antigen (HBsAg) is a reliable marker for HBV infection, but HBsAg-negative forms of HBV infection occur. The introduction of HBV DNA screening of Dutch blood donors, which were not preselected for absence of HBV core antibodies, enabled the characterization of HBsAg-negative HBV infection in healthy persons and a comparison of the HBV genomes involved. The screening of 4.4 million Dutch blood donations identified 23 HBsAg-negative, HBV DNA-positive persons. Serological testing of the index donations, follow-up samples and archived earlier samples was performed to determine the nature of each HBV DNA-only case. Despite low viral loads HBV DNA could be sequenced in 14 out of 23 donors, allowing HBV genotyping and the analysis of mutations in the HBV surface gene. Four types of HBsAg-negative HBV infection were detected: infection in the early stage before occurrence of HBsAg; suppressed infection after vaccination; HBV genotype G infection with decreased HBsAg production; and chronic occult (HBsAg negative) HBV infection. In the donors with occult HBV genotype D infection the HBV surface gene showed multiple "escape" mutations in the HBsAg a-determinant and CTL epitopes, while in an occult genotype A case the surface gene showed no mutations. HBsAg-negative forms of HBV infection in healthy blood donors explain the ongoing transmission of HBV via blood transfusion, if donor screening is limited to HBsAg. The screening of blood donors for HBV DNA and HBV core antibodies seems to cover all stages and variants of HBV infection.

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

    PubMed

    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

    2012-04-19

    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.

  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.

  19. 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.

  20. Fluoresent Binding Assays for Antigen Detection.

    DTIC Science & Technology

    1983-09-09

    Raman scattering. Light is elastically scattered in solution; in the case of aqueous solutions, Raman scattering occurs approx- imately 300 wave numbers...on either side of the exciting light. The Stokes shift of many fluorescent markers is approximately 300 wave numbers, and thus, Raman scattering is...registered by the photomultiplier using the excita- tion and emission filters for the fluorescent marker. Raman scattering can be minimized by using

  1. Presence of HBsAg and anti-HBs in patients with primary liver cancer or hepatic metastases.

    PubMed

    Nastac, E; Stoian, M; Iosipenco, M; Iordache, C

    1985-01-01

    The presence of HBsAg was detected in the serum of 1 out of 13 patients with chronic hepatitis, in 2 out of the 7 patients with liver metastases, but in none of the 10 patients with hepatocellular carcinoma (HCC) investigated. HBsAg could be visualized by orcein staining in the liver cells of 66.6% of the HCC patients, in 50% of the patients with liver metastases and in 40% of the patients with liver cirrhosis. The prevalence of anti-HBs was low both in HCC patients and in the control groups. The possible significance of these findings is discussed.

  2. 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

  3. A mimotope of pre-S2 region of surface antigen of viral hepatitis B screened by phage display.

    PubMed

    Zhang, W Y; Wan, Y; Li, D G; Tang, Y; Zhou, W

    2001-09-01

    To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino acids in length were used to screen with the serum from a hepatitis B virus infected patient in the recovery phase. After 3 rounds of biopanning, the positive phages were confirmed by competitive ELISA using HBsAg/P33. Two phagotopes were identified and one of them was confirmed as mimotope by competition experiment. Based on the mimotpe, a multiple antigenic peptide with four branches was synthesized by solid phase peptide synthesis. The antiginicity and specificity of the synthesized antigen was tested in BALB/c mice compared with the native epitope-based antigen. The results showed that the mimotope-based antigen could evoke higher titer of antibodies with the same specificity of the epitope-based antigen. Those findings indicate mimotopes can be used in antigen and vaccine design.

  4. 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.

  5. Factors important in the extraction, stability and in vitro assembly of the hepatitis B surface antigen derived from recombinant plant systems.

    PubMed

    Smith, Mark L; Keegan, Mark E; Mason, Hugh S; Shuler, Michael L

    2002-01-01

    The expression of vaccine antigens in edible plant material together with their delivery by the oral route constitutes a powerful paradigm, with the potential to dramatically reduce the cost of vaccine production and administration, in addition to improving distribution and patient compliance. These products will be subject to many of the same regulations applied to current injectable vaccines, so reliable methods to quantify antigen and ensure stability in crude plant extracts are required. As a model system the hepatitis B surface antigen (HBsAg) was expressed in soybean and tobacco cell cultures. This complex antigen consists of membrane-associated small surface antigen proteins (p24(s)), disulfide cross-linked to yield dimers and higher multimers. Although the total p24(s) extracted from plant cells was relatively unaffected by detergent concentration, the quantification of antigenically reactive product depended strongly on the ratio of detergent to cell concentration. Furthermore, 1-20% w/v sodium ascorbate improved the measured levels of monoclonal-reactive antigen 4- to 12-fold. Detergent also influenced antigen stability in cell lysates stored at 4 degrees C; under optimum conditions stability was maintained for at least 1 month, whereas excess detergent rendered the antigen susceptible to proteolytic degradation. This proteolysis could be counteracted by the addition of skim milk or its protein component, which stabilized antigenically reactive p24(s) for up to 2 months. The immunologically relevant epitopes of HBsAg are critically dependent on disulfide bonding. By altering the sodium ascorbate concentration or buffer pH the proportion of HBsAg displaying the monoclonal reactive epitopes was increased between 8- and 20-fold. In addition, under certain conditions the dimerized p24(s) could be converted to oligomeric aggregates, resembling the form of the serum-derived antigen. These simple in vitro manipulations, compatible with the goal of a minimally

  6. Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens.

    PubMed

    Le Bouguénec, C; Lalioui, L; du Merle, L; Jouve, M; Courcoux, P; Bouzari, S; Selvarangan, R; Nowicki, B J; Germani, Y; Andremont, A; Gounon, P; Garcia, M I

    2001-05-01

    Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048-5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr(+) adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr(-) adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr(-) adhesin) was found to predominate in afa-positive isolates from sepsis patients (75%); it was frequent in afa-positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr(+) strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa-positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection

  7. Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae.

    PubMed

    Okada, Munenori; Asai, Tetsuo; Futo, Satoshi; Mori, Yasuyuki; Mukai, Tetsuya; Yazawa, Shigeto; Uto, Takehiko; Shibata, Isao; Sato, Shizuo

    2005-02-25

    To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.

  8. Hepatitis B surface antigen seroprevalence among children in Papua New Guinea, 2012-2013.

    PubMed

    Kitau, Russel; Datta, Siddhartha Sankar; Patel, Minal K; Hennessey, Karen; Wannemuehler, Kathleen; Sui, Gerard; Lagani, William

    2015-03-01

    Approximately 8% of the population in Papua New Guinea (PNG) has chronic hepatitis B virus (HBV) infection. To decrease the burden of chronic HBV infection, a national 3-dose infant hepatitis B vaccination program was implemented starting in 1989, with a birth dose (BD) added to the schedule in 1992. To assess the impact of the hepatitis B vaccination program, we conducted a serosurvey among children born after vaccine introduction. During 2012-2013, a cross-sectional stratified four-stage cluster survey was conducted to estimate hepatitis B surface antigen (HBsAg) prevalence among children 4-6 years of age. We collected demographic data, vaccination history, and tested children for HBsAg. Of 2,133 participants, 2,130 children had vaccination data by either card or recall: 28% received a BD; 81% received ≥ 3 vaccine doses. Of 2,109 children providing a blood sample, 60 (2.3%) tested positive for HBsAg. This is the largest, most geographically diverse survey of hepatitis B vaccination and HBsAg seroprevalence done in PNG. Progress has been made in PNG toward the Western Pacific Regional goal to reduce the prevalence of chronic HBV infection to < 1% by 2017 among 5-year-old children. Vaccination efforts should be strengthened, including increasing BD coverage and completing the 3-dose series.

  9. Enteric trimethyl chitosan nanoparticles containing hepatitis B surface antigen for oral delivery.

    PubMed

    Farhadian, Asma; Dounighi, Naser Mohammadpour; Avadi, Mohammadreza

    2015-01-01

    Oral vaccination is the preferred route of immunization. However, the degradative condition of the gastrointestinal tract and the higher molecular size of peptides pose major challenges in developing an effective oral vaccination system. One of the most excellent methods used in the development of oral vaccine delivery system relies on the entrapment of the antigen in polymeric nanoparticles. In this work, trimethyl chitosan (TMC) nanoparticles were fabricated using ionic gelation teqnique by interaction hydroxypropyl methylcellulose phthalate (HPMCP), a pH-sensitive polymer, with TMC and the utility of the particles in the oral delivery of hepatitis B surface antigen (HBsAg) was evaluated employing solutions that simulated gastric and intestinal conditions. The particle size, morphology, zeta potential, loading capacity, loading efficiency, in vitro release behavior, structure, and morphology of nanoparticles were evaluated, and the activity of the loaded antigen was assessed. Size of the optimized TMC/HPMCP nanoparticles and that of the antigen-loaded nanoparticles were 85 nm and 158 nm, respectively. Optimum loading capacity (76.75%) and loading efficiency (86.29%) were achieved at 300 µg/mL concentration of the antigen. SEM images revealed a spherical shape as well as a smooth and near-homogenous surface of nanoparticles. Results of the in vitro release studies showed that formulation with HPMCP improved the acid stability of the TMC nanoparticles as well as their capability to preserve the loaded HBsAg from gastric destruction. The antigen showed good activity both before and after loading. The results suggest that TMC/HPMCP nanoparticles could be used in the oral delivery of HBsAg vaccine.

  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. Clinical and pathological study of asymptomatic HBsAg carriers in Chile.

    PubMed Central

    Velasco, M; González-Cerón, M; de la Fuente, C; Ruiz, A; Donoso, S; Katz, R

    1978-01-01

    A clinical, biochemical, and pathological study was performed in 38 chronic HBsAg carriers. The study group is a part of 393 carriers found among 117 705 voluntary blood donors at the National Blood Bank, Hospital del Salvador, Santiago, Chile. None of the 38 carriers had a past history of illicit drug abuse, hepatitis, or work involving a high risk of hepatitis B virus infection. Ten individuals had a normal liver biopsy, 17 reactive non-specific hepatitis, one fatty changes, four chr onic persistent hepatitis, one aggressive hepatitis, two post-necrotic cirrhosis, and three alcoholic cirrhosis. There was not a close correlation between liver function test and liver histology. The most significant laboratory finding was the postivity of alpha fetoprotein in two cases. During the follow-up the two alpha fetoprotein patients presented a hepatocarcinoma 12 and 14 months after admission to the study. PMID:680591

  12. [Research on HBsAg: comparison between two methods of the third generation, RPHA and IEA].

    PubMed

    Moretti, R; Vujovic, A; Benda, N

    1982-06-01

    Two tests of the 3rd generation were evaluated for the identification of HBsAg: the reverse hemoagglutination and the enzymatic immunoassay. The results of the study, carried out on 2,434 sera of occasional and periodic donors, have been slightly discordant. It was confirmed the greater sensibility of the IEA test over the RHA and there was a high incidence of false positive results of the IEA test probably caused by the presence in the sera of an elevated concentration of lipid and/or Hb. However, we can consider the RHA a useful test adaptable as an emergency test while the IEA method, being more sensible, can be used as a basic screening test.

  13. Renal involvement in a syndrome of vasculitis complicating HBsAg negative cirrhosis of the liver.

    PubMed

    Montoliu, J; Darnell, A; Grau, J M; Torras, A; Revert, L

    1985-01-01

    Six HBsAg negative patients with cirrhosis of the liver (CL) presented with recurrent bouts of palpable purpura in the legs due to small vessel leucocytoclastic vasculitis. In addition, all patients had renal failure, proteinuria and microhaematuria. Renal biopsy disclosed either diffuse proliferative (3 cases) or focal necrotising glomerulonephritis with crescents (2 cases). One patient had IgM-IgG mixed cryoglobulinaemia (type II). Four patients died of complications of their CL. Hepatocellular carcinoma was found in 1 case. In the patient without renal biopsy renal function improved following steroids and cyclophosphamide. The pathogenesis of this syndrome of cutaneous vasculitis with severe glomerular involvement in CL is unknown but could be immune-complex mediated.

  14. New amino acid changes in drug resistance sites and HBsAg in hepatitis B virus genotype H.

    PubMed

    Fernández-Galindo, D A; Sánchez-Ávila, F; Bobadilla-Morales, L; Gómez-Quiróz, P; Bueno-Topete, M; Armendáriz-Borunda, J; Sánchez-Orozco, L V

    2015-06-01

    Long-term treatment with retrotranscriptase (RT) inhibitors eventually leads to the development of drug resistance. Drug-related mutations occur naturally and these can be found in hepatitis B virus (HBV) carriers who have never received antiviral therapy. HBsAg are overlapped with RT domain, thus nucleot(s)ide analogues (NAs) resistance mutations and naturally-occurring mutations can cause amino acid changes in the HBsAg. Twenty-two patients with chronic hepatitis B were enrolled; three of them were previously treated with NAs and 19 were NAs-naïve treated. HBV reverse transcriptase region was sequenced; genotyping and analysis of missense mutations were performed in both RT domain and HBsAg. There was predominance of genotype H. Drug mutations were present in 18.2% of patients. Classical lamivudine resistance mutations (rtM204V/rtL180M) were present in one naïve-treatment patient infected with genotype G. New amino acid changes were identified in drug resistance sites in HBV strains from patients infected with genotype H; rtQ215E was present in two naïve-NAs treatment patients and rtI169M was identified in a patient previously treated with lamivudine. Mutations at sites rt169, rt204, and rt215 resulted in the Y161C, I195M, and C206W mutations at HBsAg. Also, new amino acid changes were identified in B-cell and T-cell epitopes and were more frequent in HBsAg compared to RT domain. In conclusion, new amino acid changes at antiviral resistance sites, B-cell and T-cell epitopes in HBV genotype H were identified in Mexican patients.

  15. CTL ELISPOT assay.

    PubMed

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  16. Serospecific antigens of Legionella pneumophila.

    PubMed Central

    Otten, S; Iyer, S; Johnson, W; Montgomery, R

    1986-01-01

    Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria. Images PMID:3017918

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

    PubMed

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

    2008-08-01

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

  18. 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

  19. Influence of chemical modification of N alpha-cocoyl-L-arginine ethyl ester on its hepatitis B surface antigen-inactivating effect.

    PubMed Central

    Sugimoto, Y; Toyoshima, S

    1980-01-01

    We have reported previously that N alpha-cocoyl-L-arginine ethyl ester (CAE) strongly inactivates hepatitis B surface antigen (HBsAg; Sugimoto and Toyoshima, Antimicrob. Agents Chemother. 16:329--332, 1979). Replacement of the L-arginine moiety of CAE by L-lysine did not decrease the HBsAg-inactivating effect of CAE, whereas replacement by some neutral amino acids and L-ornithine decreased it. Esterification of the carboxyl group of N alpha-acyl-L-arginine enhanced its inactivating effect. When the ethyl ester of CAE was converted to an amide group, the effect was appreciably decreased. Modification of the carboxyl group was essential for the inactivation. The effectiveness of N alpha-acyl-L-arginine ethyl ester depends upon the length of the acyl group, with the optimum length for the inactivation of HBsAg being C12 to C14. In addition to CAE, N alpha-lauroyl-L-lysine ethyl ester and N alpha-cocoyl-L-arginine amide were found to be strong inactivators of HBsAg. Significant inactivating effects on HBsAg were not observed in many anionic detergents containing an amino acid. These results suggest that for strongly inactivating HBsAg, a compound should contain a special amino acid, such as L-arginine, and a long acyl group and exhibit a cationic property. PMID:7447415

  20. Asymptomatic rectal mucosal lesions and hepatitis B surface antigen at sites of sexual contact in homosexual men with persistent hepatitis B virus infection.

    PubMed

    Reiner, N E; Judson, F N; Bond, W W; Francis, D P; Petersen, N J

    1982-02-01

    To ascertain why active and passive oral-anal and genital anal intercourse correlate strongly with hepatitis B virus (HBV) infection in homosexual men, we studied 22 men with HBV infection for the presence of hepatitis B surface antigen (HBsAg) in gingival and anorectal mucosa, feces, and semen. Thirteen of 22 (59%) patients had asymptomatic rectal mucosal lesions consisting of punctate bleeding points. Seventy-seven percent of swabbed specimens from lesions, 62% from feces, 59% from rectal mucosa, and 50% from anal canal mucosa contained HBsAg. Sera diluted serially and tested for HBsAg by radioimmunoassay showed that men with serum titers of 105 or greater were more likely to have HBsAg in specimens from lesions (p = 0.034), feces (p = 0.032), and normal mucosa (p = 0.001) than men with titers under 10 5. Asymptomatic rectal bleeding is frequent in homosexual men with persistent HBV infection. Rectal mucosa, feces, and anal canal mucosa become contaminated with HBsAg, creating a setting for de facto parenteral transmission when there is contact with oral or urethral mucosa.

  1. 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:…

  2. Co-Administration of Lipid Nanoparticles and Sub-Unit Vaccine Antigens Is Required for Increase in Antigen-Specific Immune Responses in Mice.

    PubMed

    Thoryk, Elizabeth A; Swaminathan, Gokul; Meschino, Steven; Cox, Kara S; Gindy, Marian; Casimiro, Danilo R; Bett, Andrew J

    2016-12-06

    A vast body of evidence suggests that nanoparticles function as potent immune-modulatory agents. We have previously shown that Merck proprietary Lipid NanoParticles (LNPs) markedly boost B-cell and T-cell responses to sub-unit vaccine antigens in mice. To further evaluate the specifics of vaccine delivery and dosing regimens in vivo, we performed immunogenicity studies in BALB/c and C57BL/6 mice using two model antigens, Hepatitis B Surface Antigen (HBsAg) and Ovalbumin (OVA), respectively. To assess the requirement for co-administration of antigen and LNP for the elicitation of immune responses, we evaluated immune responses after administering antigen and LNP to separate limbs, or administering antigen and LNP to the same limb but separated by 24 h. We also evaluated formulations combining antigen, LNP, and aluminum-based adjuvant amorphous aluminum hydroxylphosphate sulfate (MAA) to look for synergistic adjuvant effects. Analyses of antigen-specific B-cell and T-cell responses from immunized mice revealed that the LNPs and antigens must be co-administered-both at the same time and in the same location-in order to boost antigen-specific immune responses. Mixing of antigen with MAA prior to formulation with LNP did not impact the generation of antigen-specific B-cell responses, but drastically reduced the ability of LNPs to boost antigen-specific T-cell responses. Overall, our data demonstrate that the administration of LNPs and vaccine antigen together enables their immune-stimulatory properties.

  3. Co-Administration of Lipid Nanoparticles and Sub-Unit Vaccine Antigens Is Required for Increase in Antigen-Specific Immune Responses in Mice

    PubMed Central

    Thoryk, Elizabeth A.; Swaminathan, Gokul; Meschino, Steven; Cox, Kara S.; Gindy, Marian; Casimiro, Danilo R.; Bett, Andrew J.

    2016-01-01

    A vast body of evidence suggests that nanoparticles function as potent immune-modulatory agents. We have previously shown that Merck proprietary Lipid NanoParticles (LNPs) markedly boost B-cell and T-cell responses to sub-unit vaccine antigens in mice. To further evaluate the specifics of vaccine delivery and dosing regimens in vivo, we performed immunogenicity studies in BALB/c and C57BL/6 mice using two model antigens, Hepatitis B Surface Antigen (HBsAg) and Ovalbumin (OVA), respectively. To assess the requirement for co-administration of antigen and LNP for the elicitation of immune responses, we evaluated immune responses after administering antigen and LNP to separate limbs, or administering antigen and LNP to the same limb but separated by 24 h. We also evaluated formulations combining antigen, LNP, and aluminum-based adjuvant amorphous aluminum hydroxylphosphate sulfate (MAA) to look for synergistic adjuvant effects. Analyses of antigen-specific B-cell and T-cell responses from immunized mice revealed that the LNPs and antigens must be co-administered—both at the same time and in the same location—in order to boost antigen-specific immune responses. Mixing of antigen with MAA prior to formulation with LNP did not impact the generation of antigen-specific B-cell responses, but drastically reduced the ability of LNPs to boost antigen-specific T-cell responses. Overall, our data demonstrate that the administration of LNPs and vaccine antigen together enables their immune-stimulatory properties. PMID:27929422

  4. The underlying mechanisms for the "simultaneous HBsAg and anti-HBs serological profile".

    PubMed

    Pondé, R A A

    2011-11-01

    The course of HBV infection is determined by the interplay between viral replication via HBV protein production and the host's immune response. Therefore, the diagnosis of infection in clinical practice is established by the serological detection of HBV protein products as well as antibodies produced by the host. Although the serological findings for assessing the clinical course of infection are already well established, the expression of viral proteins and the dynamics of antibody production may vary during the natural course of infection. This causes the HBV infection to be occasionally associated with the presence of unusual serologic profiles, which can lead to doubts in the interpretation of results and mistaken serological diagnosis. The simultaneous detection of HBsAg and anti-HBs in the blood stream comprises an atypical serological profile, somewhat incoherent, whose significance can be complicated to establish. Outlined in this article are some immunological and molecular mechanisms which could justify the existence of this profile in which there is a great laboratorial and clinical interest.

  5. 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.

  6. Sero-Prevalence of Hepatitis B Surface Antigen Amongst Pregnant Women Attending an Antenatal Clinic, Volta Region, Ghana

    PubMed Central

    Dassah, Sylvester; Lokpo, Sylvester; Ameke, Louise; Noagbe, Mark; Adatara, Peter; Hagan, Oheneba; Binka, Fred

    2017-01-01

    Hepatitis B virus (HBV) infection remains a global challenge, although there is currently a safe and effective vaccine available. HBV prevalence in Ghana is not well documented, but vary regionally from 4.8% to 12.3% in the general population, 10.8% to 12.7% in blood donors and about 10.6% in pregnant women. This puts Ghana among the high endemic countries in Africa. The study objective was to determine the sero-prevalence of HBs antigen (Ag) and HBeAg among pregnant women in the Ho municipality. Two hundred and eigh participants (pregnant women), attending Ho Municipal antenatal clinic were enrolled into the study. This study recorded a HBsAg sero-prevalence rate of 2.4% among the pregnant women, with primigravida pregnant women recording (0.98%) and multigravida (1.42%). The prevalence of HBsAg among the pregnant women can be classified as Low Intermediate; therefore there is still the need for routine screening of pregnant women during antenatal visits. Amongst HBsAg positives, HBeAg positivity was significantly high (40% of all HBsAg positive women), which suggests high chances of carrier and vertical transmission (mother to child) state. PMID:28299162

  7. A PILOT EXTERNAL QUALITY ASSURANCE STUDY OF TRANSFUSION SCREENING FOR HIV, HCV AND HBSAG IN TWELVE AFRICAN COUNTRIES

    PubMed Central

    Bloch, Evan M; Shah, Avani; Kaidarova, Zhanna; Laperche, Syria; Lefrere, Jean-Jacques; van Hasselt, James; Zacharias, Peter; Murphy, Edward L

    2014-01-01

    Background and Objectives Serologic screening for the major transfusion transmissible viruses (TTV) is critical to blood safety and has been widely implemented. However, actual performance as measured by proficiency testing has not been well studied in Sub-Saharan Africa. Therefore, we conducted an external quality assessment of laboratories engaged in transfusion screening in the region. Materials and Methods Blinded test panels, each comprising 25 serum samples that were pedigreed for HIV, HBsAg, HCV and negative status, were sent to participating laboratories. The panels were tested using the laboratories’ routine donor screening methods and conditions. Sensitivity and specificity were calculated and multivariable analysis was used to compare performance against mode of testing, country and infrastructure. Results A total of 12 African countries and 44 laboratories participated in the study. The mean (range) sensitivities for HIV, HBsAg and HCV were 91.9% (14.3-100), 86.7% (42.9-100) and 90.1% (50-100), respectively. Mean specificities for HIV, HBsAg and HCV were 97.7%, 97% and 99.5% respectively. After adjusting for country and infrastructure, rapid tests had significantly lower sensitivity than enzyme immunoassays (EIA) for both HBsAg (p<0.0001) and HCV (p<0.05). Sensitivity also varied by country and selected infrastructure variables. Conclusion While specificity was high, sensitivity was more variable and deficient in a substantial number of testing laboratories. These findings underscore the importance of proficiency testing and quality control, particularly in Africa where TTV prevalence is high. PMID:25052195

  8. Optimization of in vitro HBV replication and HBsAg production in HuH7 cell line.

    PubMed

    Cavallone, Daniela; Moriconi, Francesco; Colombatto, Piero; Oliveri, Filippo; Bonino, Ferruccio; Brunetto, Maurizia Rossana

    2013-04-01

    The Gunther's vector-free method (GM), using PCR-amplified full length HBV-DNA (fl-HBV-DNA), is currently the best in vitro HBV replication system despite the low intracellular HBV-DNA production. The replication efficiency and HBsAg secretion of 12 isolates from HBsAg/HBeAg positive sera by GM, Monomer-Linear-Sticky-Ends-DNA (MLSE) and Monomer-Circular-Closed (MCC) were compared in HuH7 cells. Eight of twelve genomes (67%) were replication competent by GM; however direct sequencing (DS) showed that more than 80% of input DNA was undigested in spite of SapI treatment. Replication Intermediates (RI) were detected earlier (24 vs. 48h) and in higher amounts (2.51±0.32 and 6.43±0.43 fold) by MCC than GM or MLSE. By MCC 10 of 12 genomes (83%) were replication competent and 7 produced high RI levels. RI and HBsAg kinetics correlated positively in MCC (R=0.696, p=0.017 overall; R=0.928, p=0.008), but not in GM (R=-0.437, p=0.179 overall; R=-0.395, p=0.439) in genotype D isolates. In conclusion, HBV-DNA circularization prior transfection improves in vitro viral replication and replication competent HBsAg production, mimicking better the in vivo conditions.

  9. Epidemiological characteristics of the carriers with coexistence of HBsAg and anti-HBs based on a community cohort study.

    PubMed

    Pu, Z; Li, D; Wang, A; Su, H; Shao, Z; Zhang, J; Ji, Z; Gao, J; Choi, B C K; Yan, Y

    2016-04-01

    The coexistence of HBsAg and anti-HBs is an atypical serological pattern in HBV infection. There is no epidemiological characteristics of this serological pattern in the community and there is controversy over the molecular mechanisms underlying this pattern. We investigated the epidemiological characteristics of the carriers with HBsAg and anti-HBs in a longitudinal community cohort study. The prevalence of this atypical serological pattern was 2.93% (122/4169) in HBsAg-positive populations. The prevalence progressively increased with age from 40 to 70 years old. The rate of HBeAg positive and detectable HBV DNA were both significantly higher in carriers with this pattern than in carriers who were HBsAg positive but anti-HBs negative (26/122 verse 598/4047, P = 0.046; 86/122 verse 275/529,P < 0.001). After 1 year of follow-up, 85.19% of the carriers still had coexistence HBsAg and anti-HBs, 14.81% of the carriers lost their anti-HBs. Viral sequencing showed that carriers with coexistence of HBsAg and anti-HBs had higher numbers of residue changes within the S gene than carriers who were HBsAg positive but anti-HBs negative (2.42 verse 1.33 changes per 100 residues, P < 0.05). Hence, the coexistence of HBsAg and anti-HBs is a unique serological pattern which may be associated with an increased risk of adverse clinical outcome and may be related to HBsAg immune variants which have genotypic heterogeneity.

  10. 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...

  11. Posttransfusion hepatitis B transmitted by blood from a hepatitis B surface antigen-negative hepatitis B virus carrier.

    PubMed

    Larsen, J; Hetland, G; Skaug, K

    1990-06-01

    A female blood donor at our institution was implicated in three cases of clinical posttransfusion hepatitis B. She had given blood 25 times in 8 years. Twenty-seven recipients were followed up, and about one-half of those who were still living had serologic markers of hepatitis B virus (HBV) infection. The donor was repeatedly negative for hepatitis B surface antigen (HBsAg) and antibody, but she had high titers of antibody to hepatitis B core antigen. We conclude that blood from this HBsAg-negative blood donor was capable of transmitting hepatitis B.

  12. A lateral electrophoretic flow diagnostic assay.

    PubMed

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

    2015-03-21

    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.

  13. A computational framework for influenza antigenic cartography.

    PubMed

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

    2010-10-07

    Influenza viruses have been responsible for large losses of lives around the world and continue to present a great public health challenge. Antigenic characterization based on hemagglutination inhibition (HI) assay is one of the routine procedures for influenza vaccine strain selection. However, HI assay is only a crude experiment reflecting the antigenic correlations among testing antigens (viruses) and reference antisera (antibodies). Moreover, antigenic characterization is usually based on more than one HI dataset. The combination of multiple datasets results in an incomplete HI matrix with many unobserved entries. This paper proposes a new computational framework for constructing an influenza antigenic cartography from this incomplete matrix, which we refer to as Matrix Completion-Multidimensional Scaling (MC-MDS). In this approach, we first reconstruct the HI matrices with viruses and antibodies using low-rank matrix completion, and then generate the two-dimensional antigenic cartography using multidimensional scaling. Moreover, for influenza HI tables with herd immunity effect (such as those from Human influenza viruses), we propose a temporal model to reduce the inherent temporal bias of HI tables caused by herd immunity. By applying our method in HI datasets containing H3N2 influenza A viruses isolated from 1968 to 2003, we identified eleven clusters of antigenic variants, representing all major antigenic drift events in these 36 years. Our results showed that both the completed HI matrix and the antigenic cartography obtained via MC-MDS are useful in identifying influenza antigenic variants and thus can be used to facilitate influenza vaccine strain selection. The webserver is available at http://sysbio.cvm.msstate.edu/AntigenMap.

  14. Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus.

    PubMed Central

    Salfeld, J; Pfaff, E; Noah, M; Schaller, H

    1989-01-01

    The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. The single conformational determinant responsible for HBc antigenicity in the assembled core (HBc) and a linear HBe-related determinant (HBe1) were both mapped to an overlapping hydrophilic sequence around amino acid 80; a second HBe determinant (HBe2) was assigned to a location in the vicinity of amino acid 138 but found to require for its antigenicity the intramolecular participation of the extended sequence between amino acids 10 and 140. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis B virus nucleocapsid. Images PMID:2463383

  15. [A retrospective study on HBsAg clearance rate after antiviral therapy in children with HBeAg-positive chronic hepatitis B aged 1-7 years].

    PubMed

    Zhu, S S; Dong, Y; Xu, Z Q; Wang, L M; Chen, D W; Gan, Y; Wang, F C; Yan, J G; Cao, L L; Wang, P; Zhang, H F

    2016-10-20

    Objective: To investigate the HBsAg clearance rate after antiviral therapy in children with HBeAg-positive chronic hepatitis B (CHB) aged 1-7 years. Methods: A retrospective analysis was performed for the HBsAg clearance rate in 293 children who were hospitalized in 302 Hospital of PLA from June 2006 to December 2013, met the inclusion criteria, received antiviral therapy, and were followed up for at least 6 months after the withdrawal of antiviral therapy. The t-test or the rank sum test was applied according to the distribution of continuous data, and the chi-square test was used for comparison of categorical data. Results: The HBsAg positive rate of children's mothers was 91.1%. In the age groups of >1-≤2 years, >2-≤3 years, >3-≤4 years, >4-≤5 years, >5-≤6 years, and >6-≤7 years, the HBsAg clearance rates were 66.1%, 65.5%, 45.7%, 41.3%, 20.6%, and 27.6%, respectively. There were significant differences in HBsAg clearance rate between the age groups of >1-≤3 years and >3-≤5 years, >1-≤3 years and >5-≤7 years, and >3-≤5 years and >5-≤7 years (P = 0.001, 0.000, and 0.008). Of all children, 64.8% were boys, among whom 41.1% achieved HBsAg clearance, and 35.2% were girls, among whom 61.2% achieved HBsAg clearance; there was a significant difference in HBsAg clearance rate between boys and girls (P = 0.001). The children with pretreatment alanine aminotransferase (ALT) levels of ≤80 IU/L, > 80 IU/L, ≤200 IU/L, and > 200 IU/L had HBsAg clearance rates of 40.7%, 51.2%, 47.6%, and 49.4%, respectively; there were no significant differences in HBsAg clearance rate between the ALT ≤80 IU/L and ALT > 80 IU/L groups and the ALT ≤200 IU/L and ALT > 200 IU/L groups (P = 0.101 and 0.778). There was no significant difference in HBsAg clearance rate between the pretreatment HBV DNA load < 1×10(7) IU/ml and ≥1×10(7) IU/ml groups (54.9% vs 46.7%, P = 0.286). Of all children, 14.2% had genotype B and an HBsAg clearance rate of 57.1%, and 85% had

  16. Increase of the prevalence of hepatitis B virus surface antigen related to immunodeficiency inherent in acquired immune deficiency syndrome (AIDS).

    PubMed

    Ouattara, S A; Meite, M; Aron, Y; Akran, V; Gody, M; Manlan, L K; de-The, G

    1990-01-01

    During the period from January to December 1987, 2,191 serum samples were collected from different groups of the population in the Ivory Coast (1,126 healthy people selected from the general population, 416 blood donors, 112 healthy anti-HIV carriers, 173 AIDS patients, and 364 patients suffering from icterigenic hepatitis) and tested for anti-HIV (HIV-1 and HIV-2) antibodies, HBsAg, HBeAg, delta antigen (HDAg), and anti-delta (anti-HD) antibody. Anti-HIV antibodies were found in 30 (2.6%) of the general population. 55 (13.2%) blood donors, and 93 (25.5%) patients suffering from icterigenic hepatitis. HBsAg was observed in 103 (9.1%) of the general population, 45 (10.8%) blood donors, 15 (13.4%) healthy anti-HIV carriers, 59 (34.1%) AIDS patients, and 45 (40%) icterigenous hepatitis patients. The simultaneous presence of anti-HIV and HBsAg was noted in 4 (0.4%) of the general population, 8 (2%) blood donors, 15 (13.4%) healthy anti-HIV carriers, 59 (34.1%) AIDS cases, and 36 (10%) patients suffering from icterigenic hepatitis. A high prevalence of HBeAg and serological markers of infection by the delta agent were noted in the different groups. HDAg was noted only among AIDS patients or those suffering from icterigenic hepatitis, with a higher frequency among anti-HIV carriers. Our conclusion from this study is that healthy anti-HIV carriers are no more likely to be HBsAg carriers than the HIV-seronegative subjects. However, immunodeficiency induced previously by HIV infection is likely to be responsible for the high prevalence of HBsAg among AIDS patients.

  17. Development of Prototype Filovirus Recombinant Antigen Immunoassays

    PubMed Central

    Boisen, Matt L.; Oottamasathien, Darin; Jones, Abigail B.; Millett, Molly M.; Nelson, Diana S.; Bornholdt, Zachary A.; Fusco, Marnie L.; Abelson, Dafna M.; Oda, Shun-ichiro; Hartnett, Jessica N.; Rowland, Megan M.; Heinrich, Megan L.; Akdag, Marjan; Goba, Augustine; Momoh, Mambu; Fullah, Mohammed; Baimba, Francis; Gbakie, Michael; Safa, Sadiki; Fonnie, Richard; Kanneh, Lansana; Cross, Robert W.; Geisbert, Joan B.; Geisbert, Thomas W.; Kulakosky, Peter C.; Grant, Donald S.; Shaffer, Jeffery G.; Schieffelin, John S.; Wilson, Russell B.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.; Khan, S. Humarr; Pitts, Kelly R.

    2015-01-01

    Background. Throughout the 2014–2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. Methods. Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. Results. Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus–specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. Conclusions. The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins. PMID:26232440

  18. 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

  19. Detection of functional class II-associated antigen: role of a low density endosomal compartment in antigen processing

    PubMed Central

    1995-01-01

    We have developed a functional assay to identify processed antigen in subcellular fractions from antigen-presenting cells; stimulatory activity in this assay may be caused by either free peptide fragments or by complexes of peptide fragments and class II molecules present on organellar membrane sheets and vesicles. In addition, we have developed a functional assay to identify proteolytic activity in subcellular fractions capable of generating antigenic peptides from intact proteins. These techniques permit the direct identification of intracellular sites of antigen processing and class II association. Using a murine B cell line stably transfected with a phosphorylcholine (PC)-specific membrane-bound immunoglobulin (Ig), we show that PC- conjugated antigens are rapidly internalized and efficiently degraded to generate processed antigen within an early low density compartment. Proteolytic activity capable of generating antigenic peptide fragments from intact proteins is found within low density endosomes and a dense compartment consistent with lysosomes. However, neither processed peptide nor peptide-class II complexes are detected in lysosomes from antigen-pulsed cells. Furthermore, blocking the intracellular transport of internalized antigen from the low density endosome to lysosomes does not inhibit the generation of processed antigen. Therefore, antigens internalized in association with membrane Ig on B cells can be efficiently processed in low density endosomal compartments without the contribution of proteases present within denser organelles. PMID:7722450

  20. 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.

  1. Tumor antigens as related to pancreatic cancer.

    PubMed

    Chu, T M; Holyoke, E D; Douglass, H O

    1980-01-01

    Data are presented suggesting the presence of pancreas tumor-associated antigens. Slow progress has been made during the past few years in the identification of pancreatic tumor antigens that may be of clinical usefulness and it seems unlikely that many of the practical problems now being faced in identification and isolation of these antigens and in development of a specific, sensitive assay will be solved by conventional immunochemical approaches. The study of antigen and/or antibody purified from immune complexes in the host and the application of leukocyte adherence inhibition techniques to immunodiagnosis of pancreatic cancer are among the new approaches that may provide effective alternatives in the study of pancreatic tumor antigens.

  2. Antigenicity of two turkey astrovirus isolates.

    PubMed

    Tang, Y; Saif, Y M

    2004-12-01

    Astroviruses are positive-sense single-stranded RNA viruses. These viruses cause gastroenteritis in humans and in a variety of animal species, including turkey poults. Only human astroviruses are well characterized antigenically. In the current study, two turkey astrovirus isolates, TAstV1987 and TAstV2001, were antigenically compared using cross-neutralization tests in turkey embryos, as well as cross-reactivity of the two isolates by an enzyme-linked immunosorbent assay (ELISA). The antigenic relatedness values (R) were calculated using the Archetti and Horsfall formula. The R value based on the cross-neutralization tests was 0.56%, which indicates that TAstV1987 and TAstV2001 belong to different serotypes; the R value of the two viruses based on ELISA was 70.7%, which suggests these two viruses share common antigen(s).

  3. Improvement of arbovirus HA antigens by treatment with a colloidal silica gel and sonication.

    PubMed

    Traavik, T

    1977-01-01

    A remarkable increase in HA titers for weakly haemagglutinating Norwegian arbovirus strains, Uukuniemi and Runde viruses, was achieved by including treatment with the colloidal silica gel Aerosil in the antigen preparation scheme. By combining this procedure with sonication, the titers of sucrose-aceton extracted, infected suckling mouse brains could be increased several hundred times. Good antigens also were obtained from virus grown in BHK21/c 13 cell cultures and concentrated by polyethylene glycol 6000/NaCl. Rubella virus HA antigen and HBsAg were adsorbed to the gel, and excluded from a preparation by treatment with Aerosil. This indicates a limitation to the universal use of the method, presumably related to the particle size.

  4. 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

  5. Long-term persistence of hepatitis B surface antigen and antibody induced by DNA-mediated immunization results in liver and kidney lesions in mice.

    PubMed

    Zi, Xiao-Yuan; Yao, Yu-Cheng; Zhu, Hai-Ying; Xiong, Jun; Wu, Xiu-Ju; Zhang, Nan; Ba, Yue; Li, Wen-Lin; Wang, Xin-Ming; Li, Jian-Xiu; Yu, Hong-Yu; Ye, Xu-Ting; Lau, Joseph T Y; Hu, Yi-Ping

    2006-04-01

    DNA-mediated immunization has been recognized as a new approach for prevention and treatment of hepatitis B virus (HBV) infection. However, the side effects of this approach have not been well described. Here we report that DNA-mediated immunization by intramuscular injection of plasmid DNA encoding HBV surface antigen (HBsAg) induced long-term persistence of HBsAg and HBsAg-specific antibody (anti-HBs) in the sera of the immunized BALB/c mice and resulted in liver and kidney lesions. The lesions persisted for 6 months after injection. Lesions were also found in normal mice injected with the sera from immunized mice, and in HBV-transgenic mice injected with anti-HBs antibody, or sera from immunized mice. Furthermore, lesions were accompanied by deposition of circulating immune complex (CIC) of HBsAg and anti-HBs antibody in the damaged organs. These results indicate that long-term persistence of HBsAg and anti-HBs in the immunized mice can result in deposited CIC in liver and kidney, and in development of lesions. The use of DNA containing mammalian replication origins, such as the plasmids used in this study, is not appropriate for human vaccines due to safety concerns relating to persistence of DNA; nevertheless, the safety of DNA-mediated immunization protocols still needs to be carefully evaluated before practical application.

  6. Biological characteristics comparison of HBV rtA181T mutants with truncated or substituted HBsAg expression in vitro and in vivo model systems

    PubMed Central

    Zhou, Ling-Yun; Chen, En-Qiang; Wang, Meng-Lan; Chen, Lan-Lan; Liu, Cui-Ping; Zeng, Fan; Tang, Hong

    2016-01-01

    The hepatitis B virus(HBV) polymerase rtA181T mutation is selected during long-term antiviral therapy. As the polymerase gene completely overlaps with the envelope (S) gene, HBV rtA181T mutation also carries sW172 mutations. In this study, we investigated whether there were biological differences between rtA181T/sW172* (coding truncated HBsAg) and rtA181T/sW172L (coding substituted HBsAg) mutants. In cell experiments, a slight decline of viral replication was observed in both two mutants as compared to wild-type strains, but the levels of supernatant HBsAg and HBV DNA in rtA181T/sW172* were significantly lower than those in rtA181T/sW172L transfected cells. In animal experiments, we were amazed to find that viral replication in rtA181T/sW172* mutant increased and maintained significantly longer than that in rtA181T/sW172L mutant, while no significant difference was observed between rtA181T/sW172L and wild-type strains. Compared with wild-type strains, there were intracellular accumulations of HBsAg and HBcAg in rtA181/sW172* but none in rtA181/sW172L mutant strains. Importantly, we also found that truncated HBsAg could increase the activity of HBV core promoter, but substituted HBsAg could not. In summary, the characteristics of above two rtA181T mutants mentioned above were significantly different, and it is necessary and important for us to distinguish sW172* truncated mutation from sW172L substituted mutation. PMID:27976732

  7. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  8. [Atypical serological profiles in hepatitis B infections: investigation of S gene mutations in cases with concurrently positive for HBsAg and anti-HBs].

    PubMed

    Aydın, Neriman; Kırdar, Sevin; Uzun, Nilgül; Eyigör, Mete; Sayan, Murat

    2016-10-01

    Hepatitis B virus (HBV) causes different clinical manifestations, ranging from asymptomatic carriage to fulminant or chronic hepatitis. Serological tests are widely used for the diagnosis of HBV infections to detect viral markers. However, facing with atypical serological profiles in some patients leads to problems in interpreting of the results and management of the patients. The aims of this study were to investigate the atypical serologic profiles seen in patients screened for HBV infection and the S gene mutations in patients with concurrent positivity of HBsAg and anti-HBs. A total of 592 sera from patients (332 male, 260 female; age range: 13-84 years, mean age: 43.9 years) prediagnosed as HBV infection between January to September 2013, and screened for HBV markers (HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc-IgM, anti-HBc-total and HBV-DNA) were included in the study. Of those samples 364 were screened only for HBsAg and anti-HBs markers. S gene mutations were investigated by direct sequencing method in sera which were concurrent positive for HBsAg and anti-HBs. In our study, 5.2% (31/592) of the sera yielded atypical serologic profiles. Of these 13 cases were concurrently positive for HBsAg and anti-HBs; nine were HBeAg positive, anti-HBe and HBV-DNA negative; eight were HBeAg, anti-HBe and HBV-DNA positive; and one was HBsAg and anti-HBs negative, anti-HBe and HBV-DNA positive. The rate of concurrent positivity of HBsAg and anti-HBs was 3.6% (13/364), while 76.9% (10/13) of those cases were also positive for HBV-DNA. DNA sequencing was performed for seven out of 10 samples which were positive for HBsAg, anti-HBs and HBV-DNA, however three samples were not used because of the low amounts. Sequence analysis of seven samples showed S gene mutations in two samples, one was sS143L with sS193L, a HBV vaccine escape mutation, and the other was sP120R, a HBV immune escape mutation. Of the patients 2.7% (10/364) was negative for both HBsAg and anti-HBs; in which

  9. Bayesian nonparametric clustering in phylogenetics: modeling antigenic evolution in influenza.

    PubMed

    Cybis, Gabriela B; Sinsheimer, Janet S; Bedford, Trevor; Rambaut, Andrew; Lemey, Philippe; Suchard, Marc A

    2017-01-18

    Influenza is responsible for up to 500,000 deaths every year, and antigenic variability represents much of its epidemiological burden. To visualize antigenic differences across many viral strains, antigenic cartography methods use multidimensional scaling on binding assay data to map influenza antigenicity onto a low-dimensional space. Analysis of such assay data ideally leads to natural clustering of influenza strains of similar antigenicity that correlate with sequence evolution. To understand the dynamics of these antigenic groups, we present a framework that jointly models genetic and antigenic evolution by combining multidimensional scaling of binding assay data, Bayesian phylogenetic machinery and nonparametric clustering methods. We propose a phylogenetic Chinese restaurant process that extends the current process to incorporate the phylogenetic dependency structure between strains in the modeling of antigenic clusters. With this method, we are able to use the genetic information to better understand the evolution of antigenicity throughout epidemics, as shown in applications of this model to H1N1 influenza. Copyright © 2017 John Wiley & Sons, Ltd.

  10. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  11. Detection of antigenically distinct rotaviruses from infants.

    PubMed Central

    Dimitrov, D H; Estes, M K; Rangelova, S M; Shindarov, L M; Melnick, J L; Graham, D Y

    1983-01-01

    Antigenically distinct rotaviruses, i.e., viruses morphologically identical to conventional rotaviruses by electron microscopy, yet lacking the common group antigen(s) detected by an enzyme-linked immunosorbent assay, were found in 2 of 51 fecal samples from Bulgarian infants with rotavirus gastroenteritis. These antigenically distinct viruses contained 11 segments of double-stranded RNA, but they demonstrated a unique RNA migration profile after electrophoresis of the genome RNA in polyacrylamide gels. This report confirms the presence of a new group of rotaviruses in humans. The significance of these viruses is currently unknown, and specific diagnostic tests must be developed for epidemiological studies to determine their role as human and veterinary pathogens and to evaluate their impact on proposed vaccine development programs. Images PMID:6307873

  12. Antigenic Variation of Campylobacter Flagella

    DTIC Science & Technology

    1987-11-01

    Cultures were grown at 37"C zation, the flagellum is a major antigen of the campylobacter in anaerobic jars on chocolate -blood agar plates. An atmo- cell...Protein epitopes to the serospecificity of the LIO 8 serogroup. This solubilized in sample buffer was stacked in 4.5% acrylamide thermolabile serogroup...were grown for 24 h and then streaked ELISA. The enzyme-linked immunosorbent assay (ELISA) on one side of a chocolate -blood agar plate from which a was

  13. Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

    PubMed

    Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

    2013-11-01

    In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

  14. Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

    PubMed Central

    Gurramkonda, Chandrasekhar; Adnan, Ahmad; Gäbel, Thomas; Lünsdorf, Heinrich; Ross, Anton; Nemani, Satish Kumar; Swaminathan, Sathyamangalam; Khanna, Navin; Rinas, Ursula

    2009-01-01

    Background Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. Results Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. Conclusion In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries. PMID:19208244

  15. 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.

  16. 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.

  17. Antigenic Diversity of Hepatitis B Virus Strains of Genotype F in Amerindians and Other Population Groups from Venezuela

    PubMed Central

    Blitz, Linda; Pujol, Flor H.; Swenson, Paul D.; Porto, Leticia; Atencio, Ricardo; Araujo, Mary; Costa, Luciana; Monsalve, Diana Callejas; Torres, Jaime R.; Fields, Howard A.; Lambert, Steve; Van Geyt, Caroline; Norder, Helene; Magnius, Lars O.; Echevarría, José M.; Stuyver, Lieven

    1998-01-01

    The adw4 subtype of hepatitis B virus (HBV) belongs to a unique genomic group (genotype F) representing the original HBV strains from the New World. Data regarding the prevalence of this subtype among HBV carriers in South America are, however, scarce, and those concerning HBV genotype F are based on only a few samples from Latin America. In this study, serum samples were obtained from 141 hepatitis B surface antigen (HBsAg) carriers from Amerindians and urban populations from Venezuela. The HBsAg subtype was identified with monoclonal antibodies in 105 samples, and the HBV genotype was identified by reverse-phase hybridization with DNA fragments in 58 samples. The adw4 subtype was highly prevalent in the population studied (75%); among the Amerindians, the prevalence was 97%. The adw2 subtype was also present (10%), while other subtypes (ayw3 and ayw4) were only occasionally found. The HBV subtype was associated with the expected genotype in most cases (80%), and thus genotype F was highly prevalent. Sequencing of viral strains that gave genotypes unpredicted by the HBsAg subtyping confirmed seven of them as belonging to not previously described genotype-subtype associations: namely, adw2 and ayw4 within genotype F. PMID:9508289

  18. 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.

  19. Antigen-specific detection of HBsAG-containing immune complexes in the course of hepatitis B virus infection.

    PubMed Central

    Pernice, W; Sodomann, C P; Lüben, G; Seiler, F R; Sedlacek, H H

    1979-01-01

    In recent studies extrahepatic manifestations of viral hepatitis have been recognized as immune complex diseases. Hepatitis B surface antigen (HBsAg) has been successfully identified in immune complexes, but the pathogenic role of HBsAg-containing immune complexes (IC) remains questionable. The subject of the present study was the antigen-specific determination of IC in the course of hepatitis B virus infection using a new HBsAg-specific IC test (Pernice & Sedlacek, 1978). This test is based on the following principle: rabbit anti-HBs-coated polystyrole test tubes are incubated with the IC-containing test sample. The HBsAg-containing IC bind to the solid phase by their free antigenic determinants. There they can be quantified using a peroxidase-labelled anti-human IgG antibody. A good correlation was found between the level of HBsAg-containing immune complexes and the clinical state of six patients in a follow-up study. IC could be detected simultaneously with HBsAg and either decreased or disappeared before the occurrence of free anti-HBs. In the sera of an additional twenty-eight patient suffering from chronic active hepatitis, HBsAg-containing immune complexes were detected in 85% of cases. One patient suffering from polyarteritis nodosa was also positive. Occasionally, extremely high levels of IC were found in the course of these diseases. PMID:91465

  20. Identification of an immunodominant epitope in glycoproteins B and G of herpes simplex viruses (HSVs) using synthetic peptides as antigens in assay of antibodies to HSV in herpes simplex encephalitis patients.

    PubMed

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

    2014-01-01

    Herpes simplex encephalitis (HSE) is a severe viral infection of the central nervous system (CNS). Assay of antibody response is widely used in diagnostics of HSE. The aim of this study was to identify an immunodominant epitope determining the antibody response to herpes simplex viruses (HSVs) in cerebrospinal fluid (CSF) of HSE patients. The synthetic peptides that resembled type-common as well as type-specific domains of glycoproteins B (gB) and G (gG) of these viruses were evaluated for binding with IgM and IgG antibodies in CSF samples from HSE and non-HSE patients in ELISA. The QLHDLRF peptide, derived from gB of HSV was found to be an immunodominant epitope in the IgM and IgG antibody response. The patients with confirmed and suspected HSE showed in ELISA against this peptide 26% and 23% positivities for IgM, 43% and 37% positivities for IgG and 17% and 15% for both IgM and IgG antibodies, respectively. The total positivities of 86% and 75% for both IgM and IgG antibodies were obtained in the patients with confirmed and suspected HSE, respectively. These results demonstrate that a synthetic peptide-based diagnostics of HSE can be an efficient and easily accessible alternative. This is the first report describing the use of synthetic peptides derived from HSVs in diagnostics of HSE using patientsʹ CSF samples.

  1. 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

  2. Risk of underdiagnosing amebic dysentery due to false-negative Entamoeba histolytica antigen detection.

    PubMed

    Prim, Núria; Escamilla, Pilar; Solé, Roser; Llovet, Teresa; Soriano, Germán; Muñoz, Carmen

    2012-08-01

    Entamoeba histolytica antigen assays on stool are widely used to diagnose amebiasis. We report a case of confirmed amebic colitis with a false-negative antigen detection that became positive after treatment. Our results indicate that these assays may underdiagnose acute amebic infection when used alone and should be used cautiously.

  3. Human Ia-like antigens in non-lymphoid organs.

    PubMed Central

    Koyama, K; Fukunishi, T; Barcos, M; Tanigaki, N; Pressman, D

    1979-01-01

    Human Ia-like antigens in liver and kidney were shown by the immunofluorescence assay to be present mostly in the endothelial-mesenchymal cells of these organs. The parenchymal cells apparently contained no human Ia-like antigens. The antigens in liver and kidney were purified and shown to have the same subunit structure as human Ia-like antigens of cultured B-lymphoid cells. The human Ia-like antigens in non-lymphoid organs, not only in liver and kidney but also in testis, heart, muscle and brain, carried all the xenoantigenic characteristics of human Ia-like antigens expressed on lymphoid cells of B-cell lineage. Images Figure 1 PMID:389786

  4. Hexosaminidase assays.

    PubMed

    Wendeler, Michaela; Sandhoff, Konrad

    2009-11-01

    beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

  5. Factors Predicting HBsAg Seroclearance and Alanine Transaminase Elevation in HBeAg-Negative Hepatitis B Virus-Infected Patients with Persistently Normal Liver Function

    PubMed Central

    Chien, Tai-Long; Wang, Jing-Houng; Kee, Kwong-Ming; Chen, Chien-Hung; Hung, Chao-Hung; Lu, Sheng-Nan

    2016-01-01

    Background A certain proportion of hepatitis B virus (HBV)-infected patients with persistently normal alanine transaminase (ALT) levels have significant fibrosis. Using liver stiffness measurements (Fibroscan®) and laboratory data, including serum ALT, quantitative HBsAg (qHBsAg), and HBV DNA, we attempted to predict the natural histories of these patients. Methods Non-cirrhotic HBeAg-negative chronic hepatitis B patients with persistently normal ALT were followed up prospectively with the end points of HBsAg seroclearance and ALT elevation above the upper limit of normal. The factors that were predictive of the end points were identified. Results A total of 235 patients with an average age of 48.1 +/- 10.7 years were followed up for 7 years. Eight patients (3.4%) lost HBsAg, and 15 patients (6.4%) experienced ALT elevation. The overall cumulative HBsAg seroclearances were 0.4%, 1.3% and 2.3% at years 1, 3 and 5, respectively. Regarding HBsAg seroclearance, the qHBsAg (< 30 IU/ml) cutoff resulted in a hazard ratio (HR) of 19.6 with a 95% confidence interval (CI) of 2.2–166.7 (P = 0.008). The baseline ALT level (odd ratio (OR) 1.075, 95% CI 1.020–1.132, P = 0.006) and a qHBsAg above 1000 IU/ml (3.7, 1.1–12.4, P = 0.032) were associated with ALT elevation. Limited to men, the baseline liver stiffness (1.6, 1.0–2.5, P = 0.031) and a qHBsAg above 1000 IU/ml (10.4, 2.1–52.4, P = 0.004) were factors that were independently associated with ALT elevation. Conclusion A low qHBsAg level predicted HBsAg clearance. Baseline ALT and a qHBsAg above 1000 IU/ml were independent predictive factors for ALT elevation. Among the men, the independent predictive factors for ALT elevation were qHBsAg and liver stiffness. PMID:27935953

  6. Prostate Derived Ets Factor, an Immunogenic Breast Cancer Antigen

    DTIC Science & Technology

    2006-09-01

    Due to prostate restricted expression of Pse in normal tissues of mice, Pse is likely to be immunogenic in female mice. T cell responses of male and... female mice to Pse and a control antigen Her-2/neu were compared. Two different assays were used for this purpose; the ELISPOT assay and the...Cytotoxicity assay. To date, we have found that in the FVB strain of mice immunization with Pse induces specific T cell responses in female mice but not in

  7. Cell-mediated immunity to soluble and particulate inhaled antigens

    PubMed Central

    Hill, J. O.; Burrell, R.

    1979-01-01

    In order to determine the influence of an antigen's physical properties on the development of cell-mediated immunity (CMI) in the lung following aerosol immunization, human serum albumin (HSA) was prepared in either a soluble or a particulate form, the latter being coupled to respirable, carboxylated latex beads. Antigen was administered via an aerosol to groups of guinea-pigs, twice weekly for up to 4 weeks. Additional groups of animals served as unexposed and unconjugated latex controls. Lymphoid cells for CMI assays were isolated from the lung by bronchopulmonary lavage and from blood for use in mitogen- and antigen-induced lymphocyte transformation assays, as well as indirect macrophage migration inhibition tests. Particulate HSA-exposed animals yielded the highest numbers of free lung cells containing predominantly macrophages, with up to 33% lymphocytes. These were followed by the latex control, soluble HSA and unexposed control groups, respectively. Only the animals exposed to particulate HSA had evidence of antigen reactivation in the lung cell populations as measured by lymphocyte stimulation assays. In contrast, a response to polyclonal mitogens was found only in animals exposed to antigen in a soluble form. Data from macrophage depletion experiments suggest that the antigenicity of inhaled antigens may be due to the types and numbers of cells responding to the stimulus, and the subsequent role the alveolar macrophage may play in the modulation of cellular immunity. PMID:393444

  8. 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.

  9. [Study of diphtheria anatoxins in immunochemical and tissue culture assays].

    PubMed

    Gal'vidis, I A; Sviridov, V V

    2007-01-01

    Equine diphtheria antitoxins from different manufacturers were studied. Their immunochemical interaction with diphtheria toxin, toxoid, and antigens of Corynebacterium diphtheriae in ELISA and immunoblotting assays as well as biological activity in CHO cell assay were compared. The discovered differences between antitoxin samples with stated equal activity in IU/ml point to heterogeneity of antigen composition in preparations used for immunization. Mentioned methods allow to standardize antitoxins basing on their biological activity and immunochemical characteristics.

  10. 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

  11. Immunological evaluation of colonic delivered Hepatitis B surface antigen loaded TLR-4 agonist modified solid fat nanoparticles.

    PubMed

    Sahu, Kantrol Kumar; Pandey, Ravi Shankar

    2016-10-01

    Hepatitis B is one of the leading liver diseases and remains a major global health problem. Currently available vaccines provide protection but often results in weaker/minimal mucosal immunity. Thus the present study is devoted to the development and in-vivo exploration of the colonically delivered biomimetic nanoparticles which capably enhance humoral as well as cellular immune response. In present work, Hepatitis B surface antigen (HBsAg) entrapped nanoparticles containing Monophosphoryl lipid A (MPLA) (HB+L-NP) were prepared by solvent evaporation method and characterized for particle size (~210nm), shape, zeta potential (-24mV±0.68), entrapment efficiency (58.45±1.68%), in-vitro release and antigen integrity. Dose escalation study was done to confirm prophylactic immune response following defined doses of prepared nanoparticulate formulations with or without MPLA. Intramuscular administered alum based marketed HBsAg (Genevac B) was used as standard (10μg) and were able to induce significant systemic (IgG) but remarkably low mucosal immune (IgA) response. Notably, HB+L-NP (0.5ml-10μg) induced strong systemic and robust mucosal immunity (510 and 470 mIU/ml respectively, p<0.001) from which mucosal was more significant due to the involvement of Common Mucosal Immune System (CMIS). Likewise, significant cellular immune response was elicited by HB+L-NP through T-cell activation (mixed Th1 and Th2) as confirmed by significantly increased cytokines level (IL-2 and Interferon-γ) in spleen homogenates. This study supports that delivery of HBsAg to the colon may open new vista in designing oral vaccines later being one of most accepted route for potential vaccines in future.

  12. [Retrospective evaluation of HBsAg, anti-HCV, anti-HIV and syphilis reagin antibody seropositivity in blood donors at the Trabzon Farabi Hospital].

    PubMed

    Aydin, Faruk; Cubukçu, Kivanç; Yetişkul, Serpil; Yazici, Yelda; Kaklikkaya, Neşe

    2002-01-01

    Transfusion of blood and blood products is a widely used method for therapy in medicine, however it may result with the transmission of infectious agents from donor to recipient. In order to achieve safe blood transfusions and to minimize post-transfusion infections, several screening tests for infectious agents are routinely done all around the world as well as in our country. In this study, HBsAg, anti-HCV, anti-HIV and syphilis reagin antibody tests results have been retrospectively evaluated for 33.766 blood donors during January 1, 1997 to December 31, 2000 in Blood Center of Farabi Hospital, Black Sea Technical University. Testing for HBsAg, anti-HIV and anti-HCV has been done by using commercially available micro and/or macro enzyme immunoassays, and syphilis reagin antibody test by latex agglutination (RPR) method. The indeterminate results were confirmed by retesting of sera with microparticle enzyme immunoassay and Western blot methods. As a result, in 1331 (3.94%) subjects HBsAg, in 250 (0.74%) subjects anti-HCV, and in 161 (0.47%) subjects RPR were found positive. Twenty samples which have had the results in gray-zone for anti-HIV, have been found negative with the confirmation tests.

  13. Lea blood group antigen on human platelets

    SciTech Connect

    Dunstan, R.A.; Simpson, M.B.; Rosse, W.F.

    1985-01-01

    One- and two-stage radioligand assays were used to determine if human platelets possess the Lea antigen. Goat IgG anti-Lea antibody was purified by multiple adsorptions with Le(a-b-) human red blood cells, followed by affinity chromatography with synthetic Lea substance and labeling with /sup 125/I. Human IgG anti-Lea antibody was used either in a two stage radioassay with /sup 125/I-labeled mouse monoclonal IgG anti-human IgG as the second antibody or, alternatively, purified by Staph protein A chromatography, labeled with /sup 125/I, and used in a one-stage radioassay. Platelets from donors of appropriate red blood cell phenotypes were incubated with the antisera, centrifuged through phthalate esters, and assayed in a gamma scintillation counter. Dose response and saturation curve analysis demonstrate the presence of Lewis a antigen on platelets from Lea+ donors. Furthermore, platelets from an Le(a-b-) donor incubated in Le (a+b-) plasma adsorb Lea antigen in a similar manner to red blood cells. The clinical significance of these antigens in platelet transfusion remains undefined.

  14. 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.

  15. 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

  16. [Enterobacterial antigen in human peripheral blood lymphocytes].

    PubMed

    Faure-Fontenla, M A; García-Tamayo, F

    1989-11-01

    The following study has as prior history the research reports which have shown the existence of an antigenic tissue deposit in gram-negative enterobacteria. The antigens of the enterobacteria have also been found in the lymphocytic membranes and cytoplasm. Since intestinal lymphoid tissue cells can recirculate by means of the thoracic duct to the peripheral venous system, it was proposed that the circulating lymphocytes in healthy people could also contain small amounts of a common enterobacterial antigen. The study was carried out in 15 human venous blood samples, of which the lymphocytic population was separated to later be used in the preparation of 15 alcohol soluble extracts. This material was used for inhibiting the immuno-hemolysis assay in three occasions in order to show the presence of antigens shared by different enterobacterias, using as reference a fraction separated from the LPS of Escherichia coli 08. The results showed that the human lymphocytes also had antigenic determinants common to gram-negative bacteria.

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

    PubMed

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

    2014-03-01

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

  18. Thermodynamic and kinetic analysis of the interaction between hepatitis B surface antibody and antigen on a gold electrode modified with cysteamine and colloidal gold via electrochemistry.

    PubMed

    He, Yan Bin; Luo, Hong Qun; Li, Nian Bing

    2007-06-15

    Hepatitis B surface antibody (HBsAb) was immobilized to the surface of a gold electrode modified with cysteamine and colloidal gold as matrices to detect hepatitis B surface antigen (HBsAg). Differential pulse voltammetry (DPV) method was used for the investigation of the specific interaction between the immobilized HBsAb and HBsAg in solution, which was followed as a change of peak current in DPV with time. With the modified gold electrode, the differences in affinity of HBsAb with HBsAg at the temperatures of 37 and 40 degrees C were easily distinguished and the kinetic rate constants (k(ass) and k(diss)) and kinetic affinity constant K were determined from the curves of current versus time. In addition, the thermodynamic constants, DeltaG, DeltaH and DeltaS, of the interaction at 37 degrees C were calculated, which were -56.65, -64.54 and -25.45 kJ mol(-1), respectively.

  19. Application of paramagnetic beads for purifying Bacillus anthracis protective antigen.

    PubMed

    Zarzecka, A; Bartoszcze, M

    2006-10-01

    Paramagnetic beads coated with Protein G and Tosylactivated-280 dynabeads have been used to purify Bacillus anthracis protective antigen from a liquid culture. The obtained protein was used in the enzyme-linked immunosorbent assay test to detect B. anthracis protective antigen antibodies in human sera collected from immunized individuals. The purification method using paramagnetic beads is very effective. It is fast, easy and may be carried out practically in any laboratory.

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

    DTIC Science & Technology

    2015-12-01

    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...as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian

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

    PubMed

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

    2004-10-01

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

  2. 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

  3. Antigen injection (image)

    MedlinePlus

    Leprosy is caused by the organism Mycobacterium leprae . The leprosy test involves injection of an antigen just under ... if your body has a current or recent leprosy infection. The injection site is labeled and examined ...

  4. Functionalized Magnetic Nanoparticles for the Detection and Quantitative Analysis of Cell Surface Antigen

    PubMed Central

    Shahbazi-Gahrouei, Daryoush; Abdolahi, Mohammad; Zarkesh-Esfahani, Sayyed Hamid; Laurent, Sophie; Sermeus, Corine; Gruettner, Cordula

    2013-01-01

    Cell surface antigens as biomarkers offer tremendous potential for early diagnosis, prognosis, and therapeutic response in a variety of diseases such as cancers. In this research, a simple, rapid, accurate, inexpensive, and easily available in vitro assay based on magnetic nanoparticles and magnetic cell separation principle was applied to identify and quantitatively analyze the cell surface antigen expression in the case of prostate cancer cells. Comparing the capability of the assay with flow cytometry as a gold standard method showed similar results. The results showed that the antigen-specific magnetic cell separation with antibody-coated magnetic nanoparticles has high potential for quantitative cell surface antigen detection and analysis. PMID:23484112

  5. F41 pili as protective antigens of enterotoxigenic Escherichia coli that produce F41, K99, or both pilus antigens.

    PubMed Central

    Runnels, P L; Moseley, S L; Moon, H W

    1987-01-01

    Pigs suckling dams that have been vaccinated with pilus antigen are protected against challenge with enterotoxigenic Escherichia coli (ETEC) strains that express the same pilus antigen. However, some ETEC strains express more than one pilus antigen. Pregnant swine were vaccinated either with E. coli HB101 that harbored a recombinant plasmid coding for F41 expression (F41+) or with the HB101 parent strain that carries the pHC79 vector (F41-). Suckling pigs born to vaccinated dams were challenged with ETEC that expressed either K99, F41, or both pilus antigens. Production of F41 in vivo was demonstrated by immunofluorescence assay of sections of ileum and by seroconversion against F41 antigen by pigs challenged with F41+ and K99+ F41+ ETEC strains. The F41+ vaccine protected against challenge with an F41+ ETEC strain. In contrast, F41+ vaccination did not protect against challenge with K99+ or K99+ F41+ ETEC strains. The F41- vaccine did not protect against challenge with any strain used. The results indicate that K99+ F41+ ETEC strains produce F41 antigen in the small intestine during disease and that F41+ vaccination can be a protective antigen if the challenge strain expresses only F41 antigen, but that F41+ vaccination may not protect against strains that produce both K99 and F41 antigens. PMID:2880807

  6. Hepatitis D Virus Infection Among Hepatitis B Surface Antigen Carriers and in “Isolated anti-HBc” Antibodies Profile in Central Tunisia

    PubMed Central

    Mhalla, Salma; Kadri, Yosr; Alibi, Sana; Letaief, Amel; Boukadida, Jalel; Hannachi, Naila

    2016-01-01

    Background: Hepatitis D Virus (HDV) causes accelerated liver diseases in patients with Hepatitis B Virus (HBV) infection. There is lack of data about its prevalence, related risk factors and interaction with HBV carriers in our country. Objectives: The aim of this study was to estimate the prevalence of hepatitis delta and associated risk factors among Hepatitis B surface antigen (HBsAg) and “isolated anti-HBc” profile carriers in central Tunisia. Patients and Methods: In this cross-sectional study, 540 patients with positive HBsAg and 109 “isolated anti-HBc” profile receiving care in a teaching hospital were tested for the presence of HDV serum-markers using commercially available enzyme immunoassay kit. HBV-DNA was detected by nested PCR in “isolated anti-HBc” profile group. Results: Prevalence of HDV was 8.1% in HBsAg carriers group, but it was significantly higher in active than inactive hepatitis (30.2% and 4.5%, respectively, OR = 9, 95% CI: [4.48-18.58]). There was no significant association between studied risk factors and HDV infection. In the “isolated anti-HBc” profile group, prevalence of HDV was 4.6% and HBV-DNA had negative result in all patients with positive results for HDV. Conclusions: Although HDV had low prevalence in our area, it is vital to plan preventive strategies for HDV spread as well as HBV prevention. It is particularly important to suspect HDV infection in active HBV carriers to manage a particularly severe dual infection. HDV infection should be suspected even in negative HBsAg patients having “isolated anti-HBc” profile. PMID:27110257

  7. Immunogenicity of an investigational hepatitis B vaccine (hepatitis B surface antigen co-administered with an immunostimulatory phosphorothioate oligodeoxyribonucleotide) in nonresponders to licensed hepatitis B vaccine.

    PubMed

    Halperin, Scott A; Ward, Brian J; Dionne, Marc; Langley, Joanne M; McNeil, Shelly A; Smith, Bruce; Mackinnon-Cameron, Donna; Heyward, William L; Martin, J Tyler

    2013-07-01

    An additional one to three doses of hepatitis B vaccine are recommended for nonresponders to an initial standard three-dose series. We compared the safety and immunogenicity of an investigational hepatitis B surface antigen vaccine (HBsAg-1018) with a phosphorothioate oligodeoxyribonucleotide adjuvant that targets toll-like receptor-9 to a commercially available, alum-adjuvanted hepatitis B vaccine (HBsAg-Eng) in nonresponders to three previous doses (primary study) or to four to six previous doses (substudy) of HBsAg-Eng. Both vaccines were well tolerated, although HBsAg-1018 was associated with more injection-site tenderness (63.2% vs. 18.8%, p = 0.016 in the primary study and 81.8% vs. 15.4%, p = 0.003 in the substudy). No statistically significant differences in rates of seroprotection (anti-HBs concentration ≥ 10 mIU/mL) or geometric mean antibody concentrations were found in the primary study. In the substudy, a greater proportion of HBsAg-1018 recipients achieved an anti-HBs concentration ≥ 100 mIU/mL (54.5% vs. 8.3%, p = 0.027), and those responders had higher geometric mean antibody concentrations at 4 weeks (264 vs. 46.5 mIU/mL, p = 0.021) and 52 weeks (7.0 vs. 1.2 mIU/mL, p = 0.030) than HBsAg-Eng recipients. Although this study suggests that HBsAg-1018 may have improved immunogenicity in nonresponders to hepatitis B vaccine vaccination when compared with HBsAg-Eng, larger studies are required.

  8. PreS deletion mutations of hepatitis B virus in chronically infected patients with simultaneous seropositivity for hepatitis-B surface antigen and anti-HBS antibodies.

    PubMed

    Huang, Xiangyan; Qin, Yanghua; Zhang, Peng; Tang, Gusheng; Shi, Qingfen; Xu, Jun; Qi, Falian; Shen, Qian

    2010-01-01

    Hepatitis B surface antigen (HBsAg) and anti-HBs antibodies (anti-HBs) may coexist in certain chronic hepatitis B (CHB) patients. This study was designed to further explore the relationship between this coexistence and hepatitis B Virus (HBV) preS deletions. Sera of 28 patients carrying both HBsAg and anti-HBs (Group I) and those of another 28 HBsAg positive but anti-HBs negative patients (Group II) were collected from CHB patients. Direct sequencing of polymerase chain reaction products or sequencing of clones was applied to both groups to determine sequences of HBV preS and S genes. Genotyping of the S gene indicated that all sampled HBVs were either Genosubtype Ba or Genosubtype Ce. Seven samples in Group I harbored HBV preS deletion mutations. Three of the seven samples showed large deletion mutations in 3' terminus of preS1 and co-existence of the mutant type and the full-length wild type, and the remaining four samples showed deletion mutations in 5' terminus of preS2. All mutant strains were found to be genosubtype Ce. Only two samples in Group I showed G145R/A mutation. Only one sample in Group II contained preS deletion mutation. It is therefore concluded that HBV preS deletion mutations are likely to be related to the coexistence of HBsAg and anti-HBs in CHB patients (P-value = 0.024). Some immune reactions may select for the preS deletion in CHB patients with anti-HBs, the possible marker for immune selection.

  9. Telbivudine vs tenofovir in hepatitis B e antigen-negative chronic hepatitis B patients: OPTIMA roadmap study

    PubMed Central

    Krastev, Zahari; Petrova, Diana; Kotzev, Iskren; Celen, Mustafa Kemal; Mendelson, Meryl; Chandra, Richa; Pandey, Priti; Hamed, Kamal

    2016-01-01

    AIM To make efficacy and safety comparison of telbivudine-raodmap and tenofovir-roadmap in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) patients. METHODS This was the first prospective, randomised, two-arm, open-label, non-inferiority study in HBeAg-negative CHB patients that compared telbivudine and tenofovir administered as per roadmap concept. Patients were treated up to 24 wk and, depending on virologic response, continued the same therapy or received add-on therapy up to 104 wk. Eligible patients received an additional 52 wk of treatment in the extension period (i.e., up to 156 wk). Patients who developed virologic breakthrough (VB) while on monotherapy also received add-on therapy. The primary efficacy endpoint was the rate of patients achieving hepatitis B virus (HBV) DNA < 300 copies/mL at week 52. Secondary efficacy endpoints included the rates of HBV DNA < 300 and < 169 copies/mL, HBV DNA change from baseline, alanine aminotransferase normalisation, hepatitis B surface antigen (HBsAg) loss, HBsAg seroconversion, VB, and emergence of resistance at various timepoints throughout the study. Safety and estimated glomerular filtration rate (eGFR) were also analysed. RESULTS A total of 241 patients were randomised. Non-inferiority of telbivudine arm to tenofovir arm was demonstrated at week 52 (± 7 d window), with over 91% of patients in each treatment arm achieving HBV DNA level < 300 copies/mL. Both arms were similar in terms of key secondary efficacy variables at weeks 104 and 156. The percentage of patients achieving HBV DNA < 300 copies/mL remained high and was similar in the telbivudine and tenofovir arms at both weeks 104 and 156. Over 82% of patients in both arms achieved alanine aminotransferase normalisation at week 52, and this percentage remained high at weeks 104 and 156. Telbivudine treatment progressively reduced serum HBsAg levels from baseline while no change was reported in quantitative HBsAg during therapy with tenofovir

  10. 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.

  11. Hepatitis B virus and hepatitis D virus in blood donors from Argentina: circulation of HBsAg and reverse transcriptase mutants.

    PubMed

    Delfino, Cecilia María; Gentile, Emiliano Alberto; Castillo, Amalia Inés; Cuestas, María Luján; Pataccini, Gabriela; Cánepa, Camila; Malan, Richard; Blejer, Jorgelina; Berini, Carolina; Eirin, María Emilia; Pedrozo, Williams; Oubiña, José Raúl; Biglione, Mirna Marcela; Mathet, Verónica Lidia

    2014-05-01

    In Argentina, current procedures to ensure the safety of the blood supply for transfusion include the serologic detection of specific blood-borne infections. The aim of this study was to evaluate the prevalence and the genetic diversity of hepatitis B virus (HBV) and hepatitis D virus (HDV) in blood donor populations from two distantly located Argentine regions. Data from 56,983 blood donations from the Favaloro Foundation, in the city of Buenos Aires (Central Region), and the Central Blood Bank of Misiones Province (Northeast Region) were analyzed. Samples that were reactive for HBsAg were analyzed for HBV-DNA characterization and HDV serological and molecular analysis. The HBV prevalence was 0.12 % for HBsAg and 1.68 % for anti-HBc antibodies in Buenos Aires, and 0.73 % and 8.55 %, respectively, in Misiones. Seventy-seven HBsAg-reactive samples were analyzed by polymerase chain reaction for HBV-DNA. Subgenotypes A2, B2, C2, F1b and F4 (Buenos Aires) and F1b and D3 (Misiones) were detected. Several mutations within the major hydrophilic region of HBsAg, the reverse transcriptase, the basal core promoter, and the precore/core were detected. HDV genotype 1 was identified in Buenos Aires. This study confirms the circulation of several HBV subgenotypes, as well as known and newly identified variants, and the presence of HDV1 in this population. A thorough investigation has to be carried out to evaluate the clinical importance of some of the documented mutations as well as those detected in the HDV1 case.

  12. Universal screening for hepatitis B among pregnant women led to 96% vaccination coverage among newborns of HBsAg positive mothers in Denmark.

    PubMed

    Harder, Katja Majlund; Cowan, Susan; Eriksen, Mette Brandt; Krarup, Henrik B; Christensen, Peer Brehm

    2011-11-21

    In Denmark selective screening programs of pregnant women for hepatitis B missed 30-50% of high-risk groups and in late 2005 a universal screening of pregnant women for HBsAg was implemented. During a 2-year period a prospective enhanced surveillance of the universal screening was performed to examine the effectiveness of universal HBV-screening of pregnant women and HBV-immunizations of their newborn, and to provide a prevalence-estimate for HBV in Denmark. On a opt out basis all women in Denmark attending antenatal care were tested for hepatitis B serology. Vaccination data of the newborns and households of HBsAg positive pregnant women were assembled. Among 140,376 HBsAg tests of pregnant women, 371 (0.26%) were positive. The prevalence among women of Danish origin was 0.012% and 2.74% among foreign born women, highest for women from Southeast Asia (14.5%). Genotype C was the most prevalent (37%) and 13% had a HBVDNA ≥10(8) IU/ml. The prevalence estimate of chronic hepatitis B in Denmark was 0.2-0.3% in the general population. Among children born within the project period, 96% received vaccination at birth compared to 50% of siblings born prior to universal screening. During 3 years of passive follow-up two transmissions (0.5%) have been notified. Among children born of the positive mothers prior to the trial-period 7.3% had been notified. Thus the prevalence of HBV positive mothers has more than doubled in Denmark over the last 40 years, but among women of Danish origin it has decreased 10-fold. By replacing selective screening with universal, identification of newborns in need of HBV-immunization was increased from 50% to almost complete coverage, and also identifies mothers with high viral load for evaluation of pre-term treatment to interrupt in utero transmission.

  13. 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.

  14. 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.

  15. Collaborative study on antigens for immunodiagnosis of schistosomiasis*

    PubMed Central

    Mott, K. E.; Dixon, H.

    1982-01-01

    Eight research laboratories in Europe and the United States of America were selected on the basis of having published data on Schistosoma mansoni and S. japonicum antigens to participate in a study of various antigen/test combinations for immunodiagnosis of schistosomiasis. The serum bank consisted of 395 well documented sera from four endemic areas in Brazil (2 areas), Kenya, and the Philippines. Altogether, 21 S. mansoni and four S. japonicum antigen and immunoassay combinations were evaluated. S. mansoni egg antigens yielded a higher combined sensitivity than adult worm antigens, irrespective of their purity, in active S. mansoni infections before and after specific treatment. Quantitative seroreactivity of characterized S. mansoni egg antigens showed good correlation with faecal egg counts in the 5-14 year age group. No correlation between morbidity related to S. mansoni and seroreactivity was observed in any test system. Three S. japonicum egg antigens showed high sensitivity and specificity in relation to the presence or absence of eggs in the stool. The quantitative seroreactivity of the characterized S. japonicum egg antigens correlated directly with the intensity of S. japonicum infection in all age groups. The enzyme-linked immunosorbent assay (ELISA), using several different procedures, performed well with the antigens used in the study. The indium slide immunoassay (ISI), a simple qualitative visual test system using an S. mansoni egg antigen, demonstrated a high degree of sensitivity and specificity. The results did not indicate the superiority of any particular immunodiagnostic method for detecting antischistosome antibodies. This collaborative study is considered a first step towards developing and standardizing antigens for immunodiagnosis of schistosomiasis. PMID:6983926

  16. 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.

  17. 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.

  18. Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time-resolved fluorescence immunoassay.

    PubMed

    Aggerbeck, H; Nørgaard-Pedersen, B; Heron, I

    1996-04-19

    A dual, double antigen, time-resolved fluorescence immunoassay (DELFIA) for the simultaneous detection and quantitation of diphtheria (D) and tetanus (T) antibodies in sera has been developed. In the double antigen format one arm of the antibody binds to antigen coated microtitre wells and the other arm binds to labelled antigen to provide a fluorescent signal. This assay was found to be functionally specific for IgG antibodies and showed a good correlation with established toxin neutralization assays. Furthermore, the double antigen set-up was species independent, permitting the direct use of existing international references of animal origin to measure protective antibody levels in humans in international units (IU/ml). The detection limit corresponded to 0.0003 IU/ml with Eu(3+)-labelled toxoids and to 0.0035 IU/ml using Sm(3+)-labelled toxoids. The assay was fast with a high capacity making it a suitable method for serological surveillance studies.

  19. Diagnostic Antigens of Leishmania.

    DTIC Science & Technology

    1994-01-31

    braziliensis (MHOM/BR/75/M2903), L. chagasi (MJOM/BR/82/BA-2,C 1), L. donovani (MHOMiEt/67iHU3), Leishmania guyanensis (MIHOMJBR/75/M4147), L. infantum (IPT-1...comparative test to a variety of other recombinant Leishmania antigens including L. chagasi hsp70, L. braziliensis hsp83/90, L. braziliensis eIF4A, L...34 4. AD CONTRACT NO: DAMD17-92-C-2082 EC•£ 2 j 994 ’i, L TITLE: DIAGNOSTIC ANTIGENS OF LEISHMANIA L PRINCIPAL INVESTIGATOR: Steven G. Reed, Ph.D

  20. 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.

  1. Hetero-organic thymus antigens.

    PubMed

    Beletskaya, L V; Gnezditskaya, E V

    1985-01-01

    The use of sera containing antibodies to tissue-specific antigens of highly specialized organs (skeletal muscles, heart, skin, excretory glands) enabled us to detect, by immunofluorescence, cells capable of synthesizing analogous antigens (i.e. hetero-organic thymus antigens) in human and animal thymus. Detection of hetero-organic antigens in the thymus is the basis for the hypothesis that natural immunological tolerance to tissue self antigens is formed within the thymus in the course of T-lymphocyte maturation, with thymus antigens taking part in the process.

  2. Human liver nucleolar antigens.

    PubMed

    Busch, R K; Busch, H

    1981-10-01

    In an extension of previous studies on the antigens in rat liver nucleoli (R. K. Busch, R. C. Reddy, D. H. Henning, and H. Busch, Proc. Soc. Exp. Biol. Med. 160, 185 (1979); R. K. Busch and H. Busch, Tumori 63, 347 (1977); F. M. Davis, R. K. Busch, L. C. Yeoman, and H. Busch, Cancer Res. 38, 1906 (1978), rabbit antibodies were elicited to human liver nucleoli isolated by the sucrose--Mg2+ method (10). Fluorescent nucleoli were found in liver cryostat sections treated with rabbit anti-human liver nucleolar antibodies followed by fluorescein-conjugated goat anti-rabbit antibodies. In HeLa cells, fluorescence was distributed throughout the nucleus and in a nuclear network but was not localized to the nucleolus. In placental cryostat sections, an overall nuclear fluorescence was observed with some localization to nucleoli. Immunodiffusion analysis revealed two immunoprecipitin bands which appeared to be liver specific. Other immunoprecipitin bands were common to liver, placenta, and HeLa nuclear extracts. Rocket immunoelectrophoresis revealed two liver-specific antigens, one migrating to the cathode and the other to the anode Other rockets exhibited identity to antigens of other nuclear extracts. These results demonstrate the presence of human liver nucleolar-specific antigens which were not found in the HeLa and placental cells.

  3. 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.

  4. Circulating parasite antigen in patients with hydrocephalus secondary to neurocysticercosis.

    PubMed

    Garcia, H H; Gonzalez, A E; Gilman, R H; Bernal, T; Rodriguez, S; Pretell, E J; Azcurra, O; Parkhouse, R M E; Tsang, V C W; Harrison, L J S

    2002-04-01

    End stages of neurocysticercosis include residual intraparenchymal brain calcifications and hydrocephalus. Although brain calcifications alone have a benign prognosis, hydrocephalus is frequently associated with chronic inflammation and intracranial hypertension, together with a protracted clinical evolution, and may lead to patient deaths. By using a monoclonal-based antigen detection enzyme-linked immunosorbent assay, we measured the levels of circulating parasite antigen in the sera of 56 patients with neurocysticercosis: 27 with calcifications only and 29 with hydrocephalus. The assay gave positive results in 14 of 29 patients with hydrocephalus but was consistently negative in patients with calcifications. Circulating parasite antigen in hydrocephalus secondary to neurocysticercosis indicates the presence of live parasites in these patients and thus a potential benefit from antiparasitic therapy.

  5. A Rapid and Sensitive Method for the Quantitation of Legionella Pneumophila Antigen from Human Urine.

    DTIC Science & Technology

    1980-11-12

    reverse% passive hemagglutination test was developed to assay concentrations of solubl4 antigen of Legionnaires ’ Disease ( Legionella pneumophila) in...reversed passive hemagglutination test was developed to assay concentrations of soluble antigen of Legionnaires disease ( Legionella pneumophila) in...G. W. Gorman, R. E. Weaver, D. C. Mackel, and H. W. Smith. 1978. Primary isolation media for Legionnaires ’ disease bacterium. J. Clin. Hicrobiol. 8

  6. 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

  7. Contamination of passenger trains with Dermatophagoides (Acari: Pyroglyphidae) mite antigen in Japan.

    PubMed

    Uehara, K; Toyoda, Y; Konishi, E

    2000-01-01

    Passenger trains were surveyed for contamination with Dermatophagoides farinae Hughes and Dermatophagoides pteronyssinus (Trouesart) mites in Japan. A total of 492 dust samples were collected from upholstered seats in six commuter trains, one long-distance express train and three night trains in October, 1996 and January, April, and July, 1997. Mite antigen levels contained in fine dust fractions of these samples were measured by an enzyme-linked immunosorbent assay. Most samples obtained from commuter trains showed relatively high mite antigen levels of > 10 microgm(-2) (corresponding to > 100 mites). Express and night trains showed lower antigen levels per square meter, but higher mite antigen levels per gram of fine dust than commuter trains, indicating relatively high mite antigen densities. Seasonal comparisons indicated that commuter trains showed the highest mean antigen level per square meter in winter (January), whereas the highest antigen level per gram of fine dust was observed in summer (July) in express and night trains.

  8. Biomarkers for Antigen Immunotherapy in Allergy and Type 1 Diabetes

    PubMed Central

    Odegard, Jared M.; Wambre, Erik

    2015-01-01

    Allergy and type 1 diabetes are immune mediated diseases that, despite being etiologically distinct, have inappropriate activation and effector function of antigen-specific T cells in the pathogenic process. Understanding changes in frequency and phenotype of these cells is critical to improve assessment of disease diagnosis and prognosis and effectively assess immunological response to therapy. In the setting of antigen-specific therapy in allergy and type 1 diabetes, assays to monitor the immunological mechanisms of disease have been improving in recent years, and we are getting closer to an accurate understanding of how the cellular immune response is modulated during treatment. In this review, we summarize the current state of cell-based immune monitoring of antigen therapy trials. We then discuss emerging advances in antigen-specific biomarkers that are transforming our knowledge about allergy and that have the potential to dramatically impact our understanding of T cell-mediated autoimmune diseases, such as type 1 diabetes. PMID:26122171

  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. Cooperative Immunoassays: Ultrasensitive Assays with Mixed Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ehrlich, Paul H.; Moyle, William R.

    1983-07-01

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

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

    PubMed

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

    2006-01-01

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

  12. 21 CFR 866.3310 - Hepatitis A virus (HAV) serological assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Hepatitis A virus (HAV) serological assays. 866... Hepatitis A virus (HAV) serological assays. (a) Identification. HAV serological assays are devices that consist of antigens and antisera for the detection of hepatitis A virus-specific IgM, IgG, or...

  13. 21 CFR 866.3310 - Hepatitis A virus (HAV) serological assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Hepatitis A virus (HAV) serological assays. 866... Hepatitis A virus (HAV) serological assays. (a) Identification. HAV serological assays are devices that consist of antigens and antisera for the detection of hepatitis A virus-specific IgM, IgG, or...

  14. 21 CFR 866.3310 - Hepatitis A virus (HAV) serological assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Hepatitis A virus (HAV) serological assays. 866... Hepatitis A virus (HAV) serological assays. (a) Identification. HAV serological assays are devices that consist of antigens and antisera for the detection of hepatitis A virus-specific IgM, IgG, or...

  15. 21 CFR 866.3310 - Hepatitis A virus (HAV) serological assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Hepatitis A virus (HAV) serological assays. 866... Hepatitis A virus (HAV) serological assays. (a) Identification. HAV serological assays are devices that consist of antigens and antisera for the detection of hepatitis A virus-specific IgM, IgG, or...

  16. 21 CFR 866.3310 - Hepatitis A virus (HAV) serological assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Hepatitis A virus (HAV) serological assays. 866... Hepatitis A virus (HAV) serological assays. (a) Identification. HAV serological assays are devices that consist of antigens and antisera for the detection of hepatitis A virus-specific IgM, IgG, or...

  17. 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

  18. [Development of a new hydrophobic interaction chromatography absorbent and its application to the purification of recombinant hepatitis B surface antigen].

    PubMed

    Wang, Yang-Mu; Bi, Jing-Xiu; Zhao, Lan; Zhou, Wei-Bin; Li, Yan; Huang, Yong-Dong; Zhang, Yan; Lin, Hai; Su, Zhi-Guo

    2006-03-01

    A new hydrophobic absorbent based on homemade highly cross-linked agarose beads was synthesized by immobilizing butyl derivative onto the matrix linkage. The density of ligand was controlled by adjusting the concentration of butanethiol and the synthesis route was optimized by evaluating the purification efficiency of recombinant Hepatitis B surface antigen (HBsAg) expressed by Chinese hamster ovary (CHO) cell line. A high performance absorbent was finally screened out with up to 80% of HBsAg recovery and purification-fold (PF) about 20. Furthermore, the column pressure was about 0.06 MPa under the flow rate of 500cm/h, and no leaked butyl were detected after exposing the gel in common buffers, chaotropic agents, high concentrations of denaturing agents such as guanidine hydrochloride, urea and polar organic solvents. These results demonstrated that the absorbent have high physico-chemical stability, so it was available for the downstream process. Finally, after scaled up to 2L wet gel/batch, the absorbent was applied to the integration of three-step chromatography and obtained the purified CHO-HBsAg with 95% purity by SDS-PAGE and HPLC, which meet the requirements of SFDA. The purification efficiency and the reproducible ability of the absorbents were also evaluated from batch-to-batch. The results demonstrated that the absorbent met the requirement of scalable, reproducible, economic effect as well. This absorbent is a promising alternative exported HIC gel for wildly being used in Chinese pharmaceutical industries.

  19. 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

    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.

  20. 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.

  1. 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.

  2. Detection of Fasciola hepatica and Fasciola gigantica common and uncommon antigens, using rabbit hyper immune serum raised against their excretory-secretory and somatic antigens.

    PubMed

    Abdolahi Khabisi, S; Sarkari, B

    2016-12-01

    Fasciolosis is an important neglected helminth disease caused by two liver flukes, Fasciola hepatica and Fasciola gigantica. The two species of Fasciola are usually different in their morphological and molecular features. They have also common and uncommon antigens in both their somatic and excretory secretory metabolites. In this study, we compared somatic and excretory-secretory (ES) antigens of F. hepatica and F. gigantica, by using rabbit hyper immune serum raised against these antigens. Adult worms were collected from bile ducts of infected animals and species of the fluke was confirmed by RFLP-PCR. ES and somatic antigens of both species were prepared. Rabbits were subcutaneously immunized with either ES or somatic antigens to produce antibodies against these antigens. SDS-PAGE pattern of F. hepatica and F. gigantica somatic antigens was similar and both of them revealed 30 protein bands, ranging from 18 to 180 kDa. In contrast, SDS-PAGE pattern of ES antigen of the two species was different. While protein bands with molecular weight of 18, 27, 29, 48, and 62 kDa were common in both species, bands of 19, 45, 55 and 58 kDa were only noticed in F. hepatica ES antigen. Rabbit polyclonal antibodies, raised against F. hepatica and F. gigantica ES antigen, reacted with main five protein bands, 25, 27, 29, 62 and 67 kDa and polyclonal antibodies raised against somatic antigens of both species reacted with three protein bands, 25, 27 and 72 kDa. Thus, the 25, 27 and 29 kDa protein bands may serve as immunodominant antigens, which might be considered for serodiagnosis of fasciolosis. Moreover, bands of 62 and 67 kDa in ES antigen and 72 kDa in somatic antigens of both species were immunodominant and might be suitable candidate for development of serological assays for diagnosis of fasciolosis.

  3. 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.

  4. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    SciTech Connect

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-12-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

  5. Semiquantitative measure of immune responses against erythropoietic stem cell antigens

    SciTech Connect

    Harrison, D.E.

    1987-01-01

    A semiquantitative assay was developed and used to measure the effects of immune responses against 16 independent non-H-2 antigenic loci on erythropoietic stem cells. The assay compares repopulation in genetically anemic WBB6F1-W/Wv recipients that have normal immune responses, and in lethally irradiated WBB6F1 +/+ mice whose immune responses are suppressed by the irradiation. The differences in repopulating ability between these two types of recipients measure how immune responses affect erythropoietic stem cells. Stem cell repopulating abilities for the cells with antigens specified by the Thy-1, H-1, H-24, Ly-1, H-37, and H-17 loci were affected slightly, if at all. Repopulating abilities were moderately reduced by responses against antigens specified by H-15, 16, Ea-2, and Ly-2, 3 loci, and against the differences between the B6 and B10 genotypes, although marrow of these types cured W/Wv recipients. A surprising result occurred for the antigen specified by the H-8 locus, in which immune responses strongly reduced repopulating abilities, although this type of marrow cell cured W/Wv recipients. A comparison of these results with skin graft survival times suggests that the antigens specified by the H-17 and H-24 loci are strongly immunogenic on skin but not on marrow stem cells, while those specified by the H-12 and H-8 loci are strongly immunogenic on marrow stem cells but not on skin.

  6. Sensitivity and Specificity of Histoplasma Antigen Detection by Enzyme Immunoassay.

    PubMed

    Cunningham, Lauren; Cook, Audrey; Hanzlicek, Andrew; Harkin, Kenneth; Wheat, Joseph; Goad, Carla; Kirsch, Emily

    2015-01-01

    The objective of this study was to evaluate the sensitivity and specificity of an antigen enzyme immunoassay (EIA) on urine samples for the diagnosis of histoplasmosis in dogs. This retrospective medical records review included canine cases with urine samples submitted for Histoplasma EIA antigen assay between 2007 and 2011 from three veterinary institutions. Cases for which urine samples were submitted for Histoplasma antigen testing were reviewed and compared to the gold standard of finding Histoplasma organisms or an alternative diagnosis on cytology or histopathology. Sensitivity, specificity, negative predictive value, positive predictive value, and the kappa coefficient and associated confidence interval were calculated for the EIA-based Histoplasma antigen assay. Sixty cases met the inclusion criteria. Seventeen cases were considered true positives based on identification of the organism, and 41 cases were considered true negatives with an alternative definitive diagnosis. Two cases were considered false negatives, and there were no false positives. Sensitivity was 89.47% and the negative predictive value was 95.35%. Specificity and the positive predictive value were both 100%. The kappa coefficient was 0.9207 (95% confidence interval, 0.8131-1). The Histoplasma antigen EIA test demonstrated high specificity and sensitivity for the diagnosis of histoplasmosis in dogs.

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

    PubMed Central

    Song, Yunfeng; Singh, Pankaj; Nelson, Eric

    2016-01-01

    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

  8. A morphological and functional study on antigen binding and endocytosis by immunocytes.

    PubMed Central

    Goud, B; Antoine, J C; Gonatas, N K; Stieber, A; Avrameas, S

    1980-01-01

    Immunoenzymatic techniques were used to study antigen binding and endocytosis by lymph node cells of rats immunized against horseradish peroxidase, hen ovalbumin and rabbit IgG. The number of antigen-binding cells varied and depended on the type of antigen used, the time after immunization, and was higher after a booster injection. In secondary responses (4 days after booster), about 80% of antigen-binding cells were proplasmocytes and plasmocytes; by a double staining procedure it was found that 82% of these cells bore in addition to surface antigen, specific intracytoplasmic antibody as well. About 20% of antigen-binding cells were small and medium lymphocytes which did not contain detectable intracytoplasmic antibody. For ultrastructural studies of the endocytosis, peroxidase was used as the antigen. This antigen was found in cytoplasmic compartments which consisted of vesicles, cisternae and large round bodies (lysosomes?) often located near the Golgi apparatus. However, the cisternae of the Golgi apparatus, involved in the synthesis of specific antibody were not sites of retrieval of endocytosed antigen. The effect of endocytosis of antigen on the secretion and synthesis of antibody was studied by the local haemolysis plaque assay and biosynthetic labelling. No change was detected in antibody secretion and synthesis as a result of antigen endocytosis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7007216

  9. Expression of hepatitis B virus surface antigens induces defective gonad phenotypes in Caenorhabditis elegans

    PubMed Central

    Chen, Yi-Yin; Lee, Li-Wei; Hong, Wei-Ning; Lo, Szecheng J

    2017-01-01

    AIM To test whether a simple animal, Caenorhabditis elegans (C. elegans), can be used as an alternative model to study the interaction between hepatitis B virus antigens (HBsAg) and host factors. METHODS Three plasmids that were able to express the large, middle and small forms of HBsAgs (LHBsAg, MHBsAg, and SHBsAg, respectively) driven by a ubiquitous promoter (fib-1) and three that were able to express SHBsAg driven by different tissue-specific promoters were constructed and microinjected into worms. The brood size, egg-laying rate, and gonad development of transgenic worms were analyzed using microscopy. Levels of mRNA related to endoplasmic reticulum stress, enpl-1, hsp-4, pdi-3 and xbp-1, were determined using reverse transcription polymerase reaction (RT-PCRs) in three lines of transgenic worms and dithiothreitol (DTT)-treated wild-type worms. RESULTS Severe defects in egg-laying, decreases in brood size, and gonad retardation were observed in transgenic worms expressing SHBsAg whereas moderate defects were observed in transgenic worms expressing LHBsAg and MHBsAg. RT-PCR analysis revealed that enpl-1, hsp-4 and pdi-3 transcripts were significantly elevated in worms expressing LHBsAg and MHBsAg and in wild-type worms pretreated with DTT. By contrast, only pdi-3 was increased in worms expressing SHBsAg. To further determine which tissue expressing SHBsAg could induce gonad retardation, we substituted the fib-1 promoter with three tissue-specific promoters (myo-2 for the pharynx, est-1 for the intestines and mec-7 for the neurons) and generated corresponding transgenic animals. Moderate defective phenotypes were observed in worms expressing SHBsAg in the pharynx and intestines but not in worms expressing SHBsAg in the neurons, suggesting that the secreted SHBsAg may trigger a cross-talk signal between the digestive track and the gonad resulting in defective phenotypes. CONCLUSION Ectopic expression of three forms of HBsAg that causes recognizable phenotypes in

  10. 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

  11. 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

  12. 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

  13. Immune recognition of protein antigens

    SciTech Connect

    Laver, W.G.; Air, G.M.

    1985-01-01

    This book contains 33 papers. Some of the titles are: Antigenic Structure of Influenze Virus Hemagglutinin; Germ-line and Somatic Diversity in the Antibody Response to the Influenza Virus A/PR/8/34 Hemagglutinin; Recognition of Cloned Influenza A Virus Gene Products by Cytotoxic T Lymphocytes; Antigenic Structure of the Influenza Virus N2 Neuraminidase; and The Molecular and Genetic Basis of Antigenic Variation in Gonococcal Pillin.

  14. 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

  15. 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

  16. Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology

    PubMed Central

    Rotman, Ella; Webber, David M.

    2016-01-01

    ABSTRACT Many pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance. Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). The N. gonorrhoeae chromosome contains one expression locus (pilE) and many promoterless, partial-coding silent copies (pilS) that act as reservoirs for variant pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transfer pilS sequences into the pilE locus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity. IMPORTANCE Measuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of pilin antigenic variation. PMID:27381912

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

    PubMed

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

    2015-10-28

    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.

  18. Standardization of anti-DNA antibody assays.

    PubMed

    Pisetsky, David S

    2013-07-01

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

  19. RAS - Screens & Assays

    Cancer.gov

    A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

  20. Urinary antigens as markers of papillary toxicity. I. Identification and characterization of rat kidney papillary antigens with monoclonal antibodies.

    PubMed

    Falkenberg, F W; Hildebrand, H; Lutte, L; Schwengberg, S; Henke, B; Greshake, D; Schmidt, B; Friederich, A; Rinke, M; Schlüter, G; Bomhard, E

    1996-01-01

    Monoclonal antibodies were prepared in an attempt to develop diagnostic tools for the identification of toxic damage to the rat renal papilla. One IgG and five IgM monoclonal antibodies, reacting with antigens localized in the papilla were obtained. Three of the IgM class and the IgG class monoclonal antibodies were found to be specific for antigens localized in collecting ducts, two of them staining papillary collecting ducts more intensely than cortical collecting ducts. The IgG class antibody, termed Pap X 5C10, recognizes an antigen located at high density on the luminal side of papillary collecting duct epithelial cells and at lower density in cortical collecting duct cells. One of the IgM class monoclonal antibodies reacts with an antigen localized in epithelial cells as ascending and descending loops of Henle and of connecting tubules. Another of the IgM class monoclonal antibodies reacts with an antigen localized in the interstices of the inner medulla. All these monoclonal antibodies react with their antigens in native frozen as well as in Bouin-fixed and paraffin-embedded tissue slices. Molecular properties of the Pap X 5C10 antigen have been investigated by gel permeation chromatography, SDS-PAGE, Western blotting, and isoelectric focusing. The results indicate that the antigen in both its tissue-derived and urinary form is of large (150-200 kDa) molecular size and can be separated into two molecular species with isoelectric points of pH 7.2 and 7.3 respectively. In the urine the antigens recognized by the monoclonal antibodies form large complexes with Tamm-Horsfall protein. The antigen-containing complexes can be extracted from urine by adsorption to diatomaceous earth and elution with SDS-containing buffer. Using sandwich ELISA-type assays it is possible to determine the concentration of the antigens. In preliminary experiments we were able to show that at least three of the antigens are detected in the urine following toxic insults to the kidney. The

  1. Assays of Serum Testosterone.

    PubMed

    Herati, Amin S; Cengiz, Cenk; Lamb, Dolores J

    2016-05-01

    The diagnosis of male hypogonadism depends on an assessment of the clinical signs and symptoms of hypogonadism and serum testosterone level. Current clinical laboratory testosterone assay platforms include immunoassays and mass spectrometry. Despite significant advances to improve the accuracy and precision of the currently available assays, limited comparability exists between assays at the lower and upper extremes of the testosterone range. Because of this lack of comparability, there is no current gold standard assay for the assessment of total testosterone levels.

  2. Large Scale Immune Profiling of Infected Humans and Goats Reveals Differential Recognition of Brucella melitensis Antigens

    PubMed Central

    Liang, Li; Leng, Diana; Burk, Chad; Nakajima-Sasaki, Rie; Kayala, Matthew A.; Atluri, Vidya L.; Pablo, Jozelyn; Unal, Berkay; Ficht, Thomas A.; Gotuzzo, Eduardo; Saito, Mayuko; Morrow, W. John W.; Liang, Xiaowu; Baldi, Pierre; Gilman, Robert H.; Vinetz, Joseph M.; Tsolis, Renée M.; Felgner, Philip L.

    2010-01-01

    Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host. PMID:20454614

  3. Large scale immune profiling of infected humans and goats reveals differential recognition of Brucella melitensis antigens.

    PubMed

    Liang, Li; Leng, Diana; Burk, Chad; Nakajima-Sasaki, Rie; Kayala, Matthew A; Atluri, Vidya L; Pablo, Jozelyn; Unal, Berkay; Ficht, Thomas A; Gotuzzo, Eduardo; Saito, Mayuko; Morrow, W John W; Liang, Xiaowu; Baldi, Pierre; Gilman, Robert H; Vinetz, Joseph M; Tsolis, Renée M; Felgner, Philip L

    2010-05-04

    Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.

  4. Increased prevalence of Dirofilaria immitis antigen in canine samples after heat treatment.

    PubMed

    Velasquez, Luisa; Blagburn, Byron L; Duncan-Decoq, Rebecca; Johnson, Eileen M; Allen, Kelly E; Meinkoth, James; Gruntmeir, Jeff; Little, Susan E

    2014-11-15

    Canine serum samples may contain factors that prevent detection of antigen of Dirofilaria immitis on commercial assays, precluding accurate diagnosis. To determine the degree to which the presence of blocking antibodies or other inhibitors of antigen detection may interfere with our ability to detect circulating antigen in canine samples, archived plasma and serum samples (n=165) collected from dogs in animal shelters were tested for D. immitis antigen before and after heat treatment. Negative samples were also evaluated for their ability to block detection of D. immitis antigen in a sample from a positive dog. All 165 samples were negative prior to heating, but 11/154 (7.1%) became positive after heat treatment, a conversion that was documented and quantified on spectrophotometric plate assays, and 7/165 (4.2%) samples decreased detection of antigen when mixed with a known positive sample, suggesting some blocking ability was present. An additional 103 plasma and serum samples that tested positive prior to heating also were evaluated; the optical density of 14/101 (13.9%) increased by ≥50%, and one sample by as much as 15-fold, after heat treatment. Our results suggest that canine serum and plasma samples from dogs in the southeastern United States can contain inhibitors of D. immitis antigen detection, and that prevalence estimates of heartworm infection based on these assays would benefit from heat treatment of samples prior to testing.

  5. 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

  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. 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

  8. 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.

  9. Stool Test: H. Pylori Antigen

    MedlinePlus

    ... Your 1- to 2-Year-Old Stool Test: H. Pylori Antigen KidsHealth > For Parents > Stool Test: H. Pylori Antigen A A A What's in this article? ... en español Muestra de materia fecal: antígeno de H. pylori What It Is Helicobacter pylori ( H. pylori ) bacteria ...

  10. Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: development and description.

    PubMed Central

    Hendry, R M; McIntosh, K

    1982-01-01

    An indirect enzyme-linked immunosorbent assay for detection of respiratory syncytial virus (RSV) antigens was developed, using commercially available antisera. Horse anti-RSV and calf antiserum to bovine RSV were used as capture and detector antibodies, respectively. The assay could detect as few as 50 PFU of unpurified RSV per ml in infected cell culture supernatant fluids and as little as 10 ng of affinity-purified RSV antigen per ml. No cross-reactions were observed with heterologous virus types. Freeze-thaw treatment had no effect on RSV enzyme-linked immunosorbent assay titers, but viral transport medium inhibited RSV enzyme-linked immunosorbent assay titers from 10- to 100-fold. The assay can be easily performed in 24 h and is a sensitive and specific method for the detection of RSV antigens. PMID:6749894

  11. 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.

  12. Radioimmunoassays of hidden viral antigens.

    PubMed Central

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

    1982-01-01

    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-bond adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure. Images PMID:6956871

  13. 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...

  14. Implication of antigenic conversion of Helicobacter pylori lipopolysaccharides that involve interaction with surfactant protein D.

    PubMed

    Yokota, Shin-ichi; Amano, Ken-ichi; Nishitani, Chiaki; Ariki, Shigeru; Kuroki, Yoshio; Fujii, Nobuhiro

    2012-08-01

    We propose two antigenic types of Helicobacter pylori lipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that β-linked N-acetyl-D-glucosamine and β-linked D-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca(2+)-dependent manner, and this interaction was inhibited by methyl-β-D-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of β-N-acetyl-D-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.

  15. Cascade enzyme-linked immunosorbent assay (CELISA).

    PubMed

    Lee, Young-mi; Jeong, Yujin; Kang, Hyo Jin; Chung, Sang J; Chung, Bong Hyun

    2009-10-15

    Immunoassays are representative biochemical detection methods. Among them, sandwich-type immunoassays, typified by sandwich ELISA, have used in disease diagnosis or biochemical detection with high target selectivity. Horseradish peroxidase and alkaline phosphatase have been typically used for signal amplification in ELISA. Recently developed sandwich-type immunoassays such as biobarcode immunoassays, immuno-PCR, and immuno-RCA have improved sensitivity by changing mainly the signal amplification method. To develop a novel amplification method in ELISA, an enzyme-cascading system was incorporated into an ELISA, and the new assay is termed a cascading enzyme-linked immunosorbent assay (CELISA). This CELISA includes a trypsinogen-enterokinase combination as the cascading enzyme system, and was used to detect alpha-fetoprotein (AFP), which is a liver cancer marker, and prostate-specific antigen (PSA). Using a colorimetric reagent for signal generation, CELISA had 0.1-10pM limits-of-detection for AFP and PSA in whole human serum and assay buffers, depending on the platform, well plate, or microbead type used. This study represents the first example that incorporated an enzyme cascading step in an ELISA system, resulting in successful signal amplification with sensitive detection of pathogenic antigens in serum.

  16. Profiling human serum antibodies with a carbohydrate antigen microarray

    PubMed Central

    Oyelaran, Oyindasola; McShane, Lisa M.; Dodd, Lori; Gildersleeve, Jeffrey C.

    2009-01-01

    Carbohydrate antigen arrays (glycan arrays) have been recently developed for the high-throughput analysis of carbohydrate macromolecule interactions. When profiling serum, information about experimental variability, inter-individual biological variability, and intra-individual temporal variability is critical. In this report, we describe the characterization of a carbohydrate antigen array and assay for profiling human serum. Through optimization of assay conditions and development of a normalization strategy, we obtain highly reproducible results with a within-experiment coefficient of variation (CV) of 10.8% and an overall CV (across multiple batches of slides and days) of 28.5%. We also report antibody profiles for 48 human subjects and evaluate for the first time the effects of age, race, sex, geographic location, and blood type on antibody profiles for a large set of carbohydrate antigens. We found significant dependence on age and blood type of antibody levels for a variety of carbohydrates. Finally, we conducted a longitudinal study with a separate group of 7 serum donors to evaluate the variation in anti-carbohydrate antibody levels within an individual over a period ranging from 3 to 13 weeks and found that, for nearly all antigens on our array, antibody levels are generally stable over this period. The results presented here provide the most comprehensive evaluation of experimental and biological variation reported to date for a glycan array and have significant implications for studies involving human serum profiling. PMID:19624168

  17. Excretory-secretory and somatic antigens in the diagnosis of human filariasis.

    PubMed

    Kaushal, N A; Hussain, R; Ottesen, E A

    1984-06-01

    In order to compare the immunodiagnostic value of excretory-secretory (E-S) antigens derived from adult Brugia malayi worms with somatic antigens derived from adults, microfilariae (Mf) and infective larvae (L3) of these parasites, well defined serum pools from patients with filarial (brugia, bancrofti, loa and perstans) and non-filarial (ascaris, stronglyoides, toxocara, echinococcus, cysticercus and schistosoma) helminth infections were tested against antigens derived from these different life cycle stages of B. malayi in a Staphylococcus aureus radioimmunoprecipitation assay (S. aureus RIA). The adult brugia antigens proved significantly more discriminatory than those of the other parasite stages, with the homologous brugia serum pool also showing greater reactivity to adult than to L3 and Mf antigens. Similar results were obtained when individual sera from patients (rather than serum pools) were tested in the same assay. The most surprising finding was the minimal reactivity seen between the adult filarial antigens and the non-filarial serum pools despite the presence in these pools of strong antibody reactivity with their homologous antigens. The reasons underlying the unexpected specificity of this S. aureus RIA for discriminating among sera from filarial and non-filarial infections were analysed qualitatively by immunoprecipitation techniques. It was found that use of the chloramine-T method for radioiodination resulted in preferential labelling of the low molecular weight (mol. wt) proteins (10-70,000 daltons) in the B. malayi adult somatic antigen and that these antigens were bound primarily by the filarial and not the non-filarial serum pools. These findings suggest that lower mol. wt helminth antigens may show greater species specificity than those with higher mol. wt, and those with higher mol. wt, greater cross-reactivity. If substantiated by further analysis, such results would have important implications for the subsequent isolation of diagnostically

  18. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  19. Antigenic variation in Giardia lamblia.

    PubMed

    Prucca, Cesar G; Lujan, Hugo D

    2009-12-01

    Giardia lamblia undergoes antigenic variation, both in vitro and within the intestines of infected individuals. Variant-specific surface proteins (VSPs) cover the entire surface of the trophozoites and are the main antigens recognized by the host. Only 1 of about 200 VSP genes encoded by the Giardia genome is expressed on the surface of individual Giardia cells at any time; however, VSP antigen switching occurs spontaneously. In the recent year, significant advances in the knowledge of the antigen switching process have been achieved, which strongly suggests that antigenic variation in Giardia is regulated at the post-transcriptional level by a mechanism similar to RNA interference (RNAi). Several enzymes of the RNAi pathway are directly involved in VSP mRNA silencing and/or translational repression. Although several questions remain regarding how individual VSP antigens are selected for expression on the parasite surface, it is clear that an epigenetic mechanism is involved. In this review, we summarize the characteristics of this fascinating mechanism, analyse conflicting information regarding the structure of VSPs as it relates to the host's immune response, and highlight the major issues that need to be resolved to fully understand antigenic variation in this important pathogen.

  20. 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.

  1. K99 surface antigen of Escherichia coli: antigenic characterization.

    PubMed Central

    Isaacson, R E

    1978-01-01

    K99 prepared by acid precipitation hemagglutinated guinea pig erythrocytes, whereas K99 prepared by chromatography on diethylaminoethyl-Sephadex did not. K99 purified by either procedure hemagglutinated horse erythrocytes. K99 prepared by acid precipitation contained a second antigen not presnet in the K99 prepared by chromatography on diethylaminoethyl-Sephadex. This antigen could be detected by immunoprecipitation with some, but not all, sera prepared against K99-positive Escherichia coli strains. It was assumed that this second antigen is not K99 and is responsible for the guinea pig erythrocyte hemagglutination reaction. Furthermore, the second antigen has an isoelectric point of 4.2, which has been reported by Morris and co-workers to be the isoelectric point of K99. Images PMID:83300

  2. Identification of human trophoblast membrane antigens in maternal blood during pregnancy.

    PubMed Central

    O'Sullivan, M J; McIntyre, J A; Prior, M; Warriner, G; Faulk, W P

    1982-01-01

    The development of an immunoradiometric assay for the detecti