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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. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Experimental conditions affecting the sensitivity of enzyme-linked immunosorbent assay (ELISA) for detection of hepatitis B surface antigen (HBsAg)*

    PubMed Central

    Fields, Howard A.; Davis, Candace L.; Bradley, Daniel W.; Maynard, James E.

    1983-01-01

    The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for the detection of hepatitis B surface antigen (HBsAg) was improved 16 to 32 times after examination of various solid-phase supports, different antibody preparations as capture antibody, and different conditions for adsorbing capture antibody to the solid-phase. Comparisons were made by checkerboard titration analysis and by sensitivity studies, both of which demonstrated essentially equivalent results. Endpoints were determined by visual inspection and by spectrophotometry using o-phenylenediamine as substrate. The assay was as sensitive as commercially available radioimmunoassays without the requirement of affinity chromatography purified reagents, expensive instrumentation, or radioisotopes. PMID:6340847

  3. Analysis of HBsAg using high-sensitivity HBsAg assays in hepatitis B virus carriers in whom HBsAg seroclearance was confirmed by conventional assays.

    PubMed

    Ozeki, Itaru; Nakajima, Tomoaki; Suii, Hirokazu; Tatsumi, Ryoji; Yamaguchi, Masakatsu; Kimura, Mutsuumi; Arakawa, Tomohiro; Kuwata, Yasuaki; Ohmura, Takumi; Hige, Shuhei; Karino, Yoshiyasu; Toyota, Joji

    2017-09-08

    We investigated the utility of high-sensitivity hepatitis B surface antigen (HBsAg) assays compared with conventional HBsAg assays. Using serum samples from 114 hepatitis B virus (HBV) carriers in whom HBsAg seroclearance was confirmed by conventional HBsAg assays (cutoff value, 0.05 IU/mL), the amount of HBsAg was re-examined by high-sensitivity HBsAg assays (cutoff value, 0.005 IU/mL). Cases negative for HBsAg in both assays were defined as consistent cases, and cases positive for HBsAg in the high-sensitivity HBsAg assay only were defined as discrepant cases. There were 55 (48.2%) discrepant cases, and the range of HBsAg titers determined by high-sensitivity HBsAg assays was 0.005-0.056 IU/mL. Multivariate analysis showed that the presence of nucleos(t)ide analogue therapy, liver cirrhosis, and negative anti-HBs contributed to the discrepancies between the two assays. Cumulative anti-HBs positivity rates among discrepant cases were 12.7%, 17.2%, 38.8%, and 43.9% at baseline, 1 year, 3 years, and 5 years, respectively, whereas the corresponding rates among consistent cases were 50.8%, 56.0%, 61.7%, and 68.0%, respectively. HBV DNA negativity rates were 56.4% and 81.4% at baseline, 51.3% and 83.3% at 1 year, and 36.8% and 95.7% at 3 years, among discrepant and consistent cases, respectively. HBsAg reversion was observed only in discrepant cases. Re-examination by high-sensitivity HBsAg assays revealed that HBsAg was positive in approximately 50% of cases. Cumulative anti-HBs seroconversion rates and HBV DNA seroclearance rates were lower in these cases, suggesting a population at risk for HBsAg reversion. This article is protected by copyright. All rights reserved.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Clinical performance of the novel DiaSorin LIAISON(®) XL murex: HBsAg Quant, HCV-Ab, HIV-Ab/Ag assays.

    PubMed

    Krawczyk, Adalbert; Hintze, Christian; Ackermann, Jessica; Goitowski, Birgit; Trippler, Martin; Grüner, Nico; Neumann-Fraune, Maria; Verheyen, Jens; Fiedler, Melanie

    2014-01-01

    The fully automated and closed LIAISON(®)XL platform was developed for reliable detection of infection markers like hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis C virus (HCV) antibodies (Ab) or human immunodeficiency virus (HIV)-Ag/Ab. To date, less is known about the diagnostic performance of this system in direct comparison to the common Abbott ARCHITECT(®) platform. We compared the diagnostic performance and usability of the DiaSorin LIAISON(®)XL with the commonly used Abbott ARCHITECT(®) system. The qualitative performance of the above mentioned assays was compared in about 500 sera. Quantitative tests were performed for HBsAg-positive samples from patients under therapy (n=289) and in vitro expressed mutants (n=37). For HCV-Ab, a total number of 155 selected samples from patients chronically infected with different HCV genotypes were tested. The concordance between both systems was 99.4% for HBsAg, 98.81% for HCV-Ab, and 99.6% for HIV-Ab/Ag. The quantitative LIAISON(®)XL murex HBsAg assay detected all mutants in comparable amounts to the HBsAg wild type and yielded highly reliable HBsAg kinetics in patients treated with antiviral drugs. Dilution experiments using the 2nd International Standard for HBsAg (WHO) showed a high accuracy of this test. HCV-Ab from patients infected with genotypes 1-3 were equally detected in both systems. Interestingly, S/CO levels of HCV-Ab from patients infected with genotype 3 seem to be relatively low using both systems. The LIAISON(®)XL platform proved to be an excellent system for diagnostics of HBV, HCV, and HIV with equal performance compared to the ARCHITECT(®) system. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    PubMed Central

    2011-01-01

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

  12. Impact of "a" determinant mutations on detection of hepatitis B surface antigen (HBsAg) in HBV strains from Chinese patients with occult hepatitis B.

    PubMed

    Huang, Xiangyan; Ma, Chenyun; Zhang, Qiang; Shi, Qingfen; Huang, Tao; Liu, Chao; Li, Jie; Hollinger, F Blaine

    2017-10-01

    This study was designed to detect mutations that occur within the "a" determinant in the S gene of the hepatitis B virus (HBV) in patients with occult hepatitis B (OHB), and to analyze the influence of these mutations on expression and reactivity of the hepatitis B surface antigen (HBsAg). Twenty-three certified OHB samples were compared to 32 HBsAg positive samples from patients with chronic hepatitis B. The median HBV DNA levels in the OHB group were significantly lower than those in the control group (P < 0.0001). Mutations within the "a" determinant were analyzed by gene amplification and sequencing. This revealed mixed infections in which clones within a sample displayed either different mutations or mutations in association with clones that exhibited wild type amino acid patterns. Sequencing analysis also showed a significant difference between the proportions of amino acid mutations observed in the OHB and control groups. Seven recombinant S (rS) proteins with corresponding OHB mutations and three wild type alleles were expressed and purified in the Pichia pastoris expression system to preserve conformational attributes, and their reactivity analyzed using six commercial HBsAg assays. The OHB sera were HBsAg nonreactive while the rS proteins with corresponding OHB mutations were universally reactive. Thus, we postulate that the reduced binding affinity between mutated HBsAg and its antibody may not be as important in defining OHB as is the effect of specific mutations in the preS/S region of the genome that affect the synthesis and secretion of the S protein and/or the virion. © 2017 Wiley Periodicals, Inc.

  13. 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

  14. Clinical performance of a new hepatitis B surface antigen quantitative assay with automatic dilution.

    PubMed

    Liu, Ta-Wei; Yeh, Ming-Lun; Huang, Chung-Feng; Lin, I-Ling; Huang, Jee-Fu; Dai, Chia-Yen; Chen, Yao-Li; Chuang, Wan-Long; Yu, Ming-Lung

    2015-01-01

    Hepatitis B virus surface antigen (HBsAg) levels reflect disease status and can predict the clinical response to antiviral treatment; however, the emergence of HBsAg mutant strains has become a challenge. The Abbott HBsAg quantification assay provides enhanced detection of HBsAg and HBsAg mutants. We aimed to evaluate the performance of the Abbott HBsAg quantification assay with automatic sample dilutions (shortened as automatic Architect assay), compared with the Abbott HBsAg quantification assay with manual sample dilutions (shortened as manual Architect assay) and the Roche HBsAg quantification assay with automatic sample dilutions (shortened as Elecsys). A total of 130 sera samples obtained from 87 hepatitis B virus (HBV)-infected patients were collected to assess the correlation between the automatic and manual Architect assays. Among the 87 patients, 41 provided 42 sera samples to confirm the linearity and reproducibility of the automatic Architect assay, and find out the correlation among the Elecsys and two Architect assays. The coefficients of variation (0.44-9.53%) and R(2) = 0.996-1, which were both determined using values obtained from the automatic Architect assay, showed good reproducibility and linearity. Results of the two Architect assays demonstrated a feasible correlation (n = 130 samples; R = 0.898, p < 0.01). With regard to subgroups, correlations between the two Architect assays were better in the hepatitis B e antigen (HBeAg)-negative group (HBeAg-negative group vs. HBeAg-positive group: R = 0.885 vs. R = 0.865, both p < 0.01) and low HBV DNA group (low DNA group vs. high DNA group: R = 0.886 vs. R = 0.844, both p < 0.01). Significant correlations were also found between the results of the Elecsys and Architect assays (R > 0.93 in all cases). In conclusion, the correlation between the automatic and manual dilution Architect assays was feasible, particularly in the HBeAg-negative and low DNA groups. With lower labor costs and less human error

  15. 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

  16. Prevalence of hepatitis B virus surface antigen (HBsAg) in patients undergoing extraction at the University College Hospital, Ibadan.

    PubMed

    Odaibo, G N; Arotiba, J T; Fasola, A O; Obiechina, A E; Olaleye, O D; Ajagbe, H A

    2003-09-01

    Hepatitis B Virus (HBV) infection and its sequelae (liver chirrhosis and hepatic carcinoma) are endemic in Africa. The risk of transmission of the infection during dental treatment is real. This study was carried out to determine the rate of Hepatits B Surface Antigen (HBsAg) as a marker of hepatitis B virus infection in patients undergoing dental extraction in order to highlight the potential risk of nosocomial transmission among the Dental Health Workers (DHW) and their patients. Three hundred (143 males and 157 females) consecutive patients requiring dental extraction who volunteered were enrolled into this study. Their ages ranged from 11 years to 95 years with a mean of 37.2 years (SD = 16.725) and a median of 36 years. The overall HBsAg infection rate was 18.3% (55/300). A higher infection rate (23.1%) occurred among the male patients compared with 14% in females (p = 0.0086). The high rate of HBV infection found among this study population suggests that Dental Surgeons in this environment have a high risk of exposure to hepatitis B virus and should be immunized at the beginning of their professional life. Universal biosafety measures should be observed strictly in all invasive procedures.

  17. 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

  18. Prediction of disease reactivation in asymptomatic hepatitis B e antigen-negative chronic hepatitis B patients using baseline serum measurements of HBsAg and HBV-DNA.

    PubMed

    Martinot-Peignoux, Michelle; Lapalus, Martine; Laouénan, Cédric; Lada, Olivier; Netto-Cardoso, Ana Carolina Ferreira; Boyer, Nathalie; Ripault, Marie Pierre; Carvalho-Filho, Roberto; Asselah, Tarik; Marcellin, Patrick

    2013-10-01

    Differentiating 'inactive carriers' (ICs) of hepatitis B virus (HBV) from hepatitis B e antigen-negative (HBeAg[-]) patients in remission is challenging. We investigated whether serum-based monitoring of hepatitis B surface antigen (HBsAg) and HBV-DNA in asymptomatic HBeAg(-) patients could distinguish these groups. 129 HBeAg(-) chronic hepatitis B (CHB) patients (HBV genotypes A-E) with normal alanine aminotransferase (ALT) levels at baseline were classified after 1 year of follow-up as either IC (HBV-DNA ≤2000 IU/mL) or 'active carrier' (AC, HBV-DNA >2000 IU/mL) if they exhibited normal ALT throughout, or classified as 'reactivation patient' (RP) if they exhibited marked, transient increases in ALT and HBV-DNA. There were 64%, 18%, and 19% patients in the IC, AC, and RP groups, respectively. Combined HBsAg and HBV-DNA cutoffs (>1000 IU/mL and >200 IU/mL, respectively) differentiated RPs with 92% sensitivity and negative predictive value (NPV) of 96%. HBsAg sero-clearance was associated with baseline HBsAg <1000 IU/mL, annual decrease of ≥0.3 log IU/mL (NPV 95%: PPV 89%) and IFNL3 genotype CC. Applying combined HBsAg and HBV-DNA cutoffs to baseline measurements accurately differentiated RPs. These results suggest that HBsAg should be included in the monitoring of asymptomatic HBeAg(-) CHB patients. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. Quantitative measurement of serum hepatitis B surface antigen using an immunoradiometric assay in chronic hepatitis B.

    PubMed

    Kwon, Hyun-Woo; Lee, Ho-Young; Kim, Seog Gyun; Kim, Won; Jung, Yong Jin; Kang, Keon Wook; Chung, June-Key; Lee, Myung Chul; Lee, Dong Soo

    2011-03-01

    Measurement of serum hepatitis B virus surface antigen (HBsAg) levels is important for the management of chronic hepatitis B patients in terms of monitoring response to antiviral therapy. This study aimed to evaluate the diagnostic performance of a new diagnostic kit, which quantitatively measures serum HBsAg level using an immunoradiometric assay (IRMA)-based method. Measurements were compared with those obtained using a chemiluminescent microparticle immunoassay (CMIA)-based method. The blood samples of 96 patients with chronic hepatitis B were used in this study. Copy numbers of serum hepatitis B virus (HBV) DNA were determined in 23 of these samples. The correlation between and the concordance of IRMA and CMIA results were determined using Pearson's correlation coefficients. P values of 0.05 were considered to be statistically significant throughout. Laboratory diagnoses based on IRMA were wholly in agreement with those based on CMIA. Furthermore, serum HBsAg levels by IRMA were found to be highly correlated with those determined by CMIA (correlation coefficient R (2) = 0.838, p < 0.001). Serum HBsAg level and serum HBV DNA copies were found to be linearly related by both methods (R (2) = 0.067, p = 0.316 by IRMA, and R (2) = 0.101, p = 0.215 by CMIA). The diagnostic performance of the investigated IRMA method of determining HBsAg levels was found to be comparable with that of a CMIA-based method in chronic hepatitis B patients.

  4. The Thr to Met substitution of amino acid 118 in hepatitis B virus surface antigen escapes from immune-assay-based screening of blood donors.

    PubMed

    Yang, Yonglin; Nan, Yuchen; Cai, Jie; Xu, Jiling; Huang, Zuhu; Cai, Xubing

    2016-05-01

    Hepatitis B surface antigen (HBsAg) is the main diagnosis marker for hepatitis B virus (HBV) infection. In this study, a novel HBV mutant from an HBV-positive blood donor with false-negative results during HBsAg screening was identified. DNA sequencing discovered two mutations at nt 353 (A to T) and nt 349 (T to A), leading to Thr to Met and Ser to Thr substitutions at aa 118 and 117 of HBsAg, respectively. Further analysis showed that eight of ten HBsAg ELISA kits failed to detect this HBsAg mutant. A mutagenesis assay indicated that the Thr to Met substitution at aa 118 was the determinant for escape from HBsAg ELISA detection. A small-scale screening of blood donors identified two individuals infected by this unique HBV mutant, suggesting a certain level of prevalence among the general population. In conclusion, our study identified the aa 118 mutation in HBV surface antigen and provided information for improvement of HBV diagnosis products.

  5. Prevalence of antibody to Hepatitis B core antigen and Hepatitis B virus DNA in HBsAg negative healthy blood donors.

    PubMed

    Karimi, Gharib; Zadsar, Maryam; Vafaei, Nasrin; Sharifi, Zohreh; FalahTafti, Mohammad

    2016-03-05

    Hepatitis B virus is one of the most important blood born viruses. Although the sensitivity of screening tests has been considerably increased, transmission may still occur due to window period or occult hepatitis B infections (OBIs). This study was aimed at evaluating the prevalence of the anti-HBc and identifying the HBV DNA in HBsAg negative blood donors. The Blood samples from 2031 HBsAg-negative blood donors were divided into three aliquots and tested for anti-HBc, anti-HBs and HBV DNA. Serologic screening including anti-HBc and anti-HBs was performed. As a confirmatory test, all positive results for anti-HBc were retested with another kit. Two positive results were considered for anti-HBc positivity. All HBsAg negative selected donations were tested by PCR assay on pooled specimens (five samples per pool), plasma samples found to be HBsAg negative but anti-HBc positive were selected for a single-unit specimen Real-Time assay. The study population had a mean age of 33.25 ± 10.09 years were mainly composed of males (94.8 %). The seroprevalance rate was 4.9 % for Anti-HBc and 31.9 % for HBsAb. The majority (58.6 %) of Anti-HBc positive cases were regular blood donors with 42-49 years being the largest age group (41.4 %). Neither individual NAT nor pooled NAT test detected any HBV DNA. However, Screening of anti-HBc Ab is proposed as a method to identify previous contact with HBV, but there is controversy in literature data regarding the cost-benefit of exclusion of positive anti-HBc Ab in blood donors. Our data does not suggest HBc-Ab test as a screening tool in the study setting.

  6. 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.

  7. 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.

  8. [Establishment of confirmatory test for HBsAb in serum of coexistence of hepatitis B surface antigen and antibodies to HBsAg].

    PubMed

    Liu, Jia; Chen, Lin; Xu, Jju; Guo, Jing-Xia; Song, Yong-Ji; Zhao, Jing; Liu, Ai-Xia; Yang, Li-Hua; Li, Bo-An; Mao, Yuan-Li

    2011-12-01

    Establish a kind of confirmation method based on ELISA, and use to verify authenticity of HBsAb + in HBsAg + HBsAb + serum, pick and get rid of the false masculine gender the result, and avoid the mistake diagnosis. Collect 60 pieces of serum whose thick degree of HBsAg at 1000 COI above tested by ECLIA as confirm serum, mixed the confirm serum of different dilution with HBsAb positive serum to screen and verify best thick degree of HBsAg. Collected 40 pieces of HBsAg + HBsAb + serum, ELISA tested the descend rate of HBsAb COI after neutralized with confirm serum in order to confirm authenticity of HBsAb + in pieces of HBsAg + HBsAb + serum. When thick degree of HBsAg is 2000 COI, the performance of neutralization to HBsAb is best. The ELISA confirmatory test is fully consistent with the ECLIA method with true positive of 37 pieces of HBsAg + HBsAb + serum while false-positive of 3 pieces of serum. The ELISA confirm method is a simple, accurate and low cost initial validation method.

  9. Hepatitis B surface antigen (HBsAg) levels in the natural history of hepatitis B virus (HBV)-infection: a European perspective.

    PubMed

    Jaroszewicz, Jerzy; Calle Serrano, Beatriz; Wursthorn, Karsten; Deterding, Katja; Schlue, Jerome; Raupach, Regina; Flisiak, Robert; Bock, C-Thomas; Manns, Michael P; Wedemeyer, Heiner; Cornberg, Markus

    2010-04-01

    The quantifiable level of HBsAg has been suggested as a predictor of treatment response in chronic hepatitis B. However, there is limited information on HBsAg levels considering the dynamic natural course of HBV-infection. This study aimed to determine HBsAg levels in the different phases of HBV-infection in European HBsAg-positive patients. 226 HBV-monoinfected patients, not undergoing antiviral therapy, were analyzed in a cross-sectional study. Patients were categorized according to the phase of HBV-infection: HBeAg(+) immune tolerance phase (IT, n=30), immune clearance phase (IC, n=48), HBeAg(-) low-replicative phase (LR, n=68), HBeAg(-) hepatitis (ENH, n=68), and acute hepatitis B (n=12). HBsAg was quantified and correlated with HBV-DNA, HBV-genotypes and clinical parameters. In addition, 30 LR-patients were followed longitudinally. HBsAg levels were higher in IT-patients and IC-patients compared to LR-patients and ENH-patients (4.96/4.37/3.09/3.87-log(10)IU/ml, p<0.001). HBsAg showed a strong correlation with HBV-DNA during acute hepatitis B (R=0.79, p<0.01). Correlation of HBsAg and HBV-DNA was weak or missing when analyzing different phases of persistent HBV-infection separately. However, associations between HBsAg and HBV-DNA were observed in patients infected with HBV-genotype D but not with HBV-genotype A. LR-patients with HBV-reactivation during follow-up (increase of HBV-DNA >2000IU/ml) showed >3-fold higher baseline HBsAg levels with a NPV of 95% for an HBsAg cut-off of 3500IU/ml. HBsAg levels show significant differences during the natural course of HBV-infection and between HBV-genotypes. These findings may have important implications for understanding the natural history of HBV-infection and for using quantitative HBsAg as a diagnostic tool, i.e. as a marker for predicting HBV-reactivation.

  10. Comparison of antigen assay and reverse transcriptase assay for detecting human immunodeficiency virus in culture.

    PubMed Central

    Feorino, P; Forrester, B; Schable, C; Warfield, D; Schochetman, G

    1987-01-01

    We compared an antigen capture assay (Abbott Laboratories, North Chicago, Ill.) with a reverse transcriptase assay to identify and quantify human immunodeficiency virus (HIV) in culture. In direct comparisons of serial dilutions of lymphadenopathy-associated virus type 1, the antigen assay was 100-fold more sensitive than the reverse transcriptase assay in detecting the virus. The antigen assay reacted strongly with 60 different HIV isolates but did not cross-react with human T-cell lymphotropic virus type I, human T-cell lymphotropic virus type II, cytomegalovirus, varicella-zoster virus, herpes simplex virus type 1, Epstein-Barr virus, adenovirus type 5, or poliovirus type 1 or with extracts from four different control human cell lines and eight different phytohemagglutinin-stimulated normal human lymphocytes. Peripheral blood lymphocyte samples from 50 individuals were evaluated by both the antigen assay and the reverse transcriptase assay. The cells from the 34 seropositive individuals were all positive by the antigen assay (range, 3 to 9 days; average time, 5.9 days) and the reverse transcriptase assay (range, 7 to 16 days; average time, 9.6 days). Cells from the 16 seronegative individuals were negative by both assays. These results indicate that the antigen assay is an important addition to the monitoring of HIV production in the lymphocytes of infected patients. PMID:2448334

  11. Negative interference in serum HBsAg ELISA from rheumatoid factors.

    PubMed

    Xu, Lei; Yu, Zhen; Fan, Wen; Wang, Xueping; Xie, Mingshui; Xu, Yiting; Hu, Lihua; Li, Yirong

    2013-01-01

    RF(Rheumatoid factor) is usually thought to cause positive interference in immunoassay. Recently, our study showed that high-concentration RFs caused negative interference as well as positive interference in serum HBsAg(Hepatitis B surface antigen) ELISA(Enzyme-linked immunosorbent assay), but it is unclear that RF causing negative interference is an anomaly produced by a certain ELISA kit or a common property of most of HBsAg ELISA kits. Serum models were made by blending HBsAg-positive sera and high- or moderate-concentration RFs sera at the ratio of 1: 9, then one-step and two-step ELISA were adopted to determine HBsAg in serum models. No matter what kind of kit used, one-step ELISA showed that HBsAg S/CO( sample/cut off) values in serum models were significantly lower than original values. Bivariate correlations tests showed decline rates of HBsAg S/CO Values were not associated to serum RF concentrations ranging from 288 to 3560 IU/mL. HBsAg converted to be negative in 69.80% serum models with original-value ranging from 1.00 to 10.00, and in 2.68% serum models with higher original-value. RF causing decline of HBsAg S/CO value provided by one-step ELISA was more obvious than that provided by two-step ELISA. It is concluded that susceptibility of all HBsAg ELISA assays to interference from RF, leading to predominantly lower and in some cases "false-negative" results, and moreover, the lower the original HBsAg S/CO Value, the higher the false-negative rate.

  12. Negative Interference in Serum HBsAg ELISA from Rheumatoid Factors

    PubMed Central

    Wang, Xueping; Xie, Mingshui; Xu, Yiting; Hu, Lihua; Li, Yirong

    2013-01-01

    Background RF(Rheumatoid factor) is usually thought to cause positive interference in immunoassay. Recently, our study showed that high-concentration RFs caused negative interference as well as positive interference in serum HBsAg(Hepatitis B surface antigen) ELISA(Enzyme-linked immunosorbent assay), but it is unclear that RF causing negative interference is an anomaly produced by a certain ELISA kit or a common property of most of HBsAg ELISA kits. Methods Serum models were made by blending HBsAg-positive sera and high- or moderate-concentration RFs sera at the ratio of 1: 9, then one-step and two-step ELISA were adopted to determine HBsAg in serum models. Results No matter what kind of kit used, one-step ELISA showed that HBsAg S/CO( sample/cut off) values in serum models were significantly lower than original values. Bivariate correlations tests showed decline rates of HBsAg S/CO Values were not associated to serum RF concentrations ranging from 288 to 3560 IU/mL. HBsAg converted to be negative in 69.80% serum models with original-value ranging from 1.00 to 10.00, and in 2.68% serum models with higher original-value. RF causing decline of HBsAg S/CO value provided by one-step ELISA was more obvious than that provided by two-step ELISA. Conclusions It is concluded that susceptibility of all HBsAg ELISA assays to interference from RF, leading to predominantly lower and in some cases "false-negative" results, and moreover, the lower the original HBsAg S/CO Value, the higher the false-negative rate. PMID:24260439

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

    PubMed

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

    2016-02-01

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

  14. Prevalence of aflatoxin induced p53 mutation at codon 249 (R249s) in hepatocellular carcinoma patients with and without hepatitis B surface antigen (HBsAg).

    PubMed

    Chittmittrapap, Salyavit; Chieochansin, Thaweesak; Chaiteerakij, Roongruedee; Treeprasertsuk, Sombat; Klaikaew, Naruemon; Tangkijvanich, Pisit; Komolmit, Piyawat; Poovorawan, Yong

    2013-01-01

    A missense mutation in exon 7 (R249S) of the p53 tumor suppressor gene is characteristic of aflatoxin B1 (AFB1) exposure. AFB1 is believed to have a synergistic effect on hepatitis virus B (HBV) carcinogenesis. However, results of studies comparing R249S prevalence among patients are conflicting. The aim of this study was to determine the prevalence of the R249S mutation in hepatocellular carcinoma (HCC) patients with or without positive HBsAg. Paraffin embedded liver tissues were obtained from 124 HCC patients who underwent liver resection and liver biopsy in King Chulalongkorn Memorial Hospital. Restriction fragment length polymorphism (RFLP) was utilized to detect the R249S mutation. Positive results were confirmed by direct sequencing. Sixty four (52%) patients were positive for HBsAg and 18 (15%) were anti-HCV positive. 12 specimens tested positive by RFLP. Ten HCC patients (8.1%) were confirmed to be R249S positive by Sanger sequencing (AGG to AGT). Out of these 10, six were HBsAg positive, and out of the remaining 4, two were anti-HCV positive. The R249S prevalence among HCC patients with positive HBsAg was 9.4% compared to 6.7% for HBsAg negative samples. Patients with the R249S mutation were younger (55±10 vs 60±13 year-old) and tended to have a more advanced Edmonson-Steiner grade of HCC, although differences did not reach statistical significance. Our study shows moderate prevalence of aflatoxin B1-related p53 mutation (R249S) in HCC with or without HBsAg. HBsAg positive status was not associated with R249S prevalence.

  15. Lack of correlation between HBsAg and HBV DNA levels in blood donors who test positive for HBsAg and anti-HBc: implications for future HBV screening policy.

    PubMed

    Kuhns, Mary C; Kleinman, Steven H; McNamara, Anne L; Rawal, Bhupat; Glynn, Simone; Busch, Michael P

    2004-09-01

    Studies showing a significant correlation between hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) deoxyribonucleic acid (DNA) levels have focused on the HBV seroconversion window period. HBsAg levels relative to HBV DNA results in 200 HBsAg-positive, anti-hepatitis B core antigen (HBc)-reactive blood donations were analyzed using quantitative polymerase chain reaction (PCR) (detection limit 400 copies/mL), two research PCR assays with increasing sensitivities (65 copies/mL and 1.3 copies/mL, respectively), and a quantitative HBsAg assay; HBsAg and HBV DNA levels were correlated with HBV serologic profiles; and the potential for replacing HBsAg screening with nucleic acid testing (NAT) was analyzed. Serologic profiles for over 90 percent of the donor samples were consistent with chronic HBV infection. Correlation between HBsAg and HBV DNA concentrations was weak (correlation coefficient = 0.33). Thirty-six percent (72/200) of donor samples had DNA levels under 400 copies per mL. Retesting of the 72 samples by more sensitive PCR assays showed that 60 out of 200 (30%) were positive by PCR with sensitivity of 65 copies per mL, whereas 6 out of 200 (3%) required PCR sensitivity of 1.3 copies per mL for positivity. Three percent (6/200) were negative by all three NAT assays. HBV DNA levels in HBsAg-positive, anti-HBc-reactive blood donations can be extremely low. About 6 percent of donations would be negative by current minipool HBV NAT methods. About 3 percent of donations would remain undetected by sensitive single-donor NAT. These results indicate caution in any consideration of dropping HBsAg screening.

  16. Prevalence of hepatitis B surface antigen (HBsAg) in a blood donor population born prior to and after implementation of universal HBV vaccination in Shenzhen, China.

    PubMed

    Wang, Zhen; Zeng, Jinfeng; Li, Tingting; Zheng, Xin; Xu, Xiaoxuan; Ye, Xianlin; Lu, Liang; Zhu, Weigang; Yang, Baocheng; Allain, Jean-Pierre; Li, Chengyao

    2016-09-20

    Neonatal hepatitis B vaccination program at birth has been implemented nationwide since 1992 in China. However, current HBV prevalence status in blood donors has not been entirely examined, which may impact HBV safety in blood donations as the vaccinees over 18 years old progressively become the majority population of blood donors. In this study, 569,145 blood donors were screened for HBsAg by rapid tests and enzyme immunoassays, among them 475,538 blood samples with negative HBsAg were further screened for HBV DNA by nucleic acid testing between 2005 and 2014 at Shenzhen blood center. An overall 2.3 % HBsAg prevalence was found in the blood donor population during the past 10 years (2.86 % in 2005, 1.76 % in 2010, and 2.79 % in 2014, respectively). HBsAg seroconversion occurred in 0.37 % of repeat-donors. When stratified by age, the prevalence of HBsAg was found significantly higher in younger donors age 18-25 years (2.73 %) than in those 26-35 years (2.13 %), 36-45 years (2.03 %) and 46-58 years (1.71 %) (P < 0.001), unexpectedly suggesting that younger donors remained at risk of chronic HBV infection. Assuming that donors aged 18-22 born before or after 1992 were non-vaccinated and vaccinated, respectively, HBsAg prevalence was higher in first-time donors born ≥1992 (3.9 %) than prior to 1992 (3.5 %, P = 0.005). The incidence of HBV infection in the 5-year period examined was significantly lower in repeat-donors born ≥1992 (0.27 %) than prior to 1992 (0.6 %, P = 0.008). The yield of HBV DNA+/HBsAg- donors was 1:3,302, including 1:4,486 occult infections and 1:43,231 window period infections. Young blood donors born after implementation of universal HBV vaccination in China presented higher prevalence of HBsAg but lower incidence of HBsAg seroconversion than older, presumed unvaccinated, donors. HBV vaccine boosting for adolescents at 15-17 years old prior to reaching blood donor age might help improve blood safety.

  17. 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.

  18. 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.

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

    PubMed

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

    2015-12-01

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

  20. 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.

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

    SciTech Connect

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

    1995-11-01

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

  2. Antigen detection based on background fluorescence quenching immunochromatographic assay.

    PubMed

    Chen, Xiangjun; Xu, Yangyang; Yu, Jinsheng; Li, Jiutong; Zhou, Xuelei; Wu, Chuanyong; Ji, Qiuliang; Ren, Yuan; Wang, Liqun; Huang, Zhengyi; Zhuang, Hanling; Piao, Long; Head, Richard; Wang, Yajie; Lou, Jiatao

    2014-09-02

    Gold immunochromatographic assay (GICA) has been around for quite a while, but it is qualitative in the vast majority of applications. A fast, simple and quantitative GICA is in call for better medicine. In the current study, we have established a novel, quantitative GICA based on fluorescence quenching and nitrocellulose membrane background signals, called background fluorescence quenching immunochromatographic assay (bFQICA). Using model analyte alpha-fetoprotein (AFP), the present study assessed the performance of bFQICA in numerous assay aspects. With serial dilutions of the international AFP standard, standard curves for the calculation of AFP concentration were successfully established. At 10 and 100ngmL(-1) of the international AFP standard, the assay variability was defined with a coefficient of variance at 10.4% and 15.2%, respectively. For samples with extended range of AFP levels, bFQICA was able to detect AFP at as low as 1ngmL(-1). Fluorescence in bFQICA strips stayed constant over months. A good correlation between the results from bFQICA and from a well-established Roche electrochemiluminescence immunoassay was observed in 27 serum samples (r=0.98, p<0.001). In conclusion, our study has demonstrated distinctive features of bFQICA over conventional GICA, including utilization of a unique fluorescence ratio between nitrocellulose membrane background and specific signals (F1/F2) to ensure accurate measurements, combined qualitative and quantitative capabilities, and exceptionally high sensitivity for detection of very low levels of antigens. All of these features could make bFQICA attractive as a model for antigen-antibody complex based GICA, and could promote bFQICA to a broad range of applications for investigation of a variety of diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed

    Tseng, Tai-Chung; Kao, Jia-Horng

    2013-01-01

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

  4. 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

  5. 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

  6. 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

  7. Development and Technical and Clinical Validation of a Quantitative Enzyme-Linked Immunosorbent Assay for the Detection of Human Antibodies to Hepatitis B Surface Antigen in Recipients of Recombinant Hepatitis B Virus Vaccine▿

    PubMed Central

    Cambron, Pierre; Jacquet, Jeanne-Marie; Hoet, Bernard; Lievens, Marc

    2009-01-01

    Pending removal from the market of a commercial assay (the AUSAB [Abbott Laboratories] enzyme immunoassay [EIA]) for the determination of antibodies to hepatitis B surface antigen (HBsAg), a new in-house quantitative enzyme-linked immunosorbent assay (ELISA) to measure antibodies against HBsAg (anti-HBs) was developed (anti-HBs in-house). Specific anti-HBs antibodies were sandwiched between the precoated HBsAg ad and ay subtypes purified from plasma from hepatitis B virus (HBV) human carriers and the recombinant HBsAg adw2 subtype tagged with horseradish peroxidase. The assay was calibrated against the 1st International Reference Preparation for anti-hepatitis B immunoglobulin (lot 1977-W1042). Analytical sensitivity and the limit of quantitation were estimated at 0.43 mIU/ml and 2.0 mIU/ml, respectively. Overall reproducibility was 11.86%, and accuracy was estimated to be 94.89%. More than 4,000 samples from seven clinical trials were tested with the anti-HBs in-house assay and compared to results generated with AUSAB EIA and AUSAB radioimmunoassay (RIA). During the technical validation, the anti-HBs in-house assay was compared to the AUSAB RIA as a reference (n = 919). Overall assessment of concordance and Deming's regression analysis were performed. The coefficient of correlation between the AUSAB RIA and anti-HBs in-house assay was 0.9815 with a slope of 0.9187. The overall agreement between anti-HBs in-house and AUSAB RIA was 97.61%, considering the clinical cutoffs at 3.3 mIU/ml and 1.0 mIU/ml for the respective assays. From a clinical perspective, seroprotection rates and anti-HBs geometric mean antibody concentrations for individual studies calculated with either the in-house assay or the reference assays were similar. Conclusions of individual studies were confirmed. The performance characteristics of the in-house assay are acceptable. There is no evidence that use of the new assay would lead to different clinical conclusions from the reference method

  8. Viral hepatitis and rapid diagnostic test based screening for HBsAg in HIV-infected patients in rural Tanzania.

    PubMed

    Franzeck, Fabian C; Ngwale, Ramadhani; Msongole, Bernadeta; Hamisi, Marian; Abdul, Omary; Henning, Lars; Letang, Emilio; Mwaigomole, Geoffrey; Battegay, Manuel; Hatz, Christoph; Tanner, Marcel

    2013-01-01

    Co-infection with hepatitis B virus (HBV) is highly prevalent in people living with HIV in Sub-Saharan Africa. Screening for HBV surface antigen (HBsAg) before initiation of combination antiretroviral therapy (cART) is recommended. However, it is not part of diagnostic routines in HIV programs in many resource-limited countries although patients could benefit from optimized antiretroviral therapy covering both infections. Screening could be facilitated by rapid diagnostic tests for HBsAg. Operating experience with these point of care devices in HIV-positive patients in Sub-Saharan Africa is largely lacking. We determined the prevalence of HBV and Hepatitis C virus (HCV) infection as well as the diagnostic accuracy of the rapid test device Determine HBsAg in an HIV cohort in rural Tanzania. Prospectively collected blood samples from adult, HIV-1 positive and antiretroviral treatment-naïve patients in the Kilombero and Ulanga antiretroviral cohort (KIULARCO) in rural Tanzania were analyzed at the point of care with Determine HBsAg, a reference HBsAg EIA and an anti-HCV EIA. Samples of 272 patients were included. Median age was 38 years (interquartile range [IQR] 32-47), 169/272 (63%) subjects were females and median CD4+ count was 250 cells/µL (IQR 97-439). HBsAg was detected in 25/272 (9.2%, 95% confidence interval [CI] 6.2-13.0%) subjects. Of these, 7/25 (28%) were positive for HBeAg. Sensitivity of Determine HBsAg was rated at 96% (95% CI 82.8-99.6%) and specificity at 100% (95% CI, 98.9-100%). Antibodies to HCV (anti-HCV) were found in 10/272 (3.7%, 95% CI 2.0-6.4%) of patients. This study reports a high prevalence of HBV in HIV-positive patients in a rural Tanzanian setting. The rapid diagnostic test Determine HBsAg is an accurate assay for screening for HBsAg in HIV-1 infected patients at the point of care and may further help to guide cART in Sub-Saharan Africa.

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

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... is a device that employs antibodies for the detection of specific malaria parasite antigens... with malaria infection. The detection of these antigens aids in the clinical laboratory diagnosis of malaria caused by the four malaria species capable of infecting humans: Plasmodium falciparum,...

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... is a device that employs antibodies for the detection of specific malaria parasite antigens... with malaria infection. The detection of these antigens aids in the clinical laboratory diagnosis of malaria caused by the four malaria species capable of infecting humans: Plasmodium falciparum,...

  11. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... is a device that employs antibodies for the detection of specific malaria parasite antigens... with malaria infection. The detection of these antigens aids in the clinical laboratory diagnosis of malaria caused by the four malaria species capable of infecting humans: Plasmodium falciparum,...

  12. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... is a device that employs antibodies for the detection of specific malaria parasite antigens... with malaria infection. The detection of these antigens aids in the clinical laboratory diagnosis of malaria caused by the four malaria species capable of infecting humans: Plasmodium falciparum,...

  13. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... is a device that employs antibodies for the detection of specific malaria parasite antigens... with malaria infection. The detection of these antigens aids in the clinical laboratory diagnosis of malaria caused by the four malaria species capable of infecting humans: Plasmodium falciparum,...

  14. 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.

  15. 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.

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

    PubMed Central

    Jitmuang, Anupop; Panackal, Anil A.; Williamson, Peter R.; Bennett, John E.; Dekker, John P.

    2015-01-01

    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. PMID:26607986

  17. Enzyme-linked immunosorbent assay for Salmonella serology using lipopolysaccharide antigen.

    PubMed

    Smith, B P; Dilling, G W; House, J K; Konrad, H; Moore, N

    1995-10-01

    Enzyme-linked immunosorbent assay (ELISA) using Salmonella lipopolysaccharide (LPS) to measure specific IgG titers in cattle has proven useful. Serology can be used to assess vaccine responses and infection rates, to detect carriers, and to aid in epidemiologic studies. The objective of this study was to assess cross-reactions using sera from cattle vaccinated with different Salmonella serogroups. ELISA plates using lipopolysaccharide from serogroup B, C1, C3, D1 or E1 as the plate antigens were tested. LPS was extracted from Salmonella typhimurium (Serogroup B; somatic antigens 01, 4, 12), S. montevideo (C1; 06, 7), S. kentucky (C3; 08, 20), S. dublin (D1; 01, 9, 12) and S. anatum (E1; 03, 10) using the Westphal method. Fifteen cows were found to be seronegative for all 5 of these serogroup antigens. Each cow was then vaccinated 3 times at 2-week intervals with a killed Salmonella bacterin. The 15 different serotypes used for vaccination were chosen to represent a wide array of Salmonella serogroups with a wide array of somatic "O" antigens expressed, including somatic antigens 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 19, 20, 22, 23, 25, and 27. With each antigen tested, the highest ELISA titers were seen with sera from cattle vaccinated with homologous O antigens, indicating that reactions were highly O antigen-specific. Some cross reactions between subgroups sharing one O factor antigen were found; these titers were lower than those found with homologous serogroups sharing 2 or more antigens. Only serum from the cow vaccinated from S. anatum (group E; antigens 03, 10) cross-reacted at a low titer with group C1 (O somatic antigens 6, 7) and D1 (O somatic antigens 1, 9, 12) plate antigens, with which no somatic antigens were shared. We conclude from these results that Salmonella serology using LPS antigens is highly O antigen-specific and predictable.

  18. The single radial immunodiffusion assay highlights small antigenic differences among influenza virus hemagglutinins.

    PubMed Central

    Rodda, S J; Gallichio, H A; Hampson, A W

    1981-01-01

    The results of single radial immunodiffusion assays of influenza virus hemagglutinin were found to be greatly altered by small antigenic differences between test and reference strains. When such differences were present, the precise specificity of the antiserum used had a critical effect on the measured hemagglutinin antigen content obtained. PMID:6171580

  19. 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.

  20. 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

  1. Development of an enzyme-linked immunosorbent assay for studying Vibrio cholerae cell surface antigens.

    PubMed Central

    Cryz, S J; Fürer, E; Germanier, R

    1982-01-01

    An enzyme-linked immunosorbent assay for the measurement of antibodies directed against cell surface antigens of Vibrio cholerae (CSA ELISA) was developed. NaN3-killed whole cells of V. cholerae, adsorbed to polystyrene tubes, were used as immobilized antigens. The assay was capable of detecting antibodies directed against lipopolysaccharide and non-lipopolysaccharide surface antigens. In addition, the CSA ELISA was capable of detecting non-vibriocidal antibody. An antiserum raised in rabbits by immunization with live V. cholerae 1418 (Ogawa, El Tor) was capable of reacting with various heterologous strains of V. cholerae used as immobilized antigens. Therefore, common antigens shared by V. cholerae strains could be detected by using the CSA ELISA. PMID:7107860

  2. 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

  3. 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.

  4. 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

  5. Optimization of a micro-neutralisation assay and its application in antigenic characterisation of influenza viruses.

    PubMed

    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

    2015-06-13

    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, additional to evolution related to escape from host immunity, variants emerging as a result of propagation in different cell substrates can complicate 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. A 96-well-plate plaque reduction MN assay based on measurement of the Infected Cell Population (ICP) using a simple imaging technique. 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. 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. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  6. 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

  7. Enzyme immunosorbent assay for Ebola virus antigens in tissues of infected primates.

    PubMed

    Ksiazek, T G; Rollin, P E; Jahrling, P B; Johnson, E; Dalgard, D W; Peters, C J

    1992-04-01

    A sandwich enzyme immunosorbent assay (EIA) using a mixture of mouse monoclonal antibodies for antigen capture and polyclonal hyperimmune rabbit anti-Ebola virus serum for antigen detection was developed and evaluated on the tissues of monkeys naturally or experimentally infected with strains of Ebola viruses. When compared with virus isolation, the antigen detection EIA was both sensitive and specific: 44 of 45 (97.7%) liver homogenates and 38 of 41 (92.7%) spleen homogenates that were culture positive and tested by both techniques were positive for viral antigen, while 85 of 87 (97.7%) culture-negative liver homogenates and 66 of 66 culture-negative spleen homogenates were found to be antigen negative. The assay, initially developed to detect antigens of prototype African strains of Ebola virus, reliably detected related strains of Ebola virus found during two recent outbreaks of Ebola virus infection among imported, quarantined Macaca fascicularis monkeys in the United States. The assay allows economical and rapid testing of large numbers of tissue specimens. Antigen was found in homogenates of spleen and liver and in serum.

  8. Evaluation of Nonstructural 1 Antigen Assays for the Diagnosis and Surveillance of Dengue in Singapore

    PubMed Central

    Pok, Kwoon-Yong; Lai, Yee-Ling; Sng, Joshua

    2010-01-01

    Abstract Early and accurate diagnosis of dengue is imperative for disease surveillance, which helps in the control of dengue in endemic countries. In this study, we evaluated the performance of three commercially available dengue nonstructural 1 (NS1) antigen assays (Bio-Rad Platelia™ Dengue NS1 Antigen ELISA, PanBio Dengue Early ELISA, and Bio-Rad Dengue NS1 Antigen Strip test) and compared them with reverse-transcription polymerase chain reaction (RT-PCR) and other commercially available serological assays for the diagnosis of dengue. The analysis showed RT-PCR to be the most sensitive and specific (100%) diagnostic method during the first 3 days of fever. The overall sensitivity of dengue NS1 antigen assays within the same period was 81.7%, indicating their potential role as a cost-effective and convenient alternative method to RT-PCR for the diagnosis of dengue fever in a primary healthcare setting. However, reduced sensitivity in detecting secondary dengue infections was one of the drawbacks of dengue NS1 antigen assays. Nonetheless, it remains a useful assay for the early detection of dengue and hence could play an important role in routine surveillance efforts to control dengue outbreaks in Singapore. PMID:20426686

  9. Western blot assay for quantitative and qualitative antigen detection in vaccine development.

    PubMed

    Kumar, Sanjai; Zheng, Hong; Mahajan, Babita; Kozakai, Yukiko; Morin, Merribeth; Locke, Emily

    2014-05-01

    Immunological methods for quantitative measurement, antigenic characterization, and monitoring the stability of active immunogenic component(s) are a critical need in the vaccine development process. This unit describes an enhanced chemiluminescence-based western blot for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP), a major malaria candidate vaccine antigen. The most salient features of this assay are its high sensitivity and reproducibility; it can reliably detect ∼5 to 10 pg PfCSP expressed on native parasites or recombinantly expressed in Escherichia coli. Although described for a specific vaccine antigen, this assay should be applicable for any antigen-antibody combination for which relevant detection reagents are available. Detailed stepwise experimental procedures and methods for data acquisition and analysis are described. Copyright © 2014 John Wiley & Sons, Inc.

  10. 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.

  11. 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.

  12. Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

    PubMed

    Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Leitolis, Amanda; Crestani, Sandra; Foti, Leonardo; de Souza, Wayner Vieira; Gomes, Yara de Miranda; Krieger, Marco Aurélio

    2017-10-01

    Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi-specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti-T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania, a pathogen with high similarity to T. cruzi, showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD. Copyright © 2017 American Society for Microbiology.

  13. Screening putative antigens as stimulators in the Mycobacterium bovis interferon-gamma release assay for cattle.

    PubMed

    Meng, Chuang; Wan, Ting; Xu, Zhengzhong; Liu, Yan; Shan, Fa; Sun, Lin; Yin, Yuelan; Chen, Xiang; Jiao, Xinan

    2015-11-15

    Bovine tuberculosis (BTB) represents not only a significant economic concern, but also an important public health problem. Currently, interferon-gamma (IFN-γ) release assays (IGRAs) are widely used as an adjunct to the tuberculin test (TST) for the diagnosis of BTB. A great number of international studies have demonstrated that the sensitivity of the IFN-γ assay, which uses purified protein derivatives (PPDs) as diagnostic reagents, is superior to that of the TST. However, there are concerns about its specificity, largely because of the cross reactivity of common antigens shared by pathogenic and non-pathogenic mycobacterial species. The use of pathogen-specific antigens theoretically offers the most effective way to improve the specificity of IGRAs. In this study, we evaluated the potential utility of 13 purified recombinant putative antigens, which are highly specific to the Mycobacterium tuberculosis complex, as diagnostic reagents in IGRAs. A CFP-10-ESAT-6 fusion protein (abbreviated CE) displayed the greatest potential, whereas four region of difference 2 (RD2) antigens, especially Rv1985c were identified as potential candidate antigens, and can be included in an IGRA cocktail, together with CE as stimulators in the IFN-γ release assay for the diagnosis of BTB. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Detecting West Nile virus in owls and raptors by an antigen-capture assay.

    PubMed

    Gancz, Ady Y; Campbell, Douglas G; Barker, Ian K; Lindsay, Robbin; Hunter, Bruce

    2004-12-01

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

  15. Detection of an acute asymptomatic HBsAg negative hepatitis B virus infection in a blood donor by HBV DNA testing.

    PubMed

    Weber, Bernard; Mühlbacher, Annelies; Melchior, Walter

    2005-01-01

    The issue of HBV DNA screening on blood donations is controversially discussed since the economic impact of post-transfusion hepatitis B is expected to be relatively low. We report on a case of HBsAg negative unapparent acute HBV infection, which was detected by HBV NAT testing on 96-member maxi-pools with a commercially available NAT assay, which has a detection threshold of 3 IU/mL of plasma. The presence of an HBsAg escape mutant could be excluded by sequencing the amplified DNA. Follow-up testing showed the presence of an acute HBV infection (anti-HBc-IgM positive) and finally anti-HBs seroconversion. Although the reduction of the diagnostic window with NAT screening on maxi-pools may be relatively low, it may help to improve the residual risk of blood donation, especially in asymptomatic HBV infection, where the HBsAg positive period may be very short and low levels of circulating surface antigen are present. It would also permit to detect occult HBV infection in chronic carriers who are HBsAg negative. Since the viral load in chronic isolated anti-HBc positive carriers is low, there is a potential risk for failure of HBV DNA detection with pool-PCR in blood donors. Anti-HBc screening would reduce the residual risk.

  16. ViroSpot microneutralization assay for antigenic characterization of human influenza viruses.

    PubMed

    van Baalen, Carel A; Jeeninga, Rienk E; Penders, Germaine H W M; van Gent, Brenda; van Beek, Ruud; Koopmans, Marion P G; Rimmelzwaan, Guus F

    2017-01-03

    The hemagglutination inhibition (HI) assay has been used for the antigenic characterization of influenza viruses for decades. However, the majority of recent seasonal influenza A viruses of the H3N2 subtype has lost the capacity to agglutinate erythrocytes of various species. The hemagglutination (HA) activity of other A(H3N2) strains is generally sensitive to the action of the neuraminidase inhibitor oseltamivir, which indicates that the neuraminidase and not the hemagglutinin is responsible for the HA activity. These findings complicate the antigenic characterization and selection of A(H3N2) vaccine strains, calling for alternative antigenic characterization assays. Here we describe the development and use of the ViroSpot microneutralization (MN) assay as a reliable and robust alternative for the HI assay. Serum neutralization of influenza A(H3N2) reference virus strains and epidemic isolates was determined by automated readout of immunostained cell monolayers, in a format designed to minimize the influence of infectious virus doses on serum neutralization titers. Neutralization of infection was largely independent from rates of viral replication and cell-to-cell transmission, facilitating the comparison of different virus isolates. Other advantages of the ViroSpot MN assay include its relative insensitivity to variation in test dose of infectious virus, automated capture and analyses of residual infection patterns, and compatibility with standardized large scale analyses. Using this assay, a number of epidemic influenza A(H3N2) strains that failed to agglutinate erythrocytes, were readily characterized antigenically. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

    PubMed

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

    2011-10-01

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

  18. 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.

  19. Identification of immunodominant antigens in canine leptospirosis by Multi-Antigen Print ImmunoAssay (MAPIA).

    PubMed

    Thomé, Sabrina; Lessa-Aquino, Carolina; Ko, Albert Icksang; Lilenbaum, Walter; Medeiros, Marco Alberto

    2014-12-03

    The microscopic agglutination test (MAT), the standard method for serological diagnosis of leptospirosis, may present limitations regarding its sensitivity. Current studies suggest that Leptospira immunoglobulin-like (Lig) proteins and LipL32 are of particular interest as serodiagnostic markers since they are present only in pathogenic species of the Leptospira genus. The purpose of this study was to identify leptospiral immunodominant proteins that are recognized by canine sera from diseased dogs. A total of 109 dogs were studied, including seroreactive dogs (MAT ≥800) and dogs with no seroreactivity detectable by MAT. Eight recombinant fragments (31-70 kDa) of pathogenic Leptospira were tested for their use as diagnostic markers for canine leptospirosis using the Multi-antigen Print Immunoassay (MAPIA) platform: LigB [582-947aa] from L. interrogans serovar Pomona, L. interrogans serovar Copenhageni and L. kirschneri serovar Gryppotyphosa, LigB [131-649aa] from L. interrogans serovar Copenhageni, L. interrogans serovar Canicola and L. kirschneri serovar Gryppotyphosa, LigA [625-1224aa] L. interrogans serovar Copenhageni and LipL32 from L. interrogans serovar Copenhageni. The data were analyzed and ROC curves were generated. Altogether, LigB [131-649aa] L. interrogans Canicola, LigB [131-649aa] L. kirschneri Gryppotyphosa and LipL32 L. interrogans Copenhageni showed best accuracy (AUC = 0.826 to 0.869), with 70% specificity and sensitivity ranging from 89% to 95%. These results reinforce their potential as diagnostic candidates for the development of new methods for the serological diagnosis of canine leptospirosis.

  20. 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

  1. 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.

  2. 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

  3. HBsAg spontaneous seroclearance in a cohort of HBeAg-seronegative patients with chronic hepatitis B virus infection.

    PubMed

    Han, Zhen-Ge; Qie, Zhong-Hong; Qiao, Wei-Zhen

    2016-01-01

    Loss of hepatitis B surface antigen (HBsAg) is considered to reflect the resolution of a hepatitis B virus (HBV) infection. Patient characteristics and various seromarkers were evaluated to characterize factors predicting spontaneous HBsAg loss in a cohort of HBeAg-seronegative patients with presumed chronic HBV infection. Relationships between seromarkers and HBsAg loss were assessed annually and after 6 years using binary logistic regression. Among the 634 participants, 117 (18.45%) cleared HBsAg after 6 years, with a 3.08% annual seroclearance rate. Baseline HBsAg levels and platelet (PLT) counts were predictors of HBsAg seroclearance. The HBsAg level predicted HBsAg seroclearance better than the PLT count (area under the receiver operating characteristic curve (AUROC): HBsAg, 0.965 (95%CI, 0.947-0.980) versus PLT count, 0.617 (95%CI, 0.561-0.669); P < 0.001). A cutoff HBsAg level of 10 IU/ml at baseline predicted spontaneous HBsAg seroclearance at 6 years with a diagnostic accuracy of 93.4%, a sensitivity of 87.2%, a specificity of 94.8%, a positive predictive value of 79.1%, and a negative predictive value of 97.0%. HBsAg seroclearance may occur more commonly than expected. A serum HBsAg level <10 IU/ml and PLT count were accurate predictors of clearance. © 2015 The Authors. Journal of Medical Virology Published by Wiley Periodicals, Inc.

  4. 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.

  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. 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.

  7. 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.

  8. 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.

  9. Broad-spectrum enzyme-linked immunosorbent assay for detection of Legionella soluble antigens.

    PubMed Central

    Tang, P W; Toma, S

    1986-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed which detected soluble antigens from culture extracts of Legionella pneumophila serogroups 1 to 8, L. micdadei, L. bozemanii serogroups 1 and 2, L. dumoffii, L. gormanii, L. longbeachae serogroups 1 and 2, L. wadsworthii, L. oakridgensis, L. anisa, L. feeleii serogroup 1, and L. jordanis. The assay was approximately 10-fold more sensitive for the eight L. pneumophila serogroups than for the other Legionella species tested. The ELISA detected Legionella antigens in the urine specimens of 25 of 35 patients with L. pneumophila serogroup 1, 3, 4, 6, and 8; L. micdadei; and L. longbeachae serogroup 1 infections. None of the 334 urine specimens from patients with either non-Legionella pneumonia or urinary tract infections was positive. For 10 patients from whom sequential urine specimens were available, Legionella antigens were not detectable from 7 to 19 days after laboratory diagnosis. Test sensitivity was not affected by heavy bacterial contamination. This ELISA offers the detection of a broad spectrum of Legionella antigens by a single test. PMID:3771744

  10. 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.

  11. 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.

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

    PubMed

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

    1995-01-01

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

  13. A simple and inexpensive point-of-care test for hepatitis B surface antigen detection: serological and molecular evaluation.

    PubMed

    Gish, R G; Gutierrez, J A; Navarro-Cazarez, N; Giang, K; Adler, D; Tran, B; Locarnini, S; Hammond, R; Bowden, S

    2014-12-01

    Early identification of chronic hepatitis B is important for optimal disease management and prevention of transmission. Cost and lack of access to commercial hepatitis B surface antigen (HBsAg) immunoassays can compromise the effectiveness of HBV screening in resource-limited settings and among marginalized populations. High-quality point-of-care (POC) testing may improve HBV diagnosis in these situations. Currently available POC HBsAg assays are often limited in sensitivity. We evaluated the NanoSign(®) HBs POC chromatographic immunoassay for its ability to detect HBsAg of different genotypes and with substitutions in the 'a' determinant. Thirty-seven serum samples from patients with HBV infection, covering HBV genotypes A-G, were assessed for HBsAg titre with the Roche Elecsys HBsAg II quantification assay and with the POC assay. The POC assay reliably detected HBsAg at a concentration of at least 50 IU/mL for all genotypes, and at lower concentrations for some genotypes. Eight samples with substitutions in the HBV 'a' determinant were reliably detected after a 1/100 dilution. The POC strips were used to screen serum samples from 297 individuals at risk for HBV in local clinical settings (health fairs and outreach events) in parallel with commercial laboratory HBsAg testing (Quest Diagnostics EIA). POC testing was 73.7% sensitive and 97.8% specific for detection of HBsAg. Although the POC test demonstrated high sensitivity over a range of genotypes, false negatives were frequent in a clinical setting. Nevertheless, the POC assay offers advantages for testing in both developed and resource-limited countries due to its low cost (0.50$) and immediately available results. © 2014 John Wiley & Sons Ltd.

  14. 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.

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed

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

    2011-05-01

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

  17. Rapid antigen-capture assay to detect West Nile virus in dead corvids.

    PubMed

    Lindsay, Robbin; Barker, Ian k; Nayar, Gopi; Drebot, Michael; Calvin, Sharon; Scammell, Cherie; Sachvie, Cheril; Fleur, Tracy Scammell-La Fleur; Dibernardo, Antonia; Andonova, Maya; Artsob, Harvey

    2003-11-01

    The utility of the VecTest antigen-capture assay to detect West Nile virus (WNV) in field-collected dead corvids was evaluated in Manitoba and Ontario, Canada, in 2001 and 2002. Swabs were taken from the oropharynx, cloaca, or both of 109 American Crows, 31 Blue Jays, 6 Common Ravens, and 4 Black-billed Magpies from Manitoba, and 255 American Crows and 28 Blue Jays from Ontario. The sensitivity and specificity of the antigen-capture assay were greatest for samples from American Crows; oropharyngeal swabs were more sensitive than cloacal swabs, and interlaboratory variation in the results was minimal. The sensitivity and specificity of the VecTest using oropharyngeal swabs from crows were 83.9% and 93.6%, respectively, for Manitoba samples and 83.3% and 95.8%, respectively, for Ontario birds. The VecTest antigen-capture assay on oropharyngeal secretions from crows is a reliable and rapid diagnostic test that appears suitable for incorporation into a WNV surveillance program.

  18. Rapid Antigen-Capture Assay To Detect West Nile Virus in Dead Corvids

    PubMed Central

    Barker, Ian; Nayar, Gopi; Drebot, Michael; Calvin, Sharon; Scammell, Cherie; Sachvie, Cheryl; La Fleur, Tracy Scammell; Dibernardo, Antonia; Andonova, Maya; Artsob, Harvey

    2003-01-01

    The utility of the VecTest antigen-capture assay to detect West Nile virus (WNV) in field-collected dead corvids was evaluated in Manitoba and Ontario, Canada, in 2001 and 2002. Swabs were taken from the oropharynx, cloaca, or both of 109 American Crows, 31 Blue Jays, 6 Common Ravens, and 4 Black-billed Magpies from Manitoba, and 255 American Crows and 28 Blue Jays from Ontario. The sensitivity and specificity of the antigen-capture assay were greatest for samples from American Crows; oropharyngeal swabs were more sensitive than cloacal swabs, and interlaboratory variation in the results was minimal. The sensitivity and specificity of the VecTest using oropharyngeal swabs from crows were 83.9% and 93.6%, respectively, for Manitoba samples and 83.3% and 95.8%, respectively, for Ontario birds. The VecTest antigen-capture assay on oropharyngeal secretions from crows is a reliable and rapid diagnostic test that appears suitable for incorporation into a WNV surveillance program. PMID:14718083

  19. 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

  20. 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.

  1. Rapid diagnosis of Mycoplasma pneumoniae in children with pneumonia by an immuno-chromatographic antigen assay

    PubMed Central

    Li, Wei; Liu, Yujie; Zhao, Yun; Tao, Ran; Li, Yonggang; Shang, Shiqiang

    2015-01-01

    Mycoplasma pneumoniae is a particularly important pathogen that causes community acquired pneumonia in children. In this study, a rapid test was developed to diagnose M. pneumoniae by using a colloidal gold-based immuno-chromatographic assay which targets a region of the P1 gene. 302 specimens were analyzed by the colloidal gold assay in parallel with real-time PCR. Interestingly, the colloidal gold assay allowed M. pneumoniae identification, with a detection limit of 1 × 103 copies/ml. 76 samples were found to be positive in both real-time PCR and the colloidal gold assay; two specimens positive in real-time PCR were negative in the rapid colloidal gold assay. The specificity and sensitivity of the colloidal gold assay were 100% and 97.4%, respectively. These findings indicate that the newly developed immuno-chromatographic antigen assay is a rapid, sensitive and specific method for identifying M. pneumoniae, with potential clinical application in the early diagnosis of Mycoplasma pneumoniae infection. PMID:26486047

  2. 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.

  3. Hepatitis B surface antigen levels during natural history of chronic hepatitis B: a Chinese perspective study.

    PubMed

    Zeng, Lin-Yan; Lian, Jiang-Shan; Chen, Jian-Yang; Jia, Hong-Yu; Zhang, Yi-Min; Xiang, Dai-Rong; Yu, Liang; Hu, Jian-Hua; Lu, Ying-Feng; Zheng, Lin; Li, Lan-Juan; Yang, Yi-Da

    2014-07-21

    To determine the baseline hepatitis B surface antigen (HBsAg) levels during the different phases of chronic hepatitis B (CHB) patients in China. Six hundred and twenty-three hepatitis B virus or un-infected patients not receiving antiviral therapy were analyzed in a cross-sectional study. The CHB patients were classified into five phases: immune-tolerant (IT, n = 108), immune-clearance (IC, n = 161), hepatitis B e antigen negative hepatitis (ENH, n = 149), low-replicative (LR, n = 135), and liver cirrhosis (LC, n = 70). HBsAg was quantified (Abbott ARCHITECT assay) and correlated with hepatitis B virus (HBV) DNA, and serum alanine aminotransferase/aspartate aminotransferase (ALT/AST) in each phase of CHB was also determined. Median HBsAg titers were different in each phase of CHB (P < 0.001): IT (4.85 log10 IU/mL), IC (4.36 log10 IU/mL), ENH (2.95 log10 IU/mL), LR (3.18 log10 IU/mL) and LC (2.69 log10 IU/mL). HBsAg titers were highest in the IT phase and lowest in the LC phase. Serum HBsAg titers showed a strong correlation with HBV viral load in the IC phase (r = 0.683, P < 0.001). No correlation between serum HBsAg level and ALT/AST was observed. The mean baseline HBsAg levels differ significantly during the five phases of CHB, providing evidence on the natural history of HBV infection. HBsAg quantification may predict the effects of immune-modulator or oral nucleos(t)ide analogue therapy.

  4. Comparison of the reactivities of baculovirus-expressed recombinant Norwalk virus capsid antigen with those of the native Norwalk virus antigen in serologic assays and some epidemiologic observations.

    PubMed Central

    Green, K Y; Lew, J F; Jiang, X; Kapikian, A Z; Estes, M K

    1993-01-01

    Since the discovery of the Norwalk virus (NV) by immune electron microscopy (IEM) in 1972, serologic studies with this virus have relied on particle-positive fecal material from infected volunteers as the source of antigen because it has not been possible to propagate this virus in cell culture. However, the recent cloning of the NV (strain 8FIIa) genome and expression of the capsid protein in a baculovirus system to form "virus-like particles" has provided a consistent source of antigen (designated rNV). The purpose of the present study was to compare the antigenicities of these rNV particles with those of native NV antigen derived from human fecal material by using well-characterized sera obtained from earlier studies. In IEM studies, the rNV antigen reacted with NV-specific antibodies in a manner similar to that observed previously when particle-positive fecal material was used as antigen. In addition, a direct enzyme-linked immunosorbent assay, in which the rNV antigen was used as antigen, proved efficient and specific for the detection of serologic responses to NV compared with the previously established techniques of IEM and blocking antibody immunoassays in which particle-positive fecal material was used as the antigen. The availability of an unlimited source of antigen will enable serologic studies that will greatly increase our understanding of the epidemiology of NV and its role in human enteric illness. Images PMID:8396590

  5. 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.

  6. 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)

  7. 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)

  8. Herpes simplex virus type-selective enzyme-linked immunosorbent assay with Helix pomatia lectin-purified antigens.

    PubMed Central

    Svennerholm, B; Olofsson, S; Jeansson, S; Vahlne, A; Lycke, E

    1984-01-01

    Helix pomatia lectin-purified antigens with specific reactivity to herpes simplex virus type 1 (HSV-1) and HSV-2 antibodies in human sera were used in an enzyme-linked immunosorbent assay. The type specificity of the antigens was assessed by double immunodiffusion precipitation in gel against rabbit HSV-1 and HSV-2 hyperimmune sera, and by enzyme-linked immunosorbent assay with human reference sera containing antibodies to either type of HSV. Fifty-two sera from patients with documented infection with either HSV-1 or HSV-2 were assayed for HSV type-specific immunoglobulin G antibodies. The reactivity of the sera against lectin-purified antigens correlated completely with the results of virus typing. We conclude that HSV type-specific immunoglobulin G antibodies can easily be measured by enzyme-linked immunosorbent assay with the use of Helix pomatia lectin-purified HSV-1 and HSV-2 antigens. Images PMID:6321548

  9. 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.

  10. Antigenic Variation in Bacteroides forsythus Detected by a Checkerboard Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sims, Tom J.; Mancl, Lloyd A.; Braham, Pamela H.; Page, Roy C.

    1998-01-01

    Evidence indicating that multiple serotypes of Bacteroides forsythus participate in rapidly progressing periodontal infections has not been reported previously. Our aim was to develop an assay for detecting subsets of B. forsythus clinical isolates which differ in serogroup membership and subsets of patients with immunoglobulin G (IgG) responses which differ in serogroup recognition. A checkerboard enzyme-linked immunosorbent assay (ELISA) was used to assess variation in the IgG binding profiles of 22 clinical isolates in sera from 28 patients with early-onset rapidly progressive periodontitis. To accommodate the maximum number of isolates and sera in a given assay run, a multiplate assay grid with standard 96-well microtest plates was established. Single dilutions of individual sera were placed in rows crossing columns of isolate-coated wells, and antigen-specific IgG immobilized in the wells was measured as ELISA absorbance. Pooled sera and isolates were assayed in parallel to serve as negative controls for variation in IgG binding profiles. Correlation and hierarchical cluster analysis of the absorbance data matrix showed that the isolates could be sorted into at least four clusters based on variations in their IgG binding profiles across different sera. Furthermore, at least two patient clusters were defined by variations in their serum IgG antigen recognition profiles across different isolates. We conclude that multiple serogroups of B. forsythus exist and that different serogroups are dominant in the antibody response of different patients. The method applied here could be used to serologically classify clinical isolates of other species which evoke a serum antibody response in patients. PMID:9729543

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

    PubMed

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

    2009-09-01

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

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

    PubMed Central

    Yang, Ung-Suk; Liu, Bang-Hyun

    1986-01-01

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

  13. [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.

  14. HBsAg mutations related to occult hepatitis B virus infection in HIV-positive patients result in a reduced secretion and conformational changes of HBsAg.

    PubMed

    Sadeghi, Ahmadreza; Shirvani-Dastgerdi, Elham; Tacke, Frank; Yagmur, Eray; Poortahmasebi, Vahdat; Poorebrahim, Mansour; Mohraz, Minoo; Hajabdolbaghi, Mahboobeh; Rasoolinejad, Mehrnaz; Abbasian, Ladan; Jafari, Rezvaneh; Fakhari, Zahra; Norouzi, Mehdi; Ebrahimian, Arefeh; Geravand, Babak; Alavian, Seyed Moayed; Jazayeri, Seyed Mohammad

    2017-02-01

    Occult hepatitis B infection (OBI) is a frequent finding in human immunodeficiency virus (HIV)-infected patients. While several related mutations in the hepatitis B virus (HBV) genome have been reported, their distinct impact on HBsAg synthesis is largely obscure. Thirty-one (18%) out of 172 HIV-infected patients, who were selected from HBsAg-negative patients, were positive for HBV-DNA assigned as being OBI-positive. We generated a series of expression constructs of variant HBsAg with "a" determinant amino acid substitutions including P127L, P127T, S136Y, and P127T + S136Y using site-directed mutagenesis. The expression of variant HBsAg was examined by transient transfection in hepatoma cells, followed by HBsAg immunoassay and immunofluorescence stained with specific anti-HBs antibodies. The potential impact of amino acid substitutions at different positions for conformational changes in the HBsAg was investigated using bioinformatics. All variants comprising either single or combined mutations resulted in significantly reduced HBsAg detection in supernatants and in cell lysates of hepatoma cells transfected with the constructs. Moreover, intracellular immunofluorescence staining of cytoblocks showed perinuclear and cytoplasmic fluorescence of HBsAg constructs with significantly diminished fluorescent intensity in comparison to the wild type. Altered protein conformations by predictive models, indicating an impaired detection by the host's immune response as well as by commercial antibody-based test assays. Mutations in the "a" determinant region of HBV as often found in OBI remarkably impair the detection of HBsAg from serum and infected cells, emphasizing the relevance of alternative methods such as HBV-DNA quantification for high-risk groups like HIV-infected individuals. J. Med. Virol. 89:246-256, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. 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.

  16. The characteristics of hepatitis B surface antigen (HBsAg)-negative hepatitis B virus (HBV) infection in Chinese blood donors: a follow-up study of donors tested negative for HBsAg and reactive for simultaneous nucleic acid testing of HBV, hepatitis C virus, and human immunodeficiency virus.

    PubMed

    Guo, Zhaofu; Fu, Ping; Yin, Yijin; Wang, Funeng; Yin, Yiqing; Wang, Jingxing; Liu, Yu

    2017-03-01

    The real infection status of hepatitis B virus (HBV) of hepatitis B surface antigen (HBsAg)-negative yet nucleic acid test (NAT)-positive blood donors is difficult to clarify. Detailed follow-up study is needed for analyzing the infectivity of these blood donors. Blood donors who screened negative for HBsAg and reactive for simultaneous NAT of HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) were included in a follow-up epidemiologic questionnaire survey and contributed follow-up samples for further testing. The follow-up samples were tested repeatedly for the serologic markers and HBV DNA. The genotypes and sequence mutations of HBV infected by 11 HBV DNA-positive donors were analyzed through the amplification and sequencing of HBV S region. Of the 46 donors included in this study, 89.1% were infected with HBV (41/46), including one (2.2%) window period infection, three (6.5%) recovered infections, and 37 (80.4%) occult HBV infections (OBIs). The S region of HBV was successfully amplified and sequenced for seven donors, five infected with Genotype B (71.4%), one with Genotype C (14.3%), and one with Genotype D (14.3%). Mutations in the S region were detected in four donors (57.1%) CONCLUSIONS: This is the first detailed study with multiple follow-up testing of the HBV infection status among blood donors who were tested negative for HBsAg and reactive for simultaneous NAT of HBV, HCV, and HIV. Most of these donors were infected with HBV with very low viral load. Our findings indicate that it is important to improve the sensitivity of NAT so as to decrease the residual risk of transfusion-transmitted HBV infection. © 2017 AABB.

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

    PubMed

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

    2013-01-01

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

  18. Hepatitis C virus core antigen assay: an alternative method for hepatitis C diagnosis.

    PubMed

    Wang, Lijuan; Lv, Hong; Zhang, Guojun

    2017-03-01

    Background The study aimed to evaluate a fully automated chemiluminescent immunoassay and compared it with a quantitative RNA assay and anti-HCV assay to verify the utility of this automated Ag assay as an alternative method for hepatitis C diagnosis. Methods A total of 229 serum samples previously tested for anti-HCV concentrations by the Architect Anti-HCV assay, were selected for HCV RNA testing by real time RT-PCR kit (Shanghai ZJ Bio-Tec Co., Ltd) and 125 specimens were tested for HCV Ag by the Architect HCV core Antigen kit. Results The log10 HCVAg and HCV RNA concentrations were highly correlated [ r = 0.834); with HCV RNA as the comparator test, HCVAg had 100% specificity, 100% positive predictive value (PPV) and 94.8% sensitivity. We found 1 pg/mL of total HCV core Ag is equivalent to approximately 6607HCV RNA international units (IU)/mL. Receiver operator characteristic curve analysis showed that the area under the curve of HCV core Ag (0.989) was greater than HCV Ab (0.871). HCV Ag concentrations and RNA-to-Ag ratio of the groups for HCV RNA concentrations ≤10(5) and >10(5 )IU/mL were both significantly different from each other ( P < 0.05). Conclusion The Architect HCV core Ag assay may be an alternative method for hepatitis C diagnosis, performed on the same analytical platform and sample as the anti-HCV assay, shortening the diagnostic window period, demonstrating good correlation with HCV RNA assay with high specificity and positive predictive value.

  19. Homogeneous assay for detection of active Epstein-Barr nuclear antigen 1 by thrombin activity modulation.

    PubMed

    Garai-Ibabe, Gaizka; Grinyte, Ruta; Canaan, Allon; Pavlov, Valeri

    2012-07-17

    Epstein-Barr virus (EBV) has been associated with several malignancies as Burkitt's lymphoma, nasopharyngeal carcinoma, and Hodgkin's disease. In those diseases, Epstein-Barr nuclear antigen 1 (EBNA-1) is constitutively expressed. Here, we reported an innovative system to detect active EBNA-1 protein in a homogeneous assay. The system is based on the modulation of thrombin activity by a self-complementary single stranded DNA (scssDNA), which was designed and synthesized to mimic the palindromic target sites of EBNA-1 in the EBV genome. This model system showed a limit of detection of 3.75 ng mL(-1) of active EBNA-1 protein with a dynamic detection range from 3.75 to 250 ng mL(-1) with a correlation coefficient of 0.997. This new homogeneous assay for active EBNA-1 protein detection and quantification provides a very useful tool for rapid screening of EBNA-1 blockers in biomedical research.

  20. Solid-phase assays for the detection of alloantibody against human leukocyte antigens: panacea or Pandora?

    PubMed

    Roberts, T; Tumer, G; Gebel, H M; Bray, R A

    2014-10-01

    Serological assessments of antibodies directed against human leucocyte antigens (HLA) formed the basis of early histocompatibility testing (Patel & Terasaki, 1969 N Engl J Med, 280, 735). However, over the past decade, significant advances in HLA antibody detection technologies have emerged. The development and implementation of solid-phase assays has led to safer and more efficient allocation of organs by effectively distinguishing HLA from non-HLA antibodies. Although solid-phase assays are not standardized, they are widely accepted as the new 'gold standard'. However, this technology is not without its challenges. This review is intended to provide a better understanding of solid-phase HLA antibody testing and will focus on important caveats associated with this evolving technology. Examples of the limitations of the technology as well as common data misinterpretations will be shown. Both of which could pose potential harm to transplant recipients (Tait et al., Transplantation, 95, 19). © 2014 John Wiley & Sons Ltd.

  1. 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).

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

    PubMed

    Galati, Giovanni; De Vincentis, Antonio; Vespasiani-Gentilucci, Umberto; Gallo, Paolo; Vincenti, Donatella; Solmone, Maria Carmela; Dell'Unto, Chiara; Picardi, Antonio

    2014-05-17

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

  3. 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

  4. 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.

  5. 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

  6. Clinical relevance of HLA donor-specific antibodies detected by single antigen assay in kidney transplantation.

    PubMed

    Caro-Oleas, José Luis; González-Escribano, María Francisca; González-Roncero, Francisco Manuel; Acevedo-Calado, María José; Cabello-Chaves, Virginia; Gentil-Govantes, Miguel Ángel; Núñez-Roldán, Antonio

    2012-03-01

    Clinical relevance of donor-specific antibodies (DSAs) detected by a single antigen Luminex virtual crossmatch in pre-transplant serum samples from patients with a negative cytotoxicity-dependent complement crossmatch is controversial. The aim of this study was to analyse the influence of a pre-transplant positive virtual crossmatch in the outcome of kidney transplantation. A total of 892 patients who received a graft from deceased donors after a negative cytotoxicity crossmatch were included. Presence of anti-human leucocyte antigen (HLA) antibodies was investigated using a Luminex screening assay and anti-HLA specificities were assigned performing a Luminex single antigen assay. Graft survival was significantly worse among patients with anti-HLA DSA compared to both patients with anti-HLA with no DSA (P = 0.001) and patients without HLA antibodies (P < 0.001) using a log-rank test. No graft survival differences between anti-HLA with no DSA and no HLA antibodies patient groups were observed (P = 0.595). Influence of both anti-Class I and anti-Class II DSA was detected (P < 0.0001 in both cases). When the fluorescence values were stratified in four groups, no significant differences in graft survival were observed among the groups with fluorescence levels >1500 (global P > 0.05). The presence of preformed HLA DSA in transplanted patients with a negative cytotoxicity crossmatch is associated with a lower allograft survival. The detection of anti-HLA with no DSA has no influence in the graft outcome. Finally, there were no demonstrable effects of mean fluorescence intensity (MFI) values >1500 on graft survival.

  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. 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.

  9. 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.

  10. 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.

  11. Spontaneous HBsAg loss in Korean patients: relevance of viral genotypes, S gene mutations, and covalently closed circular DNA copy numbers

    PubMed Central

    Chang, Hye-Young; Park, Jun Yong; Park, Eun-Sook; Park, Yong Kwang; Han, Kwang-Hyub

    2014-01-01

    Background/Aims Occult HBV infection can persist following HBsAg loss and be transmitted, but the virological features are not well defined. Methods Here we investigated 25 Korean patients who lost HBsAg during follow up, either spontaneously or subsequent to therapy. Results Whereas subtype adr (genotype C) was found in 96% of HBsAg positive patients, 75 % of patients who lost HBsAg spontaneously were seemed to be infected with the ayw subtype with sequence similar to genotype D. Mutations in the major hydrophilic region (MHR) of HBsAg were found in 7 patients who lost HBsAg spontaneously. The mutations include T123S, M125I/N, C139R, D144E, V177A, L192F, and W196L, some of which have not been reported before. Functional analysis via transfection experiments indicate that the C139R and D144E mutations drastically reduced HBsAg antigenicity, while the Y225del mutation found in one interferon-treated patient impaired HBsAg secretion. Conclusions Lack of detectable HBsAg in patient serum could be explained by low level of ccc DNA in liver tissue, low antigenicity of the surface protein, or its secretion defect. PMID:25320728

  12. 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

  13. 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.

  14. 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.

  15. Anti-amebic antibody activity in patients, determined with antigens prepared from virulent parasites (indirect hemagglutination assay and enzyme-linked immunosorbent assay).

    PubMed

    Israeli, Eitan; Talis, Batya; Peled, Nehama; Snier, Rachella; El-On, Joseph

    2007-09-01

    The serology of amebiasis is affected by low sensitivity and specificity. To evaluate the advantage of the indirect hemagglutination assay and enzyme-linked immunosorbent assay in the diagnosis of amebiasis, using Entamoeba histolytica soluble antigen (macerated amebic antigens) prepared from four different virulent isolates, continuously cultivated in the presence of the original enteric bacteria. Using IHA and ELISA with MAA antigen we examined 147 sera samples from patients with gastrointestinal symptoms, and 11 sera from amebiasis cases (confirmed by microscopy and copro-antigen ELISA). Of 104 of the 147 (70.7%) symptomatic cases that were amebiasis positive by IHA, 81 (55.1%) were positive by MAA-ELISA. In addition, of 11 amebiasis cases confirmed by microscopy and copro-antigen ELISA, 7 (64%) were amebiasis positive by both tests. Four species of bacteria were isolated from the ameba cultures: Escherichia coli, Morganella morganii, Proteus mirabilis, and Streptococcus lactis. Elimination of the bacteria from the cultures by an antibiotics cocktail containing gentamicin, imipenem, piperacillin-tazobactam and vancomycin was the preferred method. Absorption of patients' sera to bacterial antigen prior to serological analysis had only a marginal effect. These results indicate a correlation of 61% between the ELISA developed in this study and the IHA tests in the diagnosis of amebiasis.

  16. 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

  17. Detection of target staphylococcal enterotoxin B antigen in orange juice and popular carbonated beverages using antibody-dependent antigen-capture assays.

    PubMed

    Principato, MaryAnn; Njoroge, Joyce M; Perlloni, Andrei; O' Donnell, Michael; Boyle, Thomas; Jones, Robert L

    2010-10-01

    There is a critical need for qualitative and quantitative methodologies that provide the rapid and accurate detection of food contaminants in complex food matrices. However, the sensitivity of the assay can be affected when antigen-capture is applied to certain foods or beverages that are extremely acidic. This study was undertaken to assess the effects of orange juice and popular carbonated soft drink upon the fidelity of antibody-based antigen-capture assays and to develop simple approaches that could rescue assay performance without the introduction of additional or extensive extraction procedures. We examined the effects of orange juice and a variety of popular carbonated soft drink beverages upon a quantitative Interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) assay system and a lateral flow device (LFD) adapted for the detection of staphylococcal enterotoxin B (SEB) in foods. Alterations in the performance and sensitivity of the assay were directly attributable to the food matrix, and alterations in pH were especially critical. The results demonstrate that approaches such as an alteration of pH and the use of milk as a blocking agent, either singly or in combination, will partially rescue ELISA performance. The same approaches permit lateral flow to efficiently detect antigen. Practical Application: The authors present ways to rescue an ELISA assay compromised by acidity in beverages and show that either the alteration of pH, or the use of milk as a blocking agent are not always capable of restoring the assay to its intended efficiency. However, the same methods, when employed with lateral flow technology, are rapid and extremely successful.

  18. Cost-effectiveness of detection of intestinal amebiasis by using serology and specific-amebic-antigen assays among persons with or without human immunodeficiency virus infection.

    PubMed

    Chang, Sui-Yuan; Sun, Hsin-Yun; Ji, Dar-Der; Lo, Yi-Chun; Wu, Cheng-Hsin; Wu, Pei-Ying; Liu, Wen-Chun; Hung, Chien-Ching; Chang, Shan-Chwen

    2008-09-01

    Among 345 persons who underwent indirect hemagglutination (IHA) serological assays and assays of specific amebic antigens in their stool samples, 24 of 36 (66.7%) who were seropositive for Entamoeba histolytica had intestinal amebiasis as determined by antigen assays compared with 2 of 309 (0.2%) who were seronegative (odds ratio, 307; 95% confidence interval, 64.9 to 1,451). The estimated cost to detect a case of intestinal amebiasis by serology followed by antigen assays ($52) could be reduced by 74.3% and 69.9%, respectively, compared with the costs of the concurrent use of both assays ($202) and the antigen assays alone ($173). Our finding suggests that IHA assays followed by specific-amebic-antigen assays can be cost-effective in the diagnosis of intestinal amebiasis among persons with or without human immunodeficiency virus infection who are at risk for E. histolytica infection.

  19. Correlation between the promoter basal core and precore mutations and HBsAg quantification in French blood donors infected with hepatitis B virus.

    PubMed

    Pivert, A; Servant-Delmas, A; Lunel-Fabiani, F; Le Guillou-Guillemette, H; Laperche, S; Ducancelle, A

    2015-03-01

    Hepatitis B virus (HBV) basal core promoter (BCP) and precore (PC) mutations, HBV viral load and HBV surface antigen (HBsAg) quantitation were screened to assess correlations between these HBV markers in asymptomatic chronic hepatitis B carriers in France. From January 2006 to July 2007, 200 sera were collected from patients who were discovered to be HBsAg-positive when they volunteered to give blood. Direct sequencing of precore/core gene was used to detect A1762T/G1764A mutations in the BCP and G1896A in the PC region. HBV viral load and HBsAg were quantified with two commercials assays. The prevalence of the BCP and PC mixed/mutants were 37% and 60% respectively (P = 0.0001). HBV DNA level and HBsAg titer were significantly lower in subjects harboring the mixed/mutant PC virus compared to those infected by the wild phenotype. No significant difference was observed in HBV viral loads of blood donors infected by wild or mixed/mutant BCP viruses. Mutant or mixed PC virus was associated with male gender, HBeAb-positive status and HBV/D and HBV/E genotypes. BCP mutations were associated with age, and both HBV/A-HBV/E genotypes.The genetic properties of HBV in this cohort showed that most of the blood donors had a negative HBeAg serological status and harbored the PC mutant phenotype in combination with low levels of both HBV DNA and HBsAg. As the study was conducted in healthy subjects who could be considered as asmptomatic carriers, these results suggest a possible protective effect of the G1896A mutation against severe liver lesions. © 2014 Wiley Periodicals, Inc.

  20. 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

  1. 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

  2. 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

  3. 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.

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

    USGS Publications Warehouse

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

    2000-01-01

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

  5. [Comparison of five commercial assays for the detection of Legionella pneumophila antigens in urine].

    PubMed

    de Ory, Fernando; Minguito, Teodora

    2009-02-01

    Antigenuria detection is the main approach for diagnosing Legionella infections. The aim of this study was to compare 5 commercially available methods for detecting Legionella pneumophila soluble antigens in urine. Seventy-one urine samples were tested, 62 from patients with bacterial infection and 9 from patients with respiratory syncytial virus infection. All samples were assayed for the presence of L. pneumophila by immunoenzymatic (ELISA) (Binax and Bartels), and immunochromatographic (IC) (Binax, SAS and Uni-Gold) methods. Identical results (35 positive and 17 negative) were obtained by the 5 assays in 52 samples (73.2%). Samples showing discrepant results were classified by the majority criterion, and/or other laboratory results (serology), and/or epidemiological findings. On this basis, 51 samples were ultimately classified as positive, and 20 as negative. Sensitivity values of ELISA-Binax, ELISA-Bartels, IC-Binax, IC-SAS and IC-Uni-Gold were 80.4, 100, 82.4, 86.3, and 70.6%, respectively. Corresponding values for specificity were 90, 95, 100, 95 and 100%. The results indicate that the methods compared are all adequate for diagnosing Legionella infection, although some have certain limitations regarding sensitivity.

  6. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay.

    PubMed

    Van der Poel, C L; Cuypers, H T; Reesink, H W; Weiner, A J; Quan, S; Di Nello, R; Van Boven, J J; Winkel, I; Mulder-Folkerts, D; Exel-Oehlers, P J

    1991-02-09

    A new four-antigen recombinant immunoblot assay (4-RIBA) for confirmation of hepatitis C virus (HCV) C-100 enzyme-linked immunosorbent assay (ELISA) reactivity was tested in stored serum samples (1984-86) of blood donors and recipients and compared with results from polymerase chain reaction (PCR) analysis of fresh (1990) plasma samples in donors and recipients from the original study. Of 37 HCV C-100 ELISA-positive blood products, 8 were 4-RIBA positive, of which 7 were implicated in post-transfusion non-A, non-B hepatitis (PT-NANBH) and/or PCR confirmed recipient HCV infection. Of 9 recipients with PT-NANBH, 8 were reactive in 4-RIBA (6 positive and 2 indeterminate). With fresh plasma samples, 3 donors and 6 recipients who were 4-RIBA positive were also PCR positive. 4 4-RIBA indeterminate and 78 4-RIBA negative samples of donors and recipients were PCR negative. Of 6 4-RIBA positive recipients, 5 were PCR positive four to six years later. 1.6% of the 383 recipients became chronically infected with HCV. The new 4-RIBA represents a candidate confirmation test to discriminate between infective and non-infective HCV C-100 ELISA-positive blood donors.

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

    PubMed Central

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

    2015-01-01

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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. Evaluation of clinical usefulness of second-generation HCV core antigen assay: comparison with COBAS AMPLICOR HCV MONITOR assay version 2.0.

    PubMed

    Yokosuka, Osamu; Kawai, Shigenobu; Suzuki, Yoichi; Fukai, Kenichi; Imazeki, Fumio; Kanda, Tatsuo; Tada, Motohisa; Mikata, Rintarou; Hata, Akira; Saisho, Hiromitsu

    2005-12-01

    Hepatitis C virus (HCV) is an important etiologic agent for chronic liver diseases. The aim of this study was to evaluate the clinical usefulness of second-generation HCV core antigen assay by comparing the results of the assay with those of the COBAS AMPLICOR HCV MONITOR version 2.0 (COBAS v2.0). HCV core antigen was detectable by this assay in 142/149 (95.3%) of serotype 1 (3821+/-322 fmol/l; mean+/-SD), in 56/58 (96.6%) of serotype 2 (2589+/-449 fmol/l), and in 6/6 (100%) of serotypes 1+2 (1240+/-548 fmol/l). The HCV core antigen levels measured by this assay correlated well with the HCV RNA levels by COBAS v2.0 (r=0.848, P<0.0001). In relation to the outcome of interferon monotherapy, the pretreatment HCV core antigen levels of sustained and non-sustained virological responders were 659+/-189 and 4904+/-376 fmol/l in serotype 1, 1993+/-740 and 3145+/-519 fmol/l in serotype 2. The cutoff values with the best accuracy for HCV core Ag levels to discriminate between sustained and non-sustained virological response were 699 fmol/l for serotype 1 and 292 fmol/l for serotype 2, respectively, by receiver operating characteristic curve analysis. This new assay was considered to be useful in evaluating the HCV levels in patients with chronic hepatitis C.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. Aptamer-based microchip electrophoresis assays for amplification detection of carcinoembryonic antigen.

    PubMed

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

    2015-10-23

    Carcinoembryonic antigen (CEA) as one of the most widely used tumor markers 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. The magnetic beads (MBs) are employed as carriers of double strand DNA that is formed by an aptamer of the target and a complementary DNA of the aptamer. After the aptamer in the MB-dsDNA conjugate binds with the target, the complementary DNA was released from the MB-dsDNA conjugate. The released complementary DNA hybridizes with a fluorescein amidite (FAM) labeled DNA, and forms a DNA duplex, which triggers the selective cleavage of FAM labeled DNA by nicking endonuclease Nb.BbvCI, and generating a 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), the FAM labeled DNA segment is separated and detected. 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 to 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. This method may become a useful tool for rapid analysis of CEA and other tumor markers in biomedical analysis and clinical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. 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

  1. [Comparative analysis of electro-chemiluminescence immunoassay and chemiluminescent microparticle immunoassay abilities to quantitatively assess hepatitis B surface antigen].

    PubMed

    Wang, Jie; Wang, Mao-rong

    2013-03-01

    To perform a systematic comparative analysis of two different commercial automated systems using chemiluminescence immunoassay to quantitatively detect hepatitis B virus surface antigen (HBsAg) in patient sera. The Elecsys2010 electrical chemiluminescence immunoassay (ECLIA; manufactured by Roche) and the ARCHITECT il000 chemiluminescence magnetic microparticle immunoassay (CMIA; manufactured by Abbott) were used to detect HBsAg in 100 serum samples of individuals who presented at our department with suspected hepatitis infection between January and May 2012. The manufacturer's protocols were strictly followed. The categorical data was analyzed by Chi-squared test, and linear regression analysis was used to compare the results of the two assay systems. The HBsAg detection results from the two different assay systems showed good correlation (r >or= 0.95), and had good correlation at a low (r = 0.966), medium (r = 0.974) and high (r = 0.984) cutoff values. However, the positive detection rate of CMIA was significantly higher than that of ECLIA(94% vs. 88%, P < 0.05). When the HBsAg content was below 0.10 IU/ml, the ECLIA detection rate and sensitivity were slightly higher than those of CMIA. The ARCHITECT i1000 and Elecsys 2010 immunoassay systems have good correlation in quantitative detection of HBsAg, but the former may be more sensitive.

  2. 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.

  3. Multisite Validation of Cryptococcal Antigen Lateral Flow Assay and Quantification by Laser Thermal Contrast

    PubMed Central

    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. PMID:24378231

  4. 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

  5. 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

  6. 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

  7. 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).

  8. 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

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

    PubMed Central

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

    2011-01-01

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

  10. Evaluation of a rapid immunochromatographic ODK0501 assay for detecting Streptococcus pneumoniae antigens in the sputum of pneumonia patients with positive S. pneumoniae urinary antigens.

    PubMed

    Mukae, Hiroshi; Yatera, Kazuhiro; Noguchi, Shingo; Kawanami, Toshinori; Yamasaki, Kei; Tokuyama, Susumu; Inoue, Naoyuki; Nishida, Chinatsu; Kawanami, Yukiko; Ogoshi, Takaaki; Orihashi, Takeshi; Yoshii, Chiharu; Ishimoto, Hiroshi

    2015-03-01

    A novel, rapid and noninvasive test (ODK0501, RAPIRUN(®)Streptococcus pneumoniae) uses polyclonal antibodies to detect C polysaccharide of S. pneumoniae derived from sputum samples using an immunochromatographic assay. We evaluated its usefulness in Japanese patients with pneumonia who exhibited positive urinary antigen tests for S. pneumoniae (BinaxNOW(®)S. pneumoniae). Forty adult patients with pneumonia treated between May 2011 and August 2013 were enrolled. Bacterial cultures, Gram staining and ODK0501 assays of sputum as well as urinary antigen tests for S. pneumoniae using urine samples obtained from the same patients were performed upon admission, the fourth day after starting antimicrobial treatment and at the end of the antimicrobial treatment. Twenty-seven of the 40 patients were positive for ODK0501, while a negative result for ODK0501 was associated with low-quality sputum samples according to the Geckler classification of sputum. The sensitivity and specificity of the ODK0501 assay in the 40 patients were 90.9% and 61.1%, respectively, based on the culture results. The results obtained with this kit were more favorable than those observed on Gram staining. The ODK0501 assay also showed a rapid reaction to the disappearance of S. pneumoniae in the sputum samples, while approximately 80% of the patients exhibited persistent positive results on the urinary antigen detection tests at the end of treatment. The ODK0501 test is a noninvasive, rapid and accurate tool for diagnosing respiratory infections caused by S. pneumoniae, although good quality sputum must be obtained prior to adequate treatment with antibiotics. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  11. 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.

  12. Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells.

    PubMed

    Karlsson, Annika C; Martin, Jeffrey N; Younger, Sophie R; Bredt, Barry M; Epling, Lorrie; Ronquillo, Rollie; Varma, Arjun; Deeks, Steven G; McCune, Joseph M; Nixon, Douglas F; Sinclair, Elizabeth

    2003-12-01

    The enumeration of antigen-specific T cell responses has been greatly facilitated in recent years by the development of methods based on the detection of cytokines. In particular, the enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays have become popular. Since both assays are likely to continue to be in widespread use, it is important to evaluate whether their results are comparable. In the current study, we compared the results obtained in the ELISPOT and CFC assays using peptide pools corresponding to CMV and HIV-1 proteins in chronically HIV-1-infected individuals. Analysis of T cell responses to peptide pools indicated that the CMV pp65 and HIV-1 Gag CFC and ELISPOT-derived results were statistically correlated. However, the results obtained with each assay differed in important ways: the magnitude of the response was consistently higher in the CFC assay while the CFC assay was less likely than the ELISPOT assay to detect low-level responses. Furthermore, there was a lack of numeric agreement between ELISPOT and CFC results. For studies that require the detection of low-level responses, or definition of responses as positive or negative, the ELISPOT assay may be preferable. In contrast, the CFC has a greater dynamic range and allows for phenotypic discrimination of responding cells, making it the assay of choice for most other applications.

  13. 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.

  14. 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

  15. 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

  16. 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.

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

    PubMed

    Yamanaka, Atsushi; Suzuki, Ryosuke; Konishi, Eiji

    2014-07-23

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

  18. Diagnosis of paracoccidioidomycosis by dot immunobinding assay for antibody detection using the purified and specific antigen gp43.

    PubMed Central

    Taborda, C P; Camargo, Z P

    1994-01-01

    The dot immunobinding assay, a rapid, visually read test, was adapted for serodiagnosis and follow-up of paracoccidioidomycosis (PCM). Purified gp43 antigen was tested before and after sodium metaperiodate treatment. To evaluate the assay, it was tested with sera from PCM, histoplasmosis, Jorge Lobo's disease, aspergillosis, candidiasis, and cryptococcosis patients and healthy subjects. Native gp43 gave positive results with all sera from PCM patients and weakly positive results with sera from Jorge Lobo's disease patients (31.3%). No false-positive results were obtained when periodate-treated gp43 was used as the antigen. These results indicate that the dot immunobinding test is sensitive, specific, economical, and fast for serodiagnosis and follow-up studies of PCM. Images PMID:8150974

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. [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.

  5. 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.

  6. 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

  7. 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.

  8. Polydiacetylene/Anti-HBs Complexes for Visible and Fluorescent Detection of Hepatitis B Surface Antigen on a Nitrocellulose Membrane.

    PubMed

    Roh, Jinkyu; Lee, Su Yeon; Park, Sangho; Ahn, Dong June

    2017-08-17

    The immunochromatographic assay (ICA) using a nitrocellulose (NC) membrane offers several advantages. This technique is a rapid and straightforward method in contrast to other immunoassays. Polydiacetylene (PDA) vesicles have unique optical properties, displaying red color and red fluorescence at the same time. In this system, red-phase PDA vesicles are used as a fluorescent dye as well as a surface for immobilized hepatitis B surface antibody (HBsAb). PDA has a remarkable stability compared with other fluorescent dyes. In this study, the most suitable PDA/HBsAb complexes are introduced for detecting hepatitis B surface antigen (HBsAg). Then, the PDA/HBsAb complexes affixed antibody is attached to NC membrane, which has two lines to confirm detection of HBsAg. The main advantage of this system is that the detection of HBsAg can be observed in both visible and fluorescent images due to the optical properties of polydiacetylene. Detection of HBsAg is observed up to 0.1 ng mL(-1) by fluorescent analysis and confirmed by red line on the NC membrane up to 1 ng mL(-1) (HBsAg) using the naked eye. Consequently, these results show that PDA/HBsAb complexes were successfully applied to ICA for the diagnosis of hepatitis B. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. 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.

  10. 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.

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

    PubMed

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

    2002-12-01

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

  12. Designing binding kinetic assay on the bio-layer interferometry (BLI) biosensor to characterize antibody-antigen interactions.

    PubMed

    Kamat, Vishal; Rafique, Ashique

    2017-08-10

    The Octet biosensors provide a high-throughput alternative to the well-established surface plasmon resonance (SPR) and SPR imaging (SPRi) biosensors to characterize antibody-antigen interactions. However, the utility of the Octet biosensors for accurate and reproducible measurement of binding rate constants of monoclonal antibodies (mAbs) is limited due to challenges such as analyte rebinding, and mass transport limitation (MTL). This study focuses on addressing these challenges and provides experimental conditions to reliably measure kinetics of mAb-antigen interactions. The mAb capture density of less than 0.6 nm was found to be optimal to measure a wide range of binding affinities on Octet HTX biosensor. The titration kinetic and single cycle kinetic assays performed on Octet HTX generated reproducible binding kinetic parameters and correlated with the values measured on Biacore 4000 and MASS-1. Kinetic assays performed on 0.1 nm density mAb surfaces significantly reduced MTL and enabled characterization of picomolar affinity mAbs. Finally, kinetic analysis performed on 150 antibodies to 10 antigens with molecular weights ranging from 21kD to 105kD showed concordance between Octet HTX, Biacore 4000 and MASS-1 (R(2) > 0.90). The data presented in this study suggest that under optimal experimental conditions, Octet biosensor is capable of generating kinetic values comparable to SPR/SPRi biosensors. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  13. 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

  14. A new combined HIV p24 antigen and anti-HIV-1/2/O screening assay.

    PubMed

    Polywka, Susanne; Duttmann, Hedwig; Lübben, Frank; Laufs, Rainer; Feldner, Jürgen

    2005-01-01

    It is important to shorten the window period after acute HIV infection in which infected individuals are still antibody-negative, especially in blood donors. Newly developed fourth-generation assays detect antibodies to HIV-1, including subtype O, and to HIV-2 and, simultaneously, p24 antigen of HIV-1. To evaluate this assay for daily routine work we compared it with different third-generation assays using sera from uninfected patients and patients with known HIV infection. The most interesting sera are those drawn during seroconversion from freshly infected patients. Whenever we encounter such a patient with acute HIV infection we store the serum in aliquots at -20 degrees C. Thus, we were able to establish our own seroconversion panel and use it in our laboratory for evaluation of new assays. The new test was shown to be able to detect all chronically HIV-infected individuals and four of six patients during seroconversion although in two of these patients conventional assays for HIV antibodies were still negative. The rate of unspecific reactivities was slightly higher as compared with third-generation assays.

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

    PubMed Central

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

    2016-01-01

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

  16. Hepatitis B antigen, antigen subtypes, and hepatitis B antibody in normal subjects and patients with liver disease

    PubMed Central

    Nishioka, K.; Levin, A. G.; Simons, M. J.

    1975-01-01

    The relative sensitivities of counterimmunoelectrophoresis (CIE) and haemagglutination assays for the detection of hepatitis B surface antigen (HBsAg) and antibodies (anti-HBs) were compared. Twelve scientists from ten countries in Asia, Africa and the Pacific region participated in the study. The participants provided serum samples from 15 953 subjects comprising patients with acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC), as well as blood donors and other normal individuals. For the detection of HBsAg in a reference panel serum, immune adherence haemagglutination (IAHA) was slightly more sensitive than passive haemagglutination inhibition (PHI); CIE was the least sensitive. Mean HBsAg frequencies in patients with acute hepatitis, chronic hepatitis, cirrhosis, and HCC were significantly higher than in healthy controls. Passive haemagglutination (PHA) was more sensitive than CIE for the detection of anti-HBs. The frequency of anti-HBs in patients with HCC was significantly lower than that in the other groups. Mean anti-HBs frequencies in patients with acute hepatitis, chronic hepatitis, and cirrhosis were not significantly different from that in normal subjects. Subtyping of HBsAg was performed by PHI. Among asymptomatic carriers the predominant HBsAg subtype in northeast Asia was adr.In India, ayw predominated in carriers, with the demarcation between adr and ayw occurring west of Burma. In West Africa the only subtype detected was ayw, but in East Africa the majority subtype was adw. The r subtype was found only in Asian populations east of India and in Western Pacific populations. In Papua New Guinea all four subtypes were identified. With one possible exception, the subtypes of HBsAg-positive patients with liver disease reflected the predominant type in each geographic location. PMID:1084799

  17. [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.

  18. Dendritic cell-based assays, but not mannosylation of antigen, improves detection of T-cell responses to proinsulin in type 1 diabetes

    PubMed Central

    Narendran, Parth; Elsegood, Kathryn; Leech, Nicola J; Macindoe, Wallace M; Boons, Geert-Jan; Dayan, Colin M

    2004-01-01

    In vitro detection of T-cell responses to autoantigens in type 1 diabetes is recognized as being technically challenging. We aimed to accurately measure cellular responses to proinsulin in patients with diabetes, and speculated that presentation of antigen by dendritic cells (DCs) would enhance the sensitivity of the peripheral blood assay. Antigen was mannosylated to facilitate uptake through DC surface mannose receptors to further improve the assay. Whole proinsulin, as well as mannosylated peptides of proinsulin, were combined with peripheral T cells and autologous immature DCs in a proliferative assay in a panel of newly diagnosed type 1 diabetic patients. The DC-based assay detected responses to proinsulin in five of 15 diabetic patients compared to one of 15 diabetic patients detected using the standard mononuclear cell assay. When the results of all patients were combined, the DC assay, but not the mononuclear cell assay, had a proinsulin response that was significantly higher than background (P < 0·001). The DC assay was, however, associated with high autologous mixed lymphocyte reactions that possibly masked responses in individual patients. Mannosylated antigen was taken up in larger quantities than non-mannosylated antigen, but not presented any more powerfully. Our data suggest that autologous DC-based assays are more powerful than standard peripheral blood mononuclear cell assays. However, they are compromised by high autologous mixed lymphocyte reactions and this requires addressing before they can be used as a routine readout of in vitro peripheral T-cell responses. PMID:15056379

  19. 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.

  20. 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.

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

    PubMed

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

    2014-02-01

    Hepatitis B surface antigen (HBsAg) is a crucial serum marker for the diagnosis of hepatitis B virus (HBV) infection. It is imperative to compare test results from different detection methods based on different principles. Four methods, chemiluminescent microparticle immunoassay (CMIA), electrochemiluminescent immunoassay (ECLIA), enzyme-linked immunosorbent assay (ELISA) and golden immunochromato-graphic assay (GICA) were applied to test the HBsAg level in 250 specimens. According to the EP12-A2 and EP15-A2 documents from Clinical and Laboratory Standards Institute (CLSI), the concentration at which repeated results are 50% positive (C50) of HBsAg detected by CMIA, ECLIA, ELISA and GICA was 0.05, 0.08, 0.15 and 15.0IU/ml, respectively. When the detection concentration of HBsAg was 0.5IU/ml, the imprecision degree of CMIA, ECLIA and ELISA was 8.1%, 5.9% and 14.9% respectively. When detecting high HBsAg level (≥20.0IU/ml) and HBsAg negative specimens, the consistency of the four methods was high, while for the low level (0.05-20.0IU/ml), the consistency was poor (except for the CMIA and ECLIA, P<0.05). When evaluation of the four methods in qualitative diagnosis of HBsAg level in the 116,455 specimens, there was no significant discrepancy among CMIA, CMIA and ECLIA, however, GICA was significantly different from the other 3 methods. Compared with CMIA, the false negative rate of ECLIA, ELISA and GICA was 0.2%, 1.3% and 12.3% respectively. In conclusion, GICA was only suitable for the preliminary screening of HBsAg positive individuals and ELISA can be applied to the qualitative diagnosis of HBsAg. Both CMIA and ECLIA were suitable for the quantitative determination of HBsAg. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. 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

  3. An optimized assay for the enumeration of antigen-specific memory B cells in different compartments of the human body.

    PubMed

    Cao, Yanran; Gordic, Maja; Kobold, Sebastian; Lajmi, Nesrine; Meyer, Sabrina; Bartels, Katrin; Hildebrandt, York; Luetkens, Tim; Ihloff, Anna Sophie; Kröger, Nicolaus; Bokemeyer, Carsten; Atanackovic, Djordje

    2010-06-30

    In the framework of our current study we set out to develop an optimized assay for the quantification of antigen-specific B cells in different compartments of the human body. Mononuclear cells (MNC) derived from the peripheral blood, bone marrow (BM), or human tonsils were incubated with different combinations of stimuli. The stimulated cells and culture supernatants were then applied to IgG-ELISPOT and ELISA read-out assays and tetanus toxoid (TT)-specific B cell responses were quantified. We found that a combination of CD40L, CpG, and IL21 was optimal for the induction of TT-specific IgG-producing cells from memory B cell (mBc) precursors. This cocktail of stimuli led to a proliferation-dependent induction of CD19(intermediate)CD38(high)CD138(high)IgD(negative) terminally differentiated plasma cells. Applying our optimized methodology we were also able to quantify mBc specific for cytomegalovirus and influenza virus A. Most importantly, the same method proved useful for the comparison of mBc frequencies between different compartments of the body and, accordingly, we were able to demonstrate that TT-specific mBc preferably reside within tonsillar tissue. Here, we optimized an assay for the quantification of antigen-specific B cells in different human tissues demonstrating, for example, that TT-specific mBc preferably reside in human tonsils but not in the BM or the peripheral blood. We suggest that our approach can be used for the enumeration of mBc specific for a wide variety of Ag (microbial, tumor-related, auto-antigens), which will lead to significant improvements regarding our knowledge about the biology of humoral immunity. Copyright 2010. Published by Elsevier B.V.

  4. 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.

  5. Anti-Protective Antigen IgG Enzyme-Linked Immunosorbent Assay for Diagnosis of Cutaneous Anthrax in India

    PubMed Central

    Ghosh, N.

    2012-01-01

    Anthrax caused by Bacillus anthracis is a public health problem in several developing countries whose main source of income is farming. Anthrax is a disease of herbivorous animals, and humans can be infected by handling infected animals or contaminated animal products. Specific diagnostic tests are unavailable in India for the detection and confirmation of cutaneous anthrax in humans. Here, we describe the development of an enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies against Bacillus anthracis protective antigen in the Indian population. A total of 405 serum samples collected from different groups were tested by the developed ELISA. The assay provided a specificity of 99.41% (95% confidence interval [CI], 97.89 to 99.93) and a sensitivity of 100% (CI, 94.4 to 100) using a cutoff value of 0.29 ELISA unit (EU). The positive predictive value (PPV) and negative predictive value (NPV) of the assay were 97% and 100%, respectively. The efficiency and J index for the reliability of the assay were 99.5% and 0.994, respectively. The assay can be a very useful tool for surveillance as well as for diagnosis of cutaneous anthrax cases in India. PMID:22718130

  6. Enhanced Fluorescence ELISA Based on HAT Triggering Fluorescence "Turn-on" with Enzyme-Antibody Dual Labeled AuNP Probes for Ultrasensitive Detection of AFP and HBsAg.

    PubMed

    Wu, Yudong; Guo, Weisheng; Peng, Weipan; Zhao, Qian; Piao, Jiafang; Zhang, Bo; Wu, Xiaoli; Wang, Hanjie; Gong, Xiaoqun; Chang, Jin

    2017-03-22

    At present, enzyme-linked immunosorbent assay (ELISA) is considered to be the most appropriate approach in clinical biomarker detection, with good specificity, low cost, and straightforward readout. However, unsatisfactory sensitivity severely hampers its wide application in clinical diagnosis. Herein, we designed a new kind of enhanced fluorescence enzyme-linked immunosorbent assay (FELISA) based on the human alpha-thrombin (HAT) triggering fluorescence "turn-on" signals. In this system, detection antibodies (Ab2) and HAT were labeled on the gold nanoparticles (AuNPs) to form the detection probes, and a bisamide derivative of Rhodamine110 with fluorescence quenched served as the substrate of HAT. After the sandwich immunoreaction, HAT on the sandwich structure could catalyze the cleavage of the fluorescence-quenched substrate, leading to a strong fluorescence signal for sensing ultralow levels of alpha fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg). Under the optimized reaction conditions, AFP and HBsAg were detected at the ultralow concentrations of 10(-8) ng mL(-1) and 5 × 10(-4) IU mL(-1), respectively, which were at least 10(4) times lower than those of the conventional fluorescence assay and 10(6) times lower than those of the conventional ELISA. In addition, we further discussed the efficiency of the sensitive FELISA in clinical serum samples, showing great potential in practical applications.

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

    PubMed

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

    2014-01-01

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

  8. A monoclonal antibody-based VZV glycoprotein E quantitative assay and its application on antigen quantitation in VZV vaccine.

    PubMed

    Liu, Jian; Zhu, Rui; Ye, Xiangzhong; Yang, Lianwei; Wang, Yongmei; Huang, Yanying; Wu, Jun; Wang, Wei; Ye, Jianghui; Li, Yimin; Zhao, Qinjian; Zhu, Hua; Cheng, Tong; Xia, Ningshao

    2015-06-01

    Varicella-zoster virus (VZV) is a highly infectious agent that causes varicella and herpes zoster (HZ), which may be associated with severe neuralgia. Vaccination is the most effective way to reduce the burden of the diseases. VZV glycoprotein E (gE) is the major and most immunogenic membrane protein that plays important roles in vaccine efficacy. A quantitative assay for gE content is desirable for the VZV vaccine process monitoring and product analysis. In this study, 70 monoclonal antibodies (mAbs) were obtained after immunizing mice with purified recombinant gE (rgE). The collection of mAbs was well-characterized, and a pair of high-affinity neutralization antibodies (capture mAb 4A2 and detection mAb 4H10) was selected to establish a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the native and recombinant gE. The detection limit of this assay was found to be 1.95 ng/mL. Furthermore, a reasonably good correlation between the gE content (as measured by the mAb-based quantitative ELISA) and the virus titer (as measured by the "gold standard" plaque assay) was observed when both assays were performed for tracking the kinetics of virus growth during cell culture. A total of 16 batches of lyophilized VZV vaccine were tested using the newly developed quantitative ELISA and classical plaque assay, demonstrating reasonably good correlation between gE content and virus titer. Therefore, this mAb-based gE quantitative assay serves as a rapid, stable, and sensitive method for monitoring viral antigen content, one additional quantitative method for VZV vaccine process and product characterization. This quantitative ELISA may also serve as a complementary method for virus titering.

  9. 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

  10. 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

  11. Antigen Assay with the Potential To Aid in Diagnosis of Blastomycosis

    PubMed Central

    Durkin, Michelle; Witt, John; LeMonte, Ann; Wheat, Blair; Connolly, Patricia

    2004-01-01

    We report results of an immunoassay for Blastomyces dermatitidis antigenuria. Sensitivity was 92.9%, and specificity was 79.3%. Cross-reactions occurred in 96.3% of patients with histoplasmosis, 100% of patients with paracoccidioidomycosis, 70% of patients with penicilliosis marneffei, 2.9% of patients with cryptococcosis, and 1.1% of patients with aspergillosis. Reproducibility was 96.3%. These findings support a potential role for antigen testing in blastomycosis. PMID:15472368

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Usefulness of a new immuno-radiometric assay to detect hepatitis C core antigen in a community-based population.

    PubMed

    Hayashi, K; Hasuike, S; Kusumoto, K; Ido, A; Uto, H; Kenji, N; Kohara, M; Stuver, S O; Tsubouchi, H

    2005-01-01

    A new immuno-radiometric assay (IRMA) to detect hepatitis C virus (HCV) core antigen (HCVcAg) has been developed. The aim of the present study was to investigate the sensitivity and specificity of this IRMA to measure HCV antigenemia, based on the detection of HCV RNA as the gold standard, and to assess the utility of the IRMA in a community-based population. Anti-HCV positive residents in a hyperendemic area of HCV infection in Japan were studied. Serum levels of HCVcAg were measured using IRMA, and the presence of HCV RNA was determined by a qualitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The sensitivity and the specificity of the IRMA were 96.4 and 100%, respectively. The sensitivity of the IRMA was similar between serological HCV group I (HCV genotypes 1a and 1b) (97.6%) and group II (HCV genotypes 2a and 2b) (94.0%). There was a strong correlation between serum HCVcAg level and HCV-RNA measured by a quantitative RT-PCR (r = 0.832, P < 0.0001). There also was a very strong correlation of HCVcAg level between IRMA measurements performed on serum and those performed on plasma (r = 0.984, P < 0.0001). In conclusion, this new IRMA is useful for the detection of HCV core antigen in a community-based population.

  18. 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).

  19. 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.

  20. LVAD implant as a bridge to heart transplantation is associated with allosensitization as measured by single antigen bead assay.

    PubMed

    Shankar, Nisha; Daly, Richard; Geske, Jennifer; Kushwaha, Sudhir K; Timmons, Michael; Joyce, Lyle; Stulak, John; Gandhi, Manish; Kremers, Walter; Park, Soon; Pereira, Naveen L

    2013-08-15

    Left ventricular assist devices (LVAD) as a bridge (BTT) to heart transplantation (HTX) may be limited by the formation of anti-human leukocyte antigen antibodies. Whether sensitization occurs with continuous axial flow LVAD implant as assessed by single antigen bead (SAB) assay is unknown. Cytotoxic panel-reactive antibody (PRA) and SAB assays were analyzed in HTX recipients undergoing LVAD implant as a BTT. Sensitization was defined as peak anti-human leukocyte antigen antibody values of more than 2000 mean fluorescence intensity because these values have been found to correlate with flow cytometric crossmatch results. LVADs were implanted as BTT in 30 patients. There were 7% (2 of 30) of patients before LVAD implant and no patients after LVAD implant with PRA more than 10%. However, 20% (6 of 30) of patients before LVAD and 53% (16 of 30) after LVAD were sensitized as measured by SAB (P=0.024). At HTX, 47% (14 of 30) of patients remained sensitized. A positive virtual crossmatch was observed in 28% (4 of 14) of the sensitized patients at HTX. There was no difference between the sensitized and nonsensitized groups (P>0.4 for all) in usage of blood products (6411 vs. 6339 units) and time to HTX (28,663 vs. 25,748 days), and 1 year after HTX, there were no differences in rejection (total rejection score 0.30 vs. 0.37) and survival (93% vs. 88%). Allosensitization after LVAD is common despite cytotoxic PRA being negative. One year after HTX, this sensitization does not translate into increased acute cellular or antibody-mediated rejection or reduced survival.

  1. 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.

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

    PubMed

    Chen, Longyan; Neethirajan, Suresh

    2015-04-15

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

  3. 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.

  4. A direct, automated, immuno-turbidimetric assay of free protein S antigen in plasma.

    PubMed

    Deffert, C; Esteve, F; Grimaux, M; Gouault-Heilmann, M

    2001-03-01

    A new, fully automated, one-step, immuno-turbidimetric assay of free protein S (fPS) in plasma (STA Liatest Free Protein S; Diagnostica Stago, Asnières, France) has been developed for STA analysers. This technique combines the advantages of a direct assay of fPS using two monoclonal antibodies, which specifically recognize fPS but not protein S (PS)-C4b-binding protein complexes, and the advantages of automation. The assay has good analytical performances, with intra- and inter-assay variation coefficients below 5% for normal values, and slightly higher for abnormal values. In a comparison study with a one-step enzyme-linked immunosorbent assay for fPS (Asserachrom Free Protein S; Diagnostica Stago), a correlation coefficient of 0.93 with a regression line close to 1 was found between the two techniques (n = 166 normal or PS-deficient plasma samples collected from healthy subjects and individuals with a personal or family history of thrombosis). This new technique is specific, reproducible, easy to perform, and provides a useful tool in the diagnosis of PS deficiency.

  5. 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.

  6. No response to hepatitis B vaccine in infants born to HBsAg(+) mothers is associated to the transplacental transfer of HBsAg.

    PubMed

    Wang, Jing; He, Yingli; Jin, Dongfang; Liu, Jinfeng; Zheng, Jie; Yuan, Ningxia; Bai, Yun; Yan, Taotao; Yang, Yuan; Liu, Yong; Zhang, Shulin; Zhao, Yingren; Chen, Tianyan

    2017-08-01

    No or low hepatitis B (HB) vaccine response is more frequent in infants from HBsAg(+) mothers than those from HBsAg(-). Our previous study found temporary positivity of HBsAg in infants from HBsAg(+) mothers. In this study, we hypothesized that HBsAg in infant blunt immune response to standard hepatitis B vaccination. A total of 328 consecutive HBsAg(+) mothers and their offspring were enrolled. Blood samples were taken from mothers and their infants and quantified for HBsAg, anti-HBs titer and HBV DNA load concentration; Placenta samples were collected to stain for HBsAg. First, 6.7% infants (22/328) showed anti-HBs titer lower than 10 mIU/mL after HB vaccination (non-response to HB vaccine). HBsAg(+) newborns showed higher risk of non-response than HBsAg(-) infants (13.0% versus 5.0%, p = 0.016). Infants from high HBsAg titer mothers displayed higher risk of HBsAg positivity at birth than those from low titer mothers (45.3% versus 2.8%, p < 0.001). HBsAg titer in mothers of HBsAg(+) newborns was much higher than mothers of HBsAg(-) newborns (p < 0.001). All those data supported HBsAg can be transferred through placenta. Our hypothesis was further reinforced by immunostaining with specific antibody against HBsAg, a substantial higher prevalence (87.5% versus 30.8%, p = 0.024) and stronger immunostaining (p = 0.008) was demonstrated in HBsAg(+) group comparing with placenta of the HBsAg(-) group. No response to HB vaccine in infants of HBsAg(+) mothers was associated to the transplacental transfer of HBsAg.

  7. 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

  8. Comparative Study of Different Sources of Pertussis Toxin (PT) as Coating Antigens in IgG Anti-PT Enzyme-Linked Immunosorbent Assays

    PubMed Central

    Kapasi, Aditi; Meade, Bruce D.; Plikaytis, Brian; Pawloski, Lucia; Martin, Monte D.; Yoder, Sandra; Rock, Michael T.; Coddens, Séverine; Haezebroeck, Valérie; Fievet-Groyne, Françoise; Bixler, Garvin; Jones, Charles; Hildreth, Stephen; Edwards, Kathryn M.; Messonnier, Nancy E.

    2012-01-01

    In an effort to improve the reliability and reproducibility of serological assays for Bordetella pertussis, a collaborative study was conducted to compare four different sources of pertussis toxin (PT) as coating antigens in the immunoglobulin G (IgG) anti-PT enzyme-linked immunosorbent assay (ELISA). Four sources of PT were used as coating antigens in the IgG anti-PT ELISA in four different testing laboratories (labs A to D) to determine whether the different antigen preparations and different laboratories influenced assay results. A panel of 60 sera consisting of deidentified human specimens from previous vaccination trials of healthy adults and infants and clinical specimens from outbreak settings was tested. In the four laboratories, each sample was tested three times with the four PT antigens according to the standard coating optimization and IgG anti-PT ELISA testing procedures used in that laboratory. Differences among the antigens, as well as intra- and interlaboratory variability, were evaluated. Excellent agreement was observed with the test panel results among the four antigens within each laboratory. Concordance correlation coefficient (rc) measurements among the different antigens ranged from 0.99, 0.99 to 1.00, 1.00, and 0.97 to 1.00 for labs A to D, respectively. The comparisons between pairs of laboratories also indicated a high degree of concordance for each PT preparation, with rc measurements between 0.90 and 0.98, 0.93 and 0.99, 0.92 and 0.98, and 0.93 and 0.99 for antigens 1 to 4, respectively. Relatively minor differences in results were observed among laboratories or among antigens, suggesting that the four PT antigens are quite similar and could be considered for acceptance in harmonized immunoassays used for serodiagnosis or vaccine evaluation. PMID:22116688

  9. Can hepatitis B virus DNA in semen be predicted by serum levels of hepatitis B virus DNA, HBeAg, and HBsAg in chronically infected men from infertile couples?

    PubMed

    Fei, Q J; Yang, X D; Ni, W H; Pan, C S; Huang, X F

    2015-05-01

    Hepatitis B virus (HBV) in semen is important for father-to-child transmission of HBV and has adverse effects on sperm quality. However, risk factors associated with HBV in semen remain unclear. Serum HBV DNA and hepatitis B e antigen (HBeAg) levels may pose a risk on HBV in semen. This study aims to examine whether serum HBV DNA, HBeAg, and hepatitis B surface antigen (HBsAg) level were associated with HBV DNA in semen. 151 male patients chronically infected with HBV from infertile couples were included. Serum HBsAg and HBeAg were determined using an electrochemiluminescence immune assay (ECLIA). Serum and seminal plasma HBV DNA were detected by the QIAGEN Real-Time HBV DNA assay. Of 151 patients, 143 (94.7%) were serum HBV DNA-positive and 65 (43.0%) were seminal plasma HBV DNA-positive. Serum HBV DNA and HBeAg level of seminal plasma HBV DNA-positive patients were significantly higher (p < 0.001) as compared with those of seminal plasma HBV DNA-negative patients, HBsAg level of seminal plasma HBV DNA-positive patients was significantly lower (p < 0.001) compared with that of seminal plasma HBV DNA-negative patients. The best serum HBV DNA, HBeAg, and HBsAg value for discriminating between seminal plasma HBV DNA-positive and HBV DNA-negative patients were ≥6.9 log10 IU/mL (sensitivity 100.0%, specificity 90.7%), >14.8 S/CO (sensitivity 96.9%, specificity 81.5%), and <1791.5 S/CO (sensitivity 81.5%, specificity 81.2%), respectively. The combination of serum HBV DNA and HBeAg had high diagnostic sensitivity (100.0%) and specificity (95.4%) for the presence of HBV DNA in semen. As such, these serum markers especially the combination of HBV DNA and HBeAg are useful predictors of the presence of HBV DNA in semen in HBV chronically infected men from infertile couples. © 2015 American Society of Andrology and European Academy of Andrology.

  10. 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

  11. 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

  12. 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).

  13. 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

  14. 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.

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

    PubMed Central

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

    2015-01-01

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

  16. Competitive and indirect enzyme-linked immunosorbent assays for Mycobacterium bovis infections based on MPB70 and lipoarabinomannan antigens.

    PubMed Central

    Sugden, E A; Stilwell, K; Rohonczy, E B; Martineau, P

    1997-01-01

    A competitive enzyme-linked immunosorbent assay (C-ELISA) using M. bovis BCG Tokyo culture filtrate as antigen and anti-MPB70 4C3/17 monoclonal antibody was developed for use in multiple animal species. An analysis of the C-ELISA data for cattle and bison serum panels revealed specificities of 68% to 85% and sensitivities of 85% to 89%. Receiver operator characteristics (ROC) of this data revealed areas of 81% to 92% for C-ELISA and demonstrated that C-ELISA as well as the indirect ELISA protocols, MPB70-ELISA and LAM-ELISA, discriminate M. bovis infected animals from non-infected animals for these particular panels. The kappa statistic values for agreement beyond chance between C-ELISA and MPB70-ELISA were determined after ELISA cutoffs were adjusted to minimize false positives. There were poor to excellent agreements between C-ELISA and MPB70-ELISA in all species tested (Bovidae, Cervidae, and Camelidae) that were consistently higher than the kappa statistic between C-ELISA and LAM-ELISA. The humoral response to one antigen and little or no response to the other in many animals argued for a parallel interpretation of C-ELISA and LAM-ELISA to increase sensitivity. PMID:9008794

  17. An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect isometamidium chloride in Oncorhynchus spp.

    PubMed

    Ardelli, B F; Woo, P T

    2000-02-09

    An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure isometamidium chloride in the plasma of Oncorhynchus tshawytscha and O. mykiss. Isometamidium-ovalbumin conjugate and anti-isometamidium antibodies were used to coat polystyrene plates. The peroxidase saturation technique was used to optimize the coating antigen concentration; it demonstrated low affinity of the isometamidium-ovalbumin conjugate but high affinity of the anti-isometamidium antibodies for polystyrene surface sites. The optimal conditions of antiisometamidium antibodies to coat plates was at pH 7.3 and a 1:1000 dilution (0.0012 mg ml(-1) protein). The ELISA was sensitive as it detected 0.0006 mg ml(-1) of isometamidium in fish plasma. Isometamidium diluted with saline could not be detected at concentrations less than 0.05 mg ml(-1). The results indicate that this ELISA is much more sensitive when isometamidium is bound to plasma than unbound isometamidium in saline.

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

    PubMed Central

    WANG, Naidong; YUAN, Anwen; MA, Jun; DENG, Zhibang; XUE, Liqun

    2015-01-01

    The use of serologically detectable male (SDM; also called H-Y) antigens to identify male embryos may be limited by the source of anti-SDM antibody. In the present study, novel anti-SDM B9-Fab recombinant clones (obtained by chain shuffling of an A8 original clone) were used to detect SDM antigens on murine embryos. Murine morulae and blastocysts (n=138) were flushed from the oviducts of Kunming mice and incubated with anti-SDM B9-Fab for 30 min at 37°C. With an indirect immunofluorescence assay, the membrane and inner cell mass had bright green fluorescence (presumptive males). Overall, 43.5% (60/138) were classified as presumptive males and 56.5% (78/138) as presumptive females, with 85.0 and 88.5% of these, respectively, confirmed as correct predictions (based on PCR analysis of a male-specific [Sry] sequence). We concluded that the anti-SDM B9-Fab molecule had potential for non-invasive, technically simple immunological sexing of mammalian embryos. PMID:25715803

  19. 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%.

  20. Definition of an HBsAg to DNA international unit conversion factor by enrichment of circulating hepatitis B virus forms.

    PubMed

    Désiré, N; Ngo, Y; Franetich, J-F; Dembele, L; Mazier, D; Vaillant, J-C; Poynard, T; Thibault, V

    2015-09-01

    Hepatitis B (HBV) virus infection is characterized by the overproduction of subviral particles (SVP) over infectious Dane particles (VP). Precise regulation of the ratio between these forms is unknown, but its fluctuation may have a clinical impact. An enrichment method was applied to assess the SVP/VP ratio in chronically infected patients (CHB) and to compare the sensitivity of HBs antigen (HBsAg) and DNA detection methods. Plasmas from 9 genotype A-D CHB patients were fractionated on Nycodenz(®) gradients, and both HBV DNA and HBsAg were quantified in each collected fraction using standardized techniques expressed in IU/mL. Infection of primary human hepatocytes (PHHs) was performed with crude or fractionated plasma. Independently of the genotype, all plasmas showed a similar rate-zonal separation profile characterized by a bottom DNA-enriched peak surmounted by HBsAg-enriched fractions. Inoculation of PHH with plasma-derived VP-enriched fractions led to long-lasting production of virus in cell supernatants with a SVP/VP ratio similar to that observed in patient plasmas. In the VP fraction, one IU of HBsAg corresponded to approximately 5 million IU of HBV DNA. Rate-zonal gradient separation directly applied on patient plasma allows a better insight into the distribution of VP in HBeAg-positive CHB carriers. This study highlights the sensitivity difference of the techniques classically used to monitor HBV infection and indicates that VP-associated HBsAg contributes modestly to the overall amount of total circulating HBsAg in CHB. Such a fractionation approach should help to understand the fine regulation of HBsAg production over replication at different stages of CHB. © 2015 John Wiley & Sons Ltd.

  1. 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.

  2. 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

  3. PCP can enhance dendritic cells to present the HBsAg peptide.

    PubMed

    Tang, Shang-hong; Gao, Liu-cun; Zhang, Zhi-yong; Li, Yun-ming; Bi, Qian; Song, Cao-jun; Yang, Gui-tao

    2011-02-01

    To investigate the influence of paraclorophenol (pCP) on dendritic cells loading and presenting HBsAg from peripheral blood monocytes of healthy volunteers identified as hepatitis B vaccine nonresponders. The density gradient centrifugation was performed to isolate mononuclear cells from 10 hepatitis B vaccine nonresponders. The adherent monocytes were incubated with HBsAg adding rhGM-CSF and rhIL-4 in the presence of absence of pCP for 7 days. Then the supernatant was collected for ELISA assays. The culture medium system without pCP was used as negative control and without pCP or HBsAG was named blank control. the matured DCs were co-incubated with autologous T lymphocytes for 72h and the supernatant was also collected for ELISA assays. In the presence of pCP, the level of IL-12 in supernate (265.68± 16.21) ng/L was significantly higher than the negative control (168.76±10.01) ng/L (P<0.05) and blank control (87±5.79)ng/L (P<0.05); after co-incubated with autologous T lymphocytes for 3 days, the level of IFN-γ with pCP (773.04±32.73) mg/L was also significantly higher than the negative control (573.59±26.11) mg/L (P<0.05) ans blank control (362.81±24.27)mg/L (P<0.05). pCP can effectively enhance the dendritic cells loading and presenting HBsAg from peripheral blood monocytes of healthy volunteers identified as hepatitis B vaccine nonresponders, which also can dramatically increase te autologous T lymphocytes response.7

  4. Serological diagnostics in myasthenia gravis based on novel assays and recently identified antigens.

    PubMed

    Zisimopoulou, Paraskevi; Brenner, Talma; Trakas, Nikolaos; Tzartos, Socrates J

    2013-07-01

    Myasthenia gravis (MG) is the most common immune-mediated disorder of the neuromuscular junction with a prevalence of 200-300/million population and its study has established paradigms for exploring other antibody-mediated diseases. Most MG patients (~85%) have autoantibodies against the muscle acetylcholine receptor (AChR-MG), whereas about 6% of MG patients have autoantibodies against the muscle specific kinase (MuSK-MG). Until recently no autoantibodies could be detected in the remaining patients (seronegative MG). Probably, the most sensitive assays for the detection of the autoantibodies in MG sera have been the radioimmunoprecipitation assays (RIPA) for both types of MG. However, with recent novel methods, not yet used routinely, it has been shown that the "seronegative" MG group includes patients with low levels of autoantibodies or of low affinity, against the known autoantigens, or even with antibodies to recently identified autoantigens. Since MG is heterogeneous in terms of pathophysiology, depending on the autoantigen targeted and on other factors (e.g. presence of thymoma), the serological tests are crucial in verifying the initial clinical diagnosis, whereas frequent measurement of autoantibody levels is important in monitoring the course of the disease and the efficacy of treatment. In addition, in AChR-MG, autoantibodies against the muscle proteins titin and ryanodin receptor have been identified; these antibodies are useful for the classification of MG, indicating the concomitant presence of thymoma, and as prognostic markers. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2015-08-01

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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. Enzyme-linked immunosorbent assay of antibodies to Epstein-Barr virus nuclear and early antigens in patients with infectious mononucleosis and nasopharyngeal carcinoma.

    PubMed

    Halprin, J; Scott, A L; Jacobson, L; Levine, P H; Ho, J H; Niederman, J C; Hayward, S D; Milman, G

    1986-03-01

    A sensitive enzyme-linked immunosorbent assay was used to measure titers of IgG antibodies against bacterially synthesized Epstein-Barr virus nuclear antigen and early antigen in sera from 100 healthy North Americans, 40 North American patients with infectious mononucleosis, and 48 Asian patients with nasopharyngeal carcinoma. All healthy persons previously infected with Epstein-Barr virus had antibodies to nuclear antigen, and 70% had very low but detectable antibody titers to early antigen. In contrast, patients with mononucleosis had nondetectable or very low levels of antibodies to nuclear antigen and high antibody levels to early antigen. High levels of antibody to early antigen also were seen in patients with nasopharyngeal carcinoma, and a decrease in this response during the first 12 months after diagnosis and treatment was a significant prognostic indicator of survival. The probability of survival was 75% for patients whose antibody concentration to early antigen remained constant or decreased, and near 0% for patients with increasing levels of antibody.

  11. 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

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

    PubMed Central

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

    2013-01-01

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

  13. A recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) for lungworm detection in seals.

    PubMed

    Ulrich, Sophia Arlena; Lehnert, Kristina; Siebert, Ursula; Strube, Christina

    2015-09-02

    Pinnipeds are frequently infected by the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus (Metastrongyloidea). Infections are frequently associated with secondary bacterial bronchopneumonia and are often lethal. To date, a reliable lungworm diagnosis in individual seals is only possible during necropsy as examination of faeces collected from resting places does not allow assignment to individuals. Therefore, a diagnostic tool for lungworm detection in living seals is desirable for monitoring health of seals in the wild and in captivity. Previously, an ELISA based on recombinant bovine lungworm major sperm protein (MSP) as diagnostic antigen was developed for lungworm diagnosis in cattle. In the present study, this test was adapted for detection of antibodies against lungworms in harbour (Phoca vitulina) and grey seals (Halichoerus grypus). Furthermore, sera of northern elephant seals (Mirounga angustirostris) were tested to evaluate whether the harbour/grey seal ELISA is suitable for this seal species as well. For ELISA evaluation, lungworm-positive and -negative sera of harbour and grey seals were analysed using horseradish peroxidase (HRP)-conjugated Protein A as secondary antibody. Optical density was measured and a receiver operating characteristic (ROC) analysis was performed to determine a cut-off value. Potential cross-reactions were examined by testing serum of seals positive for gastrointestinal and heart nematodes, but negative for lungworm infections. In addition, sera of northern elephant seals were analysed. Harbour and grey seal serum samples showed significant differences in optical density (OD) between serum of infected and uninfected animals resulting in a cut-off value of 0.422 OD with a specificity of 100% (95% CI: 87.23-100%) and a sensitivity of 97.83% (95% CI: 88.47-99.94%). Cross-reactions with heart or gastrointestinal nematodes were not observed. Analysis of northern elephant seal samples resulted in detection of antibodies

  14. 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.

  15. Rapid method for detection of influenza a and B virus antigens by use of a two-photon excitation assay technique and dry-chemistry reagents.

    PubMed

    Koskinen, Janne O; Vainionpää, Raija; Meltola, Niko J; Soukka, Jori; Hänninen, Pekka E; Soini, Aleksi E

    2007-11-01

    New separation-free assay methods for the rapid detection of influenza A and B virus antigens are presented. The methods employ dry-chemistry reagents and the recently developed two-photon excitation (TPX) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody onto polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay-specific reagents are dispensed into assay wells already in the manufacturing process of the test kits. The performance of the assay methods was tested with nasopharyngeal specimens using a time-resolved fluoroimmunoassay as a reference method. The results suggest that the new technique enables the rapid detection of influenza virus antigens with sensitivity and specificity comparable to that of the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of 3 orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed.

  16. Rapid Method for Detection of Influenza A and B Virus Antigens by Use of a Two-Photon Excitation Assay Technique and Dry-Chemistry Reagents▿

    PubMed Central

    Koskinen, Janne O.; Vainionpää, Raija; Meltola, Niko J.; Soukka, Jori; Hänninen, Pekka E.; Soini, Aleksi E.

    2007-01-01

    New separation-free assay methods for the rapid detection of influenza A and B virus antigens are presented. The methods employ dry-chemistry reagents and the recently developed two-photon excitation (TPX) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody onto polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay-specific reagents are dispensed into assay wells already in the manufacturing process of the test kits. The performance of the assay methods was tested with nasopharyngeal specimens using a time-resolved fluoroimmunoassay as a reference method. The results suggest that the new technique enables the rapid detection of influenza virus antigens with sensitivity and specificity comparable to that of the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of 3 orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed. PMID:17855571

  17. 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.

  18. Agreement between factor XIII activity and antigen assays in measurement of factor XIII: A French multicenter study of 147 human plasma samples.

    PubMed

    Caron, C; Meley, R; Le Cam Duchez, V; Aillaud, M F; Lavenu-Bombled, C; Dutrillaux, F; Flaujac, C; Ryman, A; Ternisien, C; Lasne, D; Galinat, H; Pouplard, C

    2017-06-01

    Factor XIII (FXIII) deficiency is a rare hemorrhagic disorder whose early diagnosis is crucial for appropriate treatment and prophylactic supplementation in cases of severe deficiency. International guidelines recommend a quantitative FXIII activity assay as first-line screening test. FXIII antigen measurement may be performed to establish the subtype of FXIII deficiency (FXIIID) when activity is decreased. The aim of this multicenter study was to evaluate the analytical and diagnostic levels of performance of a new latex immunoassay, K-Assay(®) FXIII reagent from Stago, for first-line measurement of FXIII antigen. Results were compared to those obtained with the Berichrom(®) FXIII chromogenic assay for measurement of FXIII activity. Of the 147 patient plasma samples, 138 were selected for analysis. The accuracy was very good, with intercenter reproducibility close to 7%. Five groups were defined on FXIII activity level (<5% (n = 5), 5%-30% (n = 23), 30%-60% (n = 17), 60%-120% (n = 69), above 120% (n = 24)), without statistical differences between activity and antigen levels (P value >0.05). Correlation of the K-Assay(®) with the Berichrom(®) FXIII activity results was excellent (r = 0.919). Good agreement was established by the Bland and Altman method, with a bias of +9.4% on all samples, and of -1.4% for FXIII levels lower than 30%. One patient with afibrinogenemia showed low levels of Berichrom(®) FXIII activity but normal antigen level and clot solubility as expected. The measurement of FXIII antigen using the K-Assay(®) is a reliable first-line tool for detection of FXIII deficiency when an activity assay is not available. © 2017 John Wiley & Sons Ltd.

  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. 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

  1. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Accelerated decline of GFR in diabetic nephropathy predicted by interferon release assay to tuberculosis antigens.

    PubMed

    Lane, Catherine; Ashcroft, Anthony; Bothamley, Graham; Yaqoob, Magdi M; Fan, Stanley L-S

    2011-01-01

    The QuantiFERON® test (QFT) is a diagnostic tool for active and latent tuberculosis (TB) infections. High rates of positivity to QuantiFERON® have been demonstrated in patients with chronic kidney disease (CKD) and diabetic patients. We performed a pilot study to investigate if QFT positivity in diabetic CKD patients predicted the rate of renal function decline. QFT was performed in 38 diabetic patients with CKD 4-5 not on dialysis. The rate of decline in estimated glomerular filtration rate (eGFR) was calculated. 18/38 patients had a positive QFT. Patients with a positive QFT had a steeper decline in eGFR, compared with patients with a negative QFT. Ethnicity (a marker of risk of previous TB exposure), urine protein/creatinine ratio, use of ACE inhibitors/angiotensin II receptor blockers and statins, serum C-reactive protein, vitamin D levels, HbA1c concentration and presenting GFR did not differ significantly. The finding in this small cohort needs to be replicated in a larger study because our study is susceptible to both type I and type II statistical error. We found that QFT positivity was associated with a more rapid rate of decline in GFR, but this association may be coincidental (with the difference in decline attributed to differences in the blood pressure or proteinuria of the two groups). Moreover, an association does not necessarily mean causality, although it would be interesting to speculate if we are identifying patients with latent TB who have an active interstitial nephritis. Another intriguing possibility is that this assay identifies patients with an immunological phenotype that predisposes to eGFR loss. Copyright © 2010 S. Karger AG, Basel.

  3. 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.

  4. Detection of egg drop syndrome virus antigen or genome by enzyme-linked immunosorbent assay or polymerase chain reaction.

    PubMed

    Dhinakar Raj, G; Sivakumar, S; Matheswaran, K; Chandrasekhar, M; Thiagarajan, V; Nachimuthu, K

    2003-10-01

    Mouse monoclonal antibodies (mAbs) were produced against an Indian isolate of egg drop syndrome (EDS) virus and characterized. Four hybridoma clones were secreting mAbs that bound to a 100 kDa protein, presumably the hexon protein. These mAbs were found to cross-react with two other Indian isolates of EDS virus and to the reference UK 127 strain. Three of these mAbs were mapped to the same epitope compared with the other mAb (F8), which bound to a different epitope. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed using the F8 mAbs as capture antibody and polyclonal chicken serum against EDS virus as detection antibody. A polymerase chain reaction (PCR) was used to detect the EDS viral genome. Following experimental infection of oestrogen-treated chickens with EDS virus, cloacal swabs, oviduct, uterus and spleen were collected at different days post-infection and used in both AC-ELISA and PCR, directly and after a single passage in embryonated duck eggs. The sensitivity and specificity of antigen detection by AC-ELISA or PCR was 95% and 98%, respectively. For diagnosis of EDS viral infections, PCR is recommended due to its ease and the lack of requirement of prepared reagents such as mAbs or conjugates. We recommend that PCR be performed directly on boiled tissue homogenates. Any negative samples may be passaged in embryonated duck eggs and the allantoic fluids tested by PCR before a conclusive negative diagnosis is given.

  5. 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.

  6. [The significance of serum GM and BG antigens assay for invasive fungal infections in hematological malignancies patients].

    PubMed

    Zeng, Shu-ying; Liu, Ting; Meng, Wen-tong; Chen, You-nan

    2011-01-01

    To evaluate the diagnostic value of serum galactomannan antigen (GM) and (1→3)-β-D-glucan antigen (BG) assay in invasive fungal infections (IFI) in the patients with hematologic malignancies and the role in monitoring therapeutic response. Fifty one patients with hematological malignancies met the criteria for inclusion: (1) body temperature above 38°C for 48 hours, (2) failure to respond to broad-spectrum antibiotic treatment, or (3) temperature rose again after the responded drop. Blood samples were collected twice at the first week, then once a week in at least four weeks. The double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and colorimetric assay were used for detecting GM and BG. The positive GM test is defined as two consecutive tests at different time GM value > 0.5 or > 0.8 and the positive G test is defined as BG value > 80 pg/ml. The patients were assigned into four groups as proven, probable, possible, and non-fungal infection respectively, and 21 normal volunteers were as controls. Two hundred and forty serum samples were collected from 51 patients including 2 of proven IFI, 26 probable IFI, 17 possible IFI and 6 non-fungal infection. The true-positive group including the proven and probable groups, and true negative group was the non-fungal infection group. GM tests were positive in 21 of 28 cases in true positive group, and only one of 6 cases in non-fungal infection. The sensitivity, specificity, positive predictive value and negative predictive value were 75%, 83.3%, 95.5% and 41.7%, respectively. G tests were positive in all 28 cases of the true positive group, and 4 in 6 non-fungal infection cases. The sensitivity, specificity, positive predictive value and negative predictive value were 100%, 33.3%, 87.5% and 100%, respectively. G test is more sensitive than GM test (P = 0.015), but there was no significant difference in specificity of the two tests (P = 0.242). In 19 of 21 patients with GM test positive, anti

  7. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-08-03

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

  10. 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.

  11. Optimization and Validation of an 8-Color Intracellular Cytokine Staining (ICS) Assay to Quantify Antigen-Specific T Cells Induced by Vaccination

    PubMed Central

    Horton, Helen; Thomas, Evan; Stucky, Jason; Frank, Ian; Moodie, Zoe; Huang, Yunda; Chiu, Ya-Lin; McElrath, M. Juliana; De Rosa, Stephen C.

    2009-01-01

    Candidate HIV-1 vaccines currently being evaluated in clinical trials are designed to elicit HIV-1-specific cellular immunity. Intracellular cytokine staining (ICS) assays allow sensitive, quantitative ex vivo assessments of antigen-specific T cells including immunophenotyping of responding cells and measurement of multiple effector functions. Additionally, the use of banked cryopreserved PBMC samples makes this assay attractive in the setting of large efficacy trials where it is less feasible to perform immunoassays on freshly isolated samples. Here we describe extensive studies to optimize and quantitatively validate the 8-color ICS assay for use in clinical trials of candidate vaccines, which includes measurement of viable IFN-γ, IL-2, TNF-α and IL-4 secreting CD4+ and CD8+ T cells. We show that omission of viability dye staining results in an over-estimate of the true antigen-specific T cell response by up to two-fold. After optimization, the 8-color assay was validated for specificity, precision, linearity, limit of quantitation and robustness. The assay has a lower quantitation limit, generally below 0.04%, depending on the cytokine subset. Additionally, with appropriate gating, the 8-color assay gives comparable cytokine-positive responses to those observed with the conventional 4-color assay. In conclusion, we provide the first description of a quantitatively validated ICS assay, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection. PMID:17451739

  12. Evaluation of diagnostic accuracy of two rapid stool antigen tests using an immunochromatographic assay to detect Helicobacter pylori.

    PubMed

    da Silva-Etto, Joyce Matie Kinoshita; Mattar, Rejane; Villares-Lopes, Cibele Aparecida; Marques, Sergio Barbosa; Carrilho, Flair José

    2017-05-05

    The stool antigen assay for H. pylori infection diagnosis with monoclonal antibodies is a simple and recommended technique by the Maastricht V/Florence consensus report. Recently, Pylori K-Set K-1219 (Coris Bioconcept Sprl, Belgium) and HP-F23 (Symbiosys, Brazil) have been made commercially available in Brazil. Thus, the aim of this study was to evaluate the diagnostic accuracies of these two rapid stool antigen tests by immunochromatographic assays (index tests) for the clinical practice. A total of 98 patients who underwent upper gastrointestinal endoscopy and (13)C-urea breath test entered the study. H. pylori infection status was defined by the combination of the rapid urease test and the (13)C-urea breath test (reference standard). Two observers who were aware of H. pylori status performed the reading of index tests. Diagnostic accuracy (sensitivity, specificity, positive predictive value, negative predictive value with 95% confidence intervals, positive likelihood ratio, negative likelihood ratio and kappa index measure of agreement) were determined. The index tests where in perfect agreement with the H. pylori status with kappa values of 0.87 for Pylori K-Set K-1219 and 0.92 for HP-F23. The sensitivity of HP-F23 was 97.9% (IC95%: 87.5-100) and specificity was 93.8% (IC95%; 84-97.2).The positive likelihood ratio was 15.8, and the negative likelihood ratio was 0.02. The Pylori K-Set K-1219 had a sensitivity of 87.7% (IC95%: 74.5-94.9) and a specificity of 100% (IC95%: 91.6-100); the positive likelihood ratio was ∞, and the negative likelihood ratio was 0.1. The test line on the cassette device of HP-F23 was stronger than of the Pylori K-Set K-1219. The HP-F23 test performed better in clinical practice. Nonetheless, the (13)C-urea breath test is more reliable technique. Moreover, caution must be paid to the trace or clear pale test line readings that were observed in false positive and false negative results, leading to incorrect management of the patient

  13. 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.

  14. Comparison of Real-Time PCR and Antigen Assays for Detection of Hepatitis E Virus in Blood Donors

    PubMed Central

    Knabbe, C.; Dreier, J.

    2014-01-01

    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 1.92E + 03 to 2.19E + 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 >1.0E + 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. PMID:24740079

  15. 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

    Balestrino, E A; Daniel, T M; de Latini, M D; 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.

  16. 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

  17. DNA-based vaccination induces humoral and cellular immune responses against hepatitis B virus surface antigen in mice without activation of C-myc

    PubMed Central

    Zhao, Lian San; Qin, Shan; Zhou, Tao You; Tang, Hong; Liu, Li; Lei, Bing Jun

    2000-01-01

    AIM: To develop a safe and effective DNA vaccine for inducing humoral and cellular immunological responses against hepatitis B virus surface antigen (HBsAg). METHODS: BALB/c mice were inoculated with NV-HB/s, a recombinant plasmid that had been inserted S gene of hepatitis B virus genome and could express HBsAg in eukaryotes. HBsAg expression was measured by ABC immunohistochemical assay, generation of anti-HBs by ELISA and cytotoxic T lymphocyte (CTL), by MTT method, existence of vaccine DNA by Southern blot hybridization and activation of oncogene C-myc by in situ hybridization. RESULTS: With NV-HB/s vaccination by intramuscular injection, anti-HBs was initially positive 2 wk after inoculation while all mice tested were HBsAg positive in the muscles. The titers and seroconversion rate of anti-HBs were steadily increasing as time went on and were dose-dependent. All the mice inoculated with 100 μg NV-HB/s were anti-Bs positive one month after inoculation, the titer was 1∶1024 or more. The humoral immune response was similar induced by either intramuscular or intradermal injection. CTL activities were much stronger (45.26%) in NV-HB/s DNA immunized mice as compared with those (only 6%) in plasma-derived HBsAg vaccine immunized mice. Two months after inoculation, all muscle samples were positive by Southern-blot hybridization for NV-HB/s DNA detection, but decreased to 25% and all were undetectable by in situ hybridization after 6 mo. No oncogene C-myc activation was found in the muscle of inoculation site. CONCLUSION: NV-HB/s could generate humoral and cellular immunological responses against HBsAg that had been safely expressed in situ by NV-HB/s vaccination. PMID:11819565

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

    PubMed

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

    1994-01-01

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

  19. 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.

  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. Short Communication: Low False Recent Rate of Limiting Antigen-Avidity Assay Combined with HIV-1 RNA Data in Botswana.

    PubMed

    Moyo, Sikhulile; Kotokwe, Kenanao P; Mohammed, Terence; Boleo, Corretah; Mupfumi, Lucy; Chishala, Samuel; Tsalaile, Lesedi; Bussmann, Hermann; Gaseitsiwe, Simani; Musonda, Rosemary; Makhema, Joseph; Baum, Marianna; Marlink, Richard; Engelbrecht, Susan; Essex, Max; Novitsky, Vladimir

    2017-01-01

    Cross-sectional estimation of HIV incidence could misclassify some established or chronic HIV infections as recent. Usually long-term nonprogressors, elite and viremic controllers, and individuals on ART contribute to misclassification. Local data on the false recent rate (FRR) could minimize misclassification during estimation of HIV incidence. To improve monitoring of HIV incidence, we estimated local FRR in Botswana. A total of 1,036 specimens from individuals infected for at least 1.5-2 years were sampled between 2004 and 2009 and tested using the limiting antigen (LAg)-avidity assay using a cutoff of 1.5 normalized optical density units. The FRR was 0.97% (10/1,036; 95% confidence interval [CI] 0.46-1.77). Four samples had HIV-1 RNA >1,000 cps/ml, giving an adjusted FRR of 0.39% (4/1,036; 95% CI 0.11-0.99). A combination of LAg and HIV-1 RNA load data resulted in FRR below 1% in the Botswana population.

  2. 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.

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

    PubMed Central

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

    2012-01-01

    A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases. PMID:22815145

  4. 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.

  5. Evaluation of the VecTest Malaria Antigen Panel assay for the detection of Plasmodium falciparum and P. vivax circumsporozoite protein in anopheline mosquitoes in Thailand.

    PubMed

    Sattabongkot, Jetsumon; Kiattibut, Chukree; Kumpitak, Chalermporn; Ponlawat, Alongkot; Ryan, Jeffrey R; Chan, Adeline S T; Davé, Kirti; Wirtz, Robert A; Coleman, Russell E

    2004-03-01

    We evaluated the performance of the VecTest Malaria Antigen Panel (V-MAP) assay for the detection of Plasmodium falciparum and P. vivax (variants 210 and 247) circumsporozoite protein in anopheline mosquitoes in Thailand. The V-MAP assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. The circumsporozoite (CS) ELISA was used as the reference standard. Mosquitoes evaluated in the study included field-collected specimens (n = 930) and laboratory-reared specimens that had been fed on blood collected from patients with and without Plasmodium gametocytes (n = 4,110) or on cultured P. falciparum gametocytes (n = 262). Field-collected mosquitoes were triturated individually or in pools of 2-5 and tested using 613 V-MAP assays. Laboratory-reared specimens were tested individually using 4,372 V-MAP assays. Assay performance depended on the species of Plasmodium and the number of sporozoites used as the cut-off. For P. falciparum, optimal performance was achieved using a cut-off of 150 sporozoites (sensitivity = 100%, specificity = 99.2%, and accuracy = 0.99). For P. vivax variant 210, optimal performance was also achieved using a cut-off of 150 sporozoites (sensitivity = 94.8%, specificity = 94.5%, and accuracy = 0.95). We were unable to develop a standard-curve for the CS-ELISA using P. vivax variant 247 because of a lack of sporozoites; however, using a cut-off of 30 pg P. vivax 247 antigen (mosquitoes with less than this amount of antigen were considered negative), assay performance (sensitivity = 94.3%, specificity = 99.2%, and accuracy = 0.99) was comparable to that achieved for P. falciparum and P. vivax 210. These results clearly demonstrate that the V-MAP assay performs at an acceptable level and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. [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.

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

    PubMed

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

    2015-10-01

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

  14. Comparison of human saliva and synthetic histo-blood group antigens usage as ligands in norovirus-like particle binding and blocking assays.

    PubMed

    Uusi-Kerttula, Hanni; Tamminen, Kirsi; Malm, Maria; Vesikari, Timo; Blazevic, Vesna

    2014-06-01

    Blocking of norovirus-like particle binding to their cellular ligands, histo-blood group antigens with immune sera, is considered a surrogate norovirus neutralization assay. We compared human secretor positive saliva and synthetic biotinylated carbohydrates as a source of histo-blood group antigens in binding and blocking assays. Six norovirus capsid-derived virus-like particles belonging to genogroup I (GI-1-2001 and GI-3-2002) and genogroup II (GII-4-1999, GII-4-2010 New Orleans, GII-4-2012 Sydney and GII-12-1998) noroviruses were produced by a recombinant baculovirus expression system and binding profile to saliva type A, B and O and to synthetic antigens (A trimer, B trimer, H type 1, H type 3, Lewis(a) and Lewis(b)) was identified. Good correlation between virus-like particle binding to saliva type A and synthetic A trimer (r = 0.66, p < 0.05) and saliva type B and synthetic B trimer (r = 0.75, p < 0.05) was observed. Binding of each norovirus virus-like particle to the selected histo-blood group antigens was blocked by convalescent sera from NoV-infected subjects or type-specific mouse antisera. Our results support the use of either saliva or synthetic antigens in blocking assay to measure the ability of norovirus antisera to block virus-like particle binding to the carbohydrate ligands. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  15. 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

  16. 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

  17. 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.

  18. HCV core-antigen assay as an alternative to HCV RNA quantification: A correlation study for the assessment of HCV viremia.

    PubMed

    Alonso, Roberto; Pérez-García, Felipe; López-Roa, Paula; Alcalá, Luis; Rodeño, Pilar; Bouza, Emilio

    2017-02-25

    Detection of hepatitis C virus (HCV) RNA and the HCV core antigen assay (HCV-Ag) are reliable techniques for the diagnosis of active and chronic HCV infection. Our aim was to evaluate the HCV-Ag assay as an alternative to quantification of HVC RNA. A comparison was made of the sensitivity and specificity of an HCV-Ag assay (204 serum samples) with those of a PCR assay, and the correlation between the two techniques was determined. The sensitivity and specificity of HCV-Ag was 76.6% and 100%, respectively. Both assays were extremely well correlated (Pearson coefficient=0.951). The formula (LogCV=1.15*LogAg+2.26) was obtained to calculate the viral load by PCR from HCV-Ag values. HCV-Ag was unable to detect viral loads below 5000IU/mL. Although the HCV-Ag assay was less sensitive than the PCR assay, the correlation between both assays was excellent. HCV-Ag can be useful as a first step in the diagnosis of acute or chronic HCV infection and in emergency situations. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  19. 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

  20. 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

  1. Serum Hepatitis B Virus DNA, RNA, and HBsAg: Which Correlated Better with Intrahepatic Covalently Closed Circular DNA before and after Nucleos(t)ide Analogue Treatment?

    PubMed

    Gao, Yuhua; Li, Yutang; Meng, Qinghua; Zhang, Zhanqing; Zhao, Ping; Shang, Qinghua; Li, Yue; Su, Mingze; Li, Tong; Liu, Xueen; Zhuang, Hui

    2017-10-01

    The study was designed to investigate whether serum hepatitis B virus (HBV) RNA is a strong surrogate marker for intrahepatic HBV covalently closed circular DNA (cccDNA) compared with serum HBV DNA, hepatitis B surface antigen (HBsAg), and hepatitis B e antigen (HBeAg) in HBeAg-positive chronic hepatitis B (CHB) patients. Serum HBV RNA, HBV DNA, HBsAg, HBeAg, and intrahepatic cccDNA were quantitatively detected at baseline (n = 82) and 96 weeks (n = 62) after treatment with nucleos(t)ide analogue (NUC) in HBeAg-positive CHB patients. The correlations among serum HBV RNA, HBV DNA, HBsAg, HBeAg, and intrahepatic cccDNA levels were then statistically analyzed. The results showed that pretreatment intrahepatic cccDNA levels correlated better with serum HBV DNA levels (r = 0.36, P < 0.01) than with serum HBV RNA levels (r = 0.25, P = 0.02), whereas no correlations were found between pretreatment intrahepatic cccDNA levels and HBsAg (r = 0.15, P = 0.17) or HBeAg (r = 0.07, P = 0.56) levels. At 96 weeks after NUC treatment, intrahepatic cccDNA levels correlated well with HBsAg levels (r = 0.39, P < 0.01) but not with serum HBV RNA, HBV DNA, and HBeAg levels (all P > 0.05). Besides, the decline in the intrahepatic cccDNA level from baseline to week 96 correlated better with the reduction in the serum HBsAg levels than with the decreases in the levels of the other markers (for the HBsAg decline, r = 0.38, P < 0.01; for the HBV DNA decline, r = 0.35, P = 0.01; for the HBV RNA decline, r = 0.28, P < 0.05; for the HBeAg decline, r = 0.18, P = 0.19). In conclusion, the baseline serum HBV RNA level or its decline after 96 weeks of NUC therapy correlated with the corresponding intrahepatic cccDNA level, while it was less than that seen with serum HBV DNA at baseline and HBsAg (or its decline) at 96 weeks after treatment, respectively. Copyright © 2017 American Society for Microbiology.

  2. 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

  3. Combined Use of Paracoccidioides brasiliensis Recombinant 27-Kilodalton and Purified 87-Kilodalton Antigens in an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Paracoccidioidomycosis

    PubMed Central

    Díez, Soraya; Gómez, Beatriz L.; McEwen, Juan G.; Restrepo, Angela; Hay, Rod J.; Hamilton, Andrew J.

    2003-01-01

    The diagnosis of paracoccidioidomycosis (PCM) has relied on the identification of the host's humoral response by using a variety of immunological methods, such as complement fixation and immunodiffusion. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens. The use of combinations of purified, well-characterized antigens appears preferable and may yield optimum results. Accordingly an indirect enzyme-linked immunosorbent assay (ELISA) using combinations of the previously described 27-kDa recombinant antigen and the 87-kDa heat shock protein were used for diagnosis and follow-up of patients with PCM. A total of 37 patients classified according to their clinical presentations (7 with the acute or subacute form of the disease, 22 with the chronic form of the disease, and 8 with the chronic unifocal form) were studied. Eighteen of these patients were also evaluated at every follow-up appointment. Forty serum samples from patients with other diseases and 50 serum samples from healthy individuals were also studied. Detection of anti-27-kDa and anti-87-kDa antibodies in sera of patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 92% with a specificity of 88% in comparison with normal human sera and 90% in comparison with the heterologous sera. These results demonstrated a significant increase in sensitivity and specificity compared to results when the antigens were used separately. Thus, the use of combinations of well-defined antigens appears to offer clear advantages over the use of single antigens when diagnosing PCM. PMID:12682142

  4. 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

  5. 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.

  6. 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.

  7. [The significance and place of HBsAg neutralization test in the diagnosis and algorithm of hepatitis B infection].

    PubMed

    Aytaç, Özlem; Toyran, Alparslan; Aksoy, Altan

    2017-04-01

    The diagnosis of hepatitis B virus infection is evaluated serologically, virologically, biochemically, and with histologic liver indicators. The aim of this study was to investigate the place and significance of the HBsAg neutralization test in the diagnostic algorithm for hepatitis Binfection. From the venous blood samples sent to Ankara Numune Education and Research Hospital Medical Microbiology Laboratory between September 2014 and May 2016 for HBsAg test, serum samples regarded each patient as reactive (≥ 0.9 S\\CO) , 9 ≤ S ≤ CO ≤ 30) were studied twice. A total of 105 samples which were reactive in both analyses were included in the study. After the evaluation of these samples by neutralization confirmation test, which is routinely performed in our laboratory, the samples were stored under optimal conditions and studied for HBV DNA with the real-time PCR and for HBeAg, anti-HBeAg, anti-HBc IgM, and anti-HBc total antibody assays by ELISA. The 105 samples, in which HBsAg was detected, were analyzed with the neutralization test. The presence of HBsAg was confirmed by neutralization test in 67 of 105 samples (63.8%), and of these patients, two patients (2.3%) had negative HBV DNA and anti-HBc total antibody test (false positive neutralization test). Of the 105 samples included in the study, the anti-HBc total antibody test was positive in 78 patients (74.3%). However, out of these 78 patients who were positive for the anti-HBc total antibody test, there were 13 patients (16.7%) with negative neutralization and HBV DNA test results (false positive anti-HBc total antibody test). The HBV DNA positivity was detected significantly lower in samples with HBsAg level ≤ 5 S/CO compared to the samples with HBsAg level > 5 S/CO (p= 0.020). Also, if the unit price of the neutralization test used in our study was considered, the cost was 17,00 TL while the unit price of HBV DNA test was 55,00 TL. Utilization of the neutralization test instead of HBV DNA test

  8. Development of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies to O157 Antigen of Escherichia coli

    PubMed Central

    Laegreid, William; Hoffman, Mark; Keen, James; Elder, Robert; Kwang, Jimmy

    1998-01-01

    The O157 antigen of Escherichia coli shares structural elements with lipopolysaccharide (LPS) antigens of other bacterial species, notably Brucella abortus and Yersinia enterocolitica O9, a fact that confounds the interpretation of assays for anti-O157 antibodies. To address this problem, a blocking enzyme-linked immunosorbent assay (bELISA) was designed with E. coli O157:H7 LPS as the antigen and a monoclonal antibody specific for E. coli O157, designated 13B3, as the competing antibody. The bELISA had equivalent sensitivity to, and significantly higher specificity than, the indirect ELISA (iELISA), detecting anti-O157 antibodies in sera from cattle experimentally inoculated with O157:H7. Only 13% of sera from naive heifers vaccinated for or experimentally infected with B. abortus had increased anti-O157 bELISA titers, while 61% of anti-O157 iELISA titers were increased. The bELISA is a sensitive and specific method for the detection of serum antibodies resulting from exposure to E. coli O157. PMID:9521150

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

    PubMed

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

    2007-11-01

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

  10. 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.

  11. 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.

  12. 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.

  13. In vitro use of autologous dendritic cells improves detection of T cell responses to hepatitis B virus (HBV) antigens.

    PubMed

    Carotenuto, Patrizia; Artsen, Andrè; Niesters, Hubert G; Osterhaus, Albert D; Pontesilli, Oscar

    2009-02-01

    T lymphocyte responses to hepatitis B virus (HBV) core antigen (HBcAg) are vigorous and easily detectable in vitro during recovery from acute hepatitis B but significantly weaker in patients with chronic HBV infection. In contrast, T cell responses to hepatitis B surface antigen (HBsAg) are almost undetectable during infection and even in a substantial fraction of subjects receiving vaccination with HBsAg. The aim of this study was to investigate whether the use of dendritic cells (DCs) in an in vitro assay could increase the detection of HBV-specific T cells in these conditions. Autologous monocyte-derived DCs, compared to direct HBsAg addition to the cultures, increased the stimulation of HBs- specific T cells. These were detected in 73% of healthy subjects who had recently received hepatitis B vaccine and in 43% of patients recovering from acute hepatitis B. Likewise, proliferation in response to DC-presented HBcAg was detected in both CD4(+) and CD8(+) T cells from the majority of chronic hepatitis B patients. A longitudinal evaluation of HBc-specific T cell responses during and after a 1-year treatment with pegylated interferon (IFN)-alpha showed that HBc-specific CD4(+) T cell responses had no correlation with sustained virus suppression whereas CD8(+) T cell responses were more frequently detected in patients able to control HBV replication after therapy interruption. The use of autologous DCs as antigen-presenting cells appears applicable to clinically relevant in vitro evaluation of T cell responses, particularly in those conditions characterized by low frequency of circulating antigen-specific cells and suboptimal in vivo activation. (c) 2008 Wiley-Liss, Inc.

  14. 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).

  15. [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.

  16. A low steady HBsAg seroprevalence is associated with a low incidence of HBV-related liver cirrhosis and hepatocellular carcinoma in Mexico: a systematic review.

    PubMed

    Roman, Sonia; Panduro, Arturo; Aguilar-Gutierrez, Yadira; Maldonado, Montserrat; Vazquez-Vandyck, Maclovia; Martinez-Lopez, Erika; Ruiz-Madrigal, Bertha; Hernandez-Nazara, Zamira

    2009-06-01

    To address the relationship between hepatitis B virus (HBV) endemicity and HBV-related liver diseases in Mexico. Research literature reporting on HBsAg and antibody to hepatitis B core antigen (anti-HBc) prevalence in Mexican study groups were searched in NLM Gateway, PubMed, IMBIOMED, and others. Weighted mean prevalence (WMP) was calculated from the results of each study group. A total of 50 studies were analyzed. Three nationwide surveys revealed an HBsAg seroprevalence of less than 0.3%. Horizontal transmission of HBV infection occurred mainly by sexual activity and exposure to both contaminated surgical equipment and body fluids. High-risk groups exposed to these factors included healthcare workers, pregnant women, female sex workers, hemodialysis patients, and emergency department attendees with an HBsAg WMP ranging from 1.05% (95% confidence interval [CI], 0.68-1.43) to 14.3% (95% CI, 9.5-19.1). A higher prevalence of anti-HBc in adults than those younger than 20 years was associated with the main risk factors. Anti-HBc WMP ranged from 3.13% (95% CI, 3.01-3.24) in blood donors to 27.7% (95% CI, 21.6-33.9) in hemodialysis patients. A heterogeneous distribution of HBV infection was detected, mainly in native Mexican groups with a high anti-HBc WMP of 42.0% (95% CI, 39.5-44.3) but with a low HBsAg WMP of 2.9% (95% CI 2.08-3.75). Estimations of the Mexican population growth rate and main risk factors suggest that HBsAg seroprevalence has remained steady since 1974. A low HBsAg prevalence is related to the low incidence of HBV-related liver cirrhosis and hepatocellular carcinoma (HCC) previously reported in Mexico.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. DNA-based vaccination induces humoral and cellular immune responses against hepatitis B virus surface antigen in mice without activation of C-myc.

    PubMed

    Zhao, Lian-San; Qin, Shan; Zhou, Tao-You; Tang, Hong; Liu, Li; Lei, Bing-Jun

    2000-04-01

    AIM:To develop a safe and effective DNA vaccine for inducing humoral and cellular immunological responses against hepatitis B virus surface antigen (HBsAg).METHODS:BALB/c mice were inoculated with NV-HB/s, a recombinant plasmid that had been inserted S gene of hepatitis B virus genome and could express HBsAg in eukaryotes. HBsAg expression was measured by ABC immunohis tochemical assay, generation of anti-HBs by ELISA and cytotoxic T lymphocyte (CTL), by MTT method, existence of vaccine DNA by Southern blot hybridization and activation of oncogene C-myc by in situ hybridization.RESULTS:With NV-HB/s vaccination by intramuscular injection, anti-HBs was initially positive 2 weeks after inoculation while all mice tested were HBsAg positive in the muscles.The titers and seroconversion rate of anti-HBs were steadily increasing as time went on and were dose dependent. All the mice inoculated with 100&mgr;g NV-HB/s were anti-HBs positive one month after inoculation, the titer was 1 1024 or more. The humoral immune response was similar induced by either intramuscular or intradermal injection. CTL activities were much stronger (45.26%) in NV-HB/s DNA immunized mice as compared with those (only 6%) in plasma-derived HBsAg vaccine immunized mice. Two months after inoculation, all muscle samples were positive by Southernblot hybridization for NV-HB/s DNA detection, but decreased to 25% and all were undetectable by in situ hybridiza-tion after 6 months.No oncogene C-myc activation was found in the muscle of inoculation site.CONCLUSION:NV-HB/s could generate humoral and cellular immunolo-gical responses against HBsAg that had been safely expressed in situ by NV-HB/s vaccination.

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

    PubMed

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

    2014-04-01

    In low-income-countries, screening for hepatitis C virus (HCV) infection is often based on rapid tests (RT). Their lower sensitivity compared to enzyme immunoassay (EIA) suggests that newer HCV Antigen/Antibody (Ag/Ab) combination assays might have a role in such countries. To test this idea, 1998 blood donors were tested at the University Teaching Hospital blood bank in Yaoundé, Cameroon simultaneously with a RT (HCV rapid test, Human Diagnostics, Berlin, Germany) according to standard practice (S1) and with an Ag/Ab assay (Monolisa HCV Ag/Ab Ultra, Biorad, France) (S2). All discordant, borderline and reactive samples were submitted to confirmatory testing by immunoblot and/or HCV-RNA. Of the 86 (4.3%) samples positive with one or both strategies, 29 were confirmed negative, 37 positive and 20 were false positive or resolved infection. There was a significant difference in test sensitivity (p=0.01) between S1 (70.3%) and S2 (91.9%) but not in test specificity (99.4% and 98.6%, respectively). The benefit of the Ag/Ab assay in the detection of recent HCV seronegative infections could not be evaluated since no Antigen-only donations were identified. However, better Ag/Ab test sensitivity compared to RT supports the implementation of these newer immunoassays for HCV screening in the African blood bank setting. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. 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.

  4. 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

  5. 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.

  6. Comparable between rapid one step immunochromatographic assay and ELISA in the detection of prostate specific antigen in vaginal specimens of raped women.

    PubMed

    Peonim, Vichan; Chirachariyavej, Thamrong; Atamasirikul, Kalayanee; Talthip, Jate

    2007-12-01

    Rape is a crime found in Thailand nowadays. The crime is often lacking of eyewitnesses. Therefore, examination for forensic biological evidence becomes quite important, especially investigating sperm and semen in vaginal specimens of the victim. Acid phosphatase test for semen is commonly used in Thailand but is just a presumptive test. Recently, confirmatory kit tests became available in Thailand for detecting the prostate specific antigen (PSA) from semen. This test is simpler and cheaper than ELISA. To compare the rapid one-step immunochromatographic assay with ELISA for the detection of prostate specific antigen in vaginal specimens of raped women. A diagnostic test was conducted on the vaginal specimens of raped women that were sent to the laboratory of the Pathology Department, Faculty of Medicine, Ramathibodi Hospital, Mahidol University during April-August 2006. One hundred vaginal specimens were examined for prostate specific antigen by rapid one step immunochromatographic assay and compared with ELISA. There were 85% and 83% of sensitivity, 85% and 85% of specificity, 85% and 85% of accuracy, 89% and 89% of positive predictive value, and 79% and 77% of negative predictive value from rapid one-step test kit and ELISA respectively The result showed that there was no difference on specificity, accuracy and positive predictive value between the two methods but sensitivity and negative predictive value of rapid one-step test kit was better than ELISA. The research team recommends that rapid one-step test kit for prostate specific antigen should be routine service in vaginal specimens of raped women.

  7. Comparison of multiple assays for detecting human antibodies directed against surface antigens on normal and malignant human tissue culture cells.

    PubMed

    Rosenberg, S A; Schwarz, S; Anding, H; Hyatt, C; Williams, G M

    1977-01-01

    Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cell lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual and end-point cytolysis assay and the 51Chromium release assay were equally sensitive in measuring complement mediated antibody cytoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody.

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

    PubMed

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

    2015-08-01

    Anthrax caused by Bacillus anthracis is primarily a disease of herbivorous animals, although several mammals are vulnerable to it. ELISA is the most widely accepted serodiagnostic assay for large scale surveillance of cutaneous anthrax. The aims of this study were to develop and evaluate a quantitative ELISA for determination of IgG antibodies against B. anthracis protective antigen (PA) in human cutaneous anthrax cases. Quantitative ELISA was developed using the recombinant PA for coating and standard reference serum AVR801 for quantification. A total of 116 human test and control serum samples were used in the study. The assay was evaluated for its precision, accuracy and linearity. The minimum detection limit and lower limit of quantification of the assay for anti-PA IgG were 3.2 and 4 µg/ml, respectively. The serum samples collected from the anthrax infected patients were found to have anti-PA IgG concentrations of 5.2 to 166.3 µg/ml. The intra-assay precision per cent CV within an assay and within an operator ranged from 0.99 to 7.4 per cent and 1.7 to 3.9 per cent, respectively. The accuracy of the assay was high with a per cent error of 6.5 - 24.1 per cent. The described assay was found to be linear between the range of 4 to 80 ng/ml (R [2] = 0.9982; slope = 0.9186; intercept = 0.1108). The results suggested that the developed assay could be a useful tool for quantification of anti-PA IgG response in human after anthrax infection or vaccination.

  9. 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.

  10. 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.

  11. 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

  12. Comparison of Binax NOW urine antigen test and pneumococcal DNA assay using qPCR before and after nasopharyngeal swabbing in healthy Malawian children.

    PubMed

    Lees, E A; Ho, D K K; Guiver, M; Mankhambo, L A; French, N; Carrol, E D

    2015-11-01

    Diagnosis of invasive pneumococcal disease is challenging. We compared Binax NOW pneumococcal urinary antigen test with blood pneumococcal PCR in healthy Malawian children with and without pneumococcal carriage, and we found a high false-positive rate with Binax NOW. Blood pneumococcal PCR positivity was 66/88 (75%) compared to 5/27 (18%) when nasopharyngeal swabbing was performed first compared to after blood sampling for pneumococcal blood PCR. We speculate that nasopharyngeal swabbing may be causing a breach of mucosal integrity, leading to invasion into the bloodstream. These findings need to be confirmed with autolysin-based PCR assays.

  13. 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.

  14. 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

  15. 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

  16. Clinical Significance of Quantitative HBsAg Titres and its Correlation With HBV DNA Levels in the Natural History of Hepatitis B Virus Infection.

    PubMed

    Karra, Vijay K; Chowdhury, Soumya J; Ruttala, Rajesh; Polipalli, Sunil K; Kar, Premashis

    2016-09-01

    Quantification of serum hepatitis B antigen (HBsAg) is an important test that marks active infection with hepatitis B and helps in the prediction of the clinical outcome and management of hepatitis B virus (HBV) infection. Correlation with HBV DNA quantitative levels may help in developing strategies for antiviral treatment. This study is aimed to evaluate HBsAg titres in various phase of HBV infection in HBsAg positive patients, and its correlation with HBV DNA viral load levels. 976 HBV related patients were analysed in this retrospective cross-sectional study. Patients were categorised on the basis of the phase of HBV infection: immune tolerant phase (IT, n = 123), immune clearance phase (IC, n = 192), low-replicative phase (LR, n = 476), and HBeAg-negative hepatitis (ENH, n = 185). HBsAg titres were quantified and correlated with HBV-DNA levels and clinical parameters. Median HBsAg titres were different between each phases of HBV infection (P < 0.001): (4.62 log10 IU/ml), IC (3.88 log10 IU/ml), LR (2.76 log10 IU/ml) and ENH (2.94 log10 IU/ml). HBsAg and HBV DNA levels showed significant correlation in the whole group (r = 0.694, P < 0.001), and this was also observed in different phases of HBV infection. Strong correlation in IT phase (r = 0.603, P < 0.001) and IC phase (r = 0.523, P < 0.001), moderate in LR phase (r = 0.362, P < 0.001) and weak in ENH (r = 0.110, P = 0.04). No correlation was observed between serum HBsAg levels and biochemical parameters. The study demonstrated significant difference in the median baseline values of serum HBsAg titres in different phases of HBV infection and provides additional information in understanding the natural history of HBV-infection.

  17. Lymphocyte response to hepatitis B surface antigen. Findings in hepatitis and Indian childhood cirrhosis.

    PubMed

    Chandra, R K

    1975-07-01

    The lymphocyte delayed hypersensitivity response to phytohaemagglutinin (PHA) and hepatitis B antigen (HBsAG) was evaluated by two in vitro tests-leucocyte migration inhibition and DNA synthesis. Patients convalescing from HBsAG-positive hepatits showed the presence of a state of cell-mediated immune responsiveness to the antigen. In Indian childhood cirrhosis, there was a failure of response to HBsAG and a slight but significant depression of reaction to PHA. It is suggested that the lack of immune reactivity to HBsAG, perhaps determined genetically, may be a significant factor in the evolution of cirrhosis in Indian children.

  18. Lymphocyte response to hepatitis B surface antigen. Findings in hepatitis and Indian childhood cirrhosis.

    PubMed Central

    Chandra, R K

    1975-01-01

    The lymphocyte delayed hypersensitivity response to phytohaemagglutinin (PHA) and hepatitis B antigen (HBsAG) was evaluated by two in vitro tests-leucocyte migration inhibition and DNA synthesis. Patients convalescing from HBsAG-positive hepatits showed the presence of a state of cell-mediated immune responsiveness to the antigen. In Indian childhood cirrhosis, there was a failure of response to HBsAG and a slight but significant depression of reaction to PHA. It is suggested that the lack of immune reactivity to HBsAG, perhaps determined genetically, may be a significant factor in the evolution of cirrhosis in Indian children. PMID:810095

  19. 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

  20. Development of quantitative immunochromatographic assay for rapid and sensitive detection of carbohydrate antigen 19-9 (CA 19-9) in human plasma.

    PubMed

    Baryeh, Kwaku; Takalkar, Sunitha; Lund, Michelle; Liu, Guodong

    2017-09-05

    A quantitative immunochromatographic assay (QIA) was developed by using gold nanoparticle (GNP)-based lateral flow strip biosensor (LFSB) and a portable strip reader for rapid and sensitive quantitation of Carbohydrate Antigen 19-9 (CA 19-9) in human plasma. CA 19-9 is a biomarker that has been associated with cancers (such as pancreatic and colorectal cancers) and various non-cancerous diseases. The principle is based on sandwich-type immunoreactions between gold nanoparticle (GNP)-labelled detection antibody, anti-CA 19-9 capture antibody and CA 19-9to capture the GNPs on the test zone of LFSB. The accumulation of GNPs on the test zone gave a red line whose intensity was read with a portable strip reader to quantify the concentration of CA 19-9. Assay parameters including the membrane type, antibody concentration, amount of GNP-anti-CA 19-9 conjugates and the components of the running buffer were optimized to obtain the best sensitivity and reproducibility of the assay. The detection limit of the assay was determined to be 5UmL(-1) (S/N=3) with a linear range of 5UmL(-1)-100UmL(-1). CA 19-9 concentrations in healthy human and pancreatic cancer patient plasma samples were successfully evaluated using the developed quantitative immunochromatographic assay (QIA), and the results were in accordance with that obtained with enzyme linked immunosorbent assay (ELISA). The developed assay shows great promise for clinical application and biomedical diagnosis, particularly in limited resource settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. 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.

  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. [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.

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

    PubMed

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

    1990-01-01

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

  5. 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.

  6. 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.

  7. 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

  8. Human immunodeficiency virus (HIV) seropositivity and hepatitis B surface antigenemia (HBSAG) among blood donors in Benin city, Edo state, Nigeria.

    PubMed

    Umolu, Patience Idia; Okoror, Lawrence Ehis; Orhue, Philip

    2005-03-01

    Human Immunodeficiency Virus and Hepatitis B virus are blood borne pathogens that can be transmitted through blood transfusion and could pose a huge problem in areas where mechanisms of ensuring blood safety are suspect. This study became necessary in a population where most of the blood for transfusion is from commercial blood donors. A total of 130 donors comprising 120 commercial donors and 10 voluntary donors were tested for antibodies to human immunodeficiency virus and hepatitis B surface antigen in Benin city using Immunocomb HIV - 1 and 2 Biospot kit and Quimica Clinica Aplicada direct latex agglutination method respectively. Thirteen (10%) samples were HIV seropositive and 7(5.8%) were HBsAg positive. The age bracket 18 - 25years had the highest numbers of donors and also had the highest number of HBsAg positive cases (7.8%) while the age group 29 - 38years had highest number of HIV seropositive cases. High prevalence of HIV antibodies and Hepatitis B surface antigen was found among commercial blood donors. Appropriate and compulsory screening of blood donors using sensitive methods, must be ensured to prevent post transfusion hepatitis and HIV.

  9. 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

  10. Hepatitis B Surface Antigen Vector Delivers Protective Cytotoxic T-Lymphocyte Responses to Disease-Relevant Foreign Epitopes†

    PubMed Central

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

    2006-01-01

    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. PMID:16571814

  11. 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.

  12. 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.

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

    PubMed

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

    2006-06-01

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

  14. 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

  15. Novel HBsAg mutations correlate with hepatocellular carcinoma, hamper HBsAg secretion and promote cell proliferation in vitro.

    PubMed

    Salpini, Romina; Surdo, Matteo; Warner, Nadia; Cortese, Maria Francesca; Colledge, Danny; Soppe, Sally; Bellocchi, Maria Concetta; Armenia, Daniele; Carioti, Luca; Continenza, Fabio; Di Carlo, Domenico; Saccomandi, Patrizia; Mirabelli, Carmen; Pollicita, Michela; Longo, Roberta; Romano, Sara; Cappiello, Giuseppina; Spanò, Alberto; Trimoulet, Pascale; Fleury, Herve; Vecchiet, Jacopo; Iapadre, Nerio; Barlattani, Angelo; Bertoli, Ada; Mari, Terenzio; Pasquazzi, Caterina; Missale, Gabriele; Sarrecchia, Cesare; Orecchini, Elisa; Michienzi, Alessandro; Andreoni, Massimo; Francioso, Simona; Angelico, Mario; Verheyen, Jens; Ceccherini-Silberstein, Francesca; Locarnini, Stephen; Perno, Carlo Federico; Svicher, Valentina

    2017-02-28

    An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation. This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry. Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001). Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.

  16. Serum hepatitis B surface antigen levels in the natural history of chronic hepatitis B infection.

    PubMed

    Jang, J W; Yoo, S H; Kwon, J H; You, C R; Lee, S; Lee, J H; Chung, K W

    2011-12-01

    BACKGROUND  The production of hepatitis B surface antigen (HBsAg) may evolve during long-lasting virus-host interactions in chronic hepatitis B (CHB). The impact of age on HBsAg production remains unclear. AIM  To determine the age-specific distribution patterns of HBsAg and related factors during the natural course of CHB infection. METHODS  Seven hundred and sixty-eight untreated HBsAg carriers were enrolled in the study. The parameters and distribution patterns of HBsAg were evaluated in relation to age and immune phases. RESULTS  The HBsAg levels were significantly lower in the HBeAg-negative stage, with the lowest levels in inactive carriers. The HBsAg tended to decrease from hepatitis to cirrhosis and to hepatocellular carcinoma, and from Child-Pugh class A to B and to C. Age and HBV DNA were independently associated with HBsAg levels. In HBeAg-positive patients, the HBsAg levels were distributed in a triphasic-like decline pattern by 2 logs across age strata. For HBeAg-negative patients, the titres in inactive carriers exhibited a 2-log reduction, but remained unchanged over age strata in patients with HBeAg-negative hepatitis. The ratios of HBsAg/HBV-DNA were highest, but steadily decreased with age in inactive carriers, whereas the levels remained largely unchanged over the entire age strata in patients with HBeAg-negative hepatitis. CONCLUSIONS  Age and HBV DNA levels are independent parameters of HBsAg levels. During the natural course of CHB infection, HBsAg levels decrease with age and disease progression, but the patterns are significantly different between the immune phases of CHB. This information may contribute to our understanding of the immunopathogenesis of chronic hepatitis B and management involving HBsAg quantification. © 2011 Blackwell Publishing Ltd.

  17. Structural characterization of plant-derived hepatitis B surface antigen employed in oral immunization studies.

    PubMed

    Smith, Mark L; Richter, Lizabeth; Arntzen, Charles J; Shuler, Michael L; Mason, Hugh S

    2003-09-08

    Several subunit vaccine antigens have been successfully expressed in plants and recently the hepatitis B surface antigen (HBsAg), expressed in potatoes, was shown to be orally immunogenic in animal studies. However, to date, a detailed analysis of the plant-derived antigen is lacking. Herein, we comprehensively characterize the structure and post-translational processing of HBsAg from potato tuber and two plant cell suspension cultures. The HBsAg was found to accumulate intracellularly as tubular structures, with a complex size distribution, differing substantially from the virus-like particle (VLP) preparations of the current commercial vaccines. Extensive disulfide-bond cross-linking, which is important for immunogenicity, was evident and 21-37% of total HBsAg protein displayed epitopes which correlate with vaccine potency. The significance of these results with regard to the production of cost-effective orally delivered vaccines is discussed.

  18. An improved Fc function assay utilizing CMV antigen-coated red blood cells generated with synthetic function-spacer-lipid constructs.

    PubMed

    Georgakopoulos, T; Komarraju, S; Henry, S; Bertolini, J

    2012-01-01

    The Fc function assessment of immunoglobulin products is performed to confirm that the biological activity of immunoglobulin products is not affected by manufacturing and storage conditions. The European Pharmacopoeia (EP) test is cumbersome and time-consuming especially with respect to the derivatization of red blood cells (RBC) with rubella antigen via tanninization. We investigated the KODE biosurface engineering technology for inserting specific antigens into red blood cell membranes to create kodecytes and evaluated their suitability for use in the Fc function assay. Human RBC were derivatized with function-spacer-lipid (FSL) constructs comprising of a peptide epitope of a cytomegalovirus (CMV) surface protein, conjugated to a spacer and lipid tail. These kodecytes were used in an Fc function assay based on the monitoring of complement-mediated haemolysis of immunoglobulin-coated red cells. Optimization of FSL construct, immunoglobulin and complement concentration was undertaken. Fc values obtained for various immunoglobulin batches were compared with those from the EP-based method. Carbohydrate-bearing FSLs Galili (Galα1-3Galβ1-4GlcNAcβ), Galα1-4GlcNAcβ and GalNAcα1-3Galβ were also evaluated. Fc function indices determined with CMV peptide construct-coated red cells were comparable to those obtained with the EP-based method with respect to specificity and precision. Carbohydrate-bearing FSLs revealed Fc indices lower than expected. The use of CMV kodecytes was shown to be a convenient means of generating red cells for the determination of Fc function of immunoglobulin products and offers the possibility of significantly reducing the time required to perform this assay. © 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.

  19. Comparison of three luminescent immunoassays for hepatitis B virus surface antigen quantification during the natural history of chronic hepatitis B virus infection.

    PubMed

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

    2014-11-01

    Hepatitis B surface antigen (HBsAg) quantification has garnered attention because of its high predictive value in determining treatment responses. The HBsAg quantification assays, such as Architect and Elecsys, are commercially available, and more assays are in development. We aimed to compare the results of the Architect and Elecsys assays with those of a new assay, WTultra. The WTultra HBsAg assay is a sandwich chemiluminescent microplate enzyme immunoassay and provides an alternative choice which is more cost-effective and potentially applicable in developing or resource-constrained countries and areas. A total of 411 serum samples were collected from patients during various phases of chronic hepatitis B (CHB) infection. The samples were assessed using the three assays, and the results were compared and analyzed. The results for the Architect, Elecsys, and WTultra assays were well correlated according to the overall results for the samples (correlation coefficients, rArchitect versus WTultra = 0.936, rArchitect versus Elecsys = 0.952, and rWTultra versus Elecsys = 0.981) and the various infection phases (rArchitect versus WTultra ranging from 0.67 to 0.975, rArchitect versus Elecsys ranging from 0.695 to 0.982, and rWTultra versus Elecsys ranging from 0.877 to 0.99). Additionally, consistent results were observed according to genotype (genotype B: rArchitect versus WTultra = 0.976, rArchitect versus Elecsys = 0.978, and rWTultra versus Elecsys = 0.979; genotype C: rArchitect versus WTultra = 0.950, rArchitect versus Elecsys = 0.963, and rWTultra versus Elecsys = 0.981) and hepatitis B virus (HBV) DNA levels (rArchitect = 0.540, rWTultra = 0.553, and rElecsys = 0.580). In conclusion, the Elecsys and WTultra assays were well correlated with the Architect assay, irrespective of the CHB infection phase or genotype. All of these assays are reliable for HBsAg quantification. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  20. 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.

  1. Western blot assay using recombinant p26 antigen for detection of equine infectious anemia virus-specific antibodies.

    PubMed

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

    2007-12-01

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

  2. 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.

  3. 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.

  4. 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

  5. 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

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

    PubMed

    Frambo, Andreas A Besong; Atashili, Julius; Fon, Peter Nde; Ndumbe, Peter Martins

    2014-06-25

    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. 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. 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). 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.

  7. 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

  8. 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.

  9. 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

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

    PubMed Central

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

    2007-01-01

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

  11. Factors affecting the natural decay of hepatitis B surface antigen in children with chronic hepatitis B virus infection during long-term follow-up.

    PubMed

    Chiu, Yu-Chun; Liao, Shu-Fen; Wu, Jia-Feng; Lin, Chun-Yin; Lee, Wen-Chung; Chen, Huey-Ling; Ni, Yen-Hsuan; Hsu, Hong-Yuan; Chang, Mei-Hwei

    2014-10-01

    To investigate the factors predicting spontaneous clearance of hepatitis B surface antigen (HBsAg) in a long-term, prospectively followed cohort from childhood into adult life. Children with chronic hepatitis B virus (HBV) infection without treatment were followed longitudinally every 6 months. At each visit, liver profiles and HBV markers were assessed. Hepatitis B vaccination history and the maternal HBV markers also were studied. A total of 349 children (205 male) were followed for 20.6 ± 4.4 years with initial ages of 8.4 ± 3.9 years; 42 (12.0%) cleared HBsAg spontaneously. The HBsAg titers decayed with age, with an average annual clearance rate of 0.58%. Children had a lower annual HBsAg decay rate if their mothers are HBsAg carriers (P < .001). Hepatitis B e antigen-seroconversion is a favorable predictor for spontaneous HBsAg clearance (P = .04). Those with HBsAg titer ≤1000 IU/mL at enrollment during childhood have a higher rate of HBsAg clearance (hazard ratio = 5.23; P < .001). Using HBsAg titer ≤1000 IU/mL to predict HBsAg clearance, the sensitivity is 38.1%, specificity is 90.6%, positive predictive value is 35.6%, and negative predictive value is 91.4%. During long-term follow-up, spontaneous HBsAg clearance is most likely to occur in a patient born to a non-HBsAg-carrier mother, is a hepatitis B e antigen-seroconverter, and had an initial HBsAg level ≤1000 IU/mL. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. 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

  13. Hepatitis B surface antigen in late hepatitis B infection.

    PubMed

    Zeng, Da-wu; Zhu, Yue-yong; Huang, Qian; Zhang, Jie-min; Wu, Yin-lian; Dong, Jing; Jiang, Jia-ji; Liu, Yu-rui

    2015-03-01

    Hepatitis B surface antigen (HBsAg) levels are used to evaluate and monitor clinical phases of chronic hepatitis B infection but their clinical significance is unclear in the late complications, cirrhosis of the liver and hepatocellular carcinoma. This study aimed to evaluate HBsAg levels across the whole natural history of hepatitis B virus infection, including late complications. This retrospective, cross-sectional study enrolled 838 treatment-naive patients diagnosed with chronic hepatitis B infection at First Affiliated Hospital of Fujian Medical University between 2009 and 2012. Patients were classified into six groups: immunotolerance, immunoclearance, low replicative, negative hepatitis e (HBeAg) phases, liver cirrhosis, and hepatocellular carcinoma. Main outcome measures were serum HBsAg, HBeAg, HBV DNA, total bilirubin, albumin, alanine and aspartate aminotransferase, and quantitative correlation of HBsAg with HBV DNA. HBsAg levels declined significantly between clinical phases of infection (all P < 0.001) and were significantly lower in decompensated than in compensated cirrhosis (2.90 vs. 3.30, P < 0.001) but not significantly different between early versus advanced hepatocellular carcinoma. Significant positive correlations were observed between serum HBsAg and HBV DNA at immunoclearance and HBeAg negative phases, compensated and decompensated liver cirrhosis and advanced but not early hepatocellular carcinoma (all P < 0.001). HBsAg and HBV DNA were significantly higher in HBeAg positive patients with advanced hepatocellular carcinoma (P < 0.001). HBsAg levels differ significantly between chronic hepatitis B infection phases, decreasing progressively from chronic infection to cirrhosis and hepatocellular carcinoma. Significant correlations are found between serum HBsAg and HBV DNA. © 2014 Wiley Periodicals, Inc.

  14. Combined antigen-specific interferon-γ and interleukin-2 release assay (FluoroSpot) for the diagnosis of Mycobacterium tuberculosis infection.

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2013-04-01

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

  17. 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.

  18. 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.

  19. 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.

  20. Pretransplant human leukocyte antigen antibodies detected by single-antigen bead assay are a risk factor for long-term kidney graft loss even in the absence of donor-specific antibodies.

    PubMed

    Richter, Rudolf; Süsal, Caner; Köhler, Stefanie; Qidan, Sara; Schödel, Alicia; Holschuh, Lisa; Brzoska, Martin; Asbe-Vollkopf, Aida; Büttner, Stefan; Betz, Christoph; Herrmann, Eva; Gauer, Stefan; Seifried, Erhard; Geiger, Helmut; Seidl, Christian; Hauser, Ingeborg A

    2016-09-01

    Clinical relevance of ELISA- and single-antigen bead assay (SAB)-detected pretransplant HLA antibodies (SAB-HLA-Ab) for kidney graft survival was evaluated retrospectively in 197 patients transplanted between 2002 and 2009 at the University Clinic Frankfurt. Having adjusted for retransplantation and delayed graft function, a significantly increased risk for death-censored graft loss was found in patients with pretransplant SAB-HLA-Ab [HR: 4.46; 95% confidence interval (CI): 1.47-13.48; P = 0.008]. The risk for increased graft loss was also significant in patients with pretransplant SAB-HLA-Ab but without SAB-detected donor-specific Ab (SAB-DSA) (HR: 4.91; 95% CI of 1.43-16.991; P = 0.012). ELISA was not sufficient to identify pretransplant immunized patients with an increased risk for graft loss. In immunized patients, graft loss was predominantly present in patients who received transplants with a mismatch on the HLA-DR locus. In conclusion, even if our study is limited due to small sample size, the results show an increased risk for long-term graft loss in patients with pretransplant SAB-HLA, even in the absence of DSA. SAB-HLA-Ab-positive patients, being negative in ELISA or CDC assay, might profit from a well-HLA-DR-matched graft and intensified immunosuppression. © 2016 Steunstichting ESOT.

  1. 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.

  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. Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy.

    PubMed

    Zhuang, Xiaolei; Watts, Norman R; Palmer, Ira W; Kaufman, Joshua D; Dearborn, Altaira D; Trenbeath, Joni L; Eren, Elif; Steven, Alasdair C; Rader, Christoph; Wingfield, Paul T

    2017-10-06

    Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli, had unprecedentedly high binding affinities (Kd ∼10(-12) m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

  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. Validation of a stopping rule at week 12 using HBsAg and HBV DNA for HBeAg-negative patients treated with peginterferon alfa-2a.

    PubMed

    Rijckborst, Vincent; Hansen, Bettina E; Ferenci, Peter; Brunetto, Maurizia R; Tabak, Fehmi; Cakaloglu, Yilmaz; Lanza, A Galeota; Messina, Vincenzo; Iannacone, Claudio; Massetto, Benedetta; Regep, Loredana; Colombo, Massimo; Janssen, Harry L A; Lampertico, Pietro

    2012-05-01

    It was recently demonstrated that none of the hepatitis B e antigen (HBeAg)-negative patients without any serum hepatitis B surface antigen (HBsAg) decline and with <2log hepatitis B virus (HBV) DNA decline at week 12 of a 48-week peginterferon alfa-2a (PEG-IFN) treatment course achieved a sustained response (SR). We aimed at validating this stopping rule in two independent trials. HBeAg-negative patients receiving 48 or 96 weeks of PEG-IFN in the phase III registration trial (N=85) and PegBeLiver study (N=75) were stratified according to the presence of any HBsAg decline and/or 2log HBV DNA decline at week 12. SR was defined as HBV DNA <2000IU/ml and normal alanine aminotransferase 24 weeks after treatment. The original PARC trial included 102 patients (genotype A/D/other: 14/81/7), 25 (25%) had an SR. The validation dataset consisted of 160 patients (genotype A/B/C/D/other: 10/18/34/91/7), 57 (36%) achieved an SR. The stopping rule performed well across the two studies (p=0.001) and its negative predictive value [NPV] was 95% in the validation dataset harbouring genotypes A-D. Its performance was best for genotype D. Moreover, among the 34 patients treated for 96 weeks, none of the 7 (21%) without HBsAg decline and with <2log HBV DNA decline at week 12 achieved an SR (NPV 100%). We confirmed in two independent studies that the combination of HBsAg and HBV DNA levels at week 12 identifies HBeAg-negative patients with a very low chance of SR to either 48 or 96 weeks of PEG-IFN therapy. Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  6. Micropropagation of transgenic lettuce containing HBsAg as a method of mass-scale production of standardised plant material for biofarming purposes.

    PubMed

    Pniewski, Tomasz; Czyż, Marcin; Wyrwa, Katarzyna; Bociąg, Piotr; Krajewski, Paweł; Kapusta, Józef

    2017-01-01

    Micropropagation protocol of transgenic lettuce bearing S-, M- and L-HBsAg was developed for increased production of uniformised material for oral vaccine preparation. Effective manufacturing of plant-based biopharmaceuticals, including oral vaccines, depends on sufficient content of a protein of interest in the initial material and its efficient conversion into an administrable formulation. However, stable production of plants with a uniformised antigen content is equally important for reproducible processing. This can be provided by micropropagation techniques. Here, we present a protocol for micropropagation of transgenic lettuce lines bearing HBV surface antigens: S-, M- and L-HBsAg. These were multiplied through axillary buds to avoid the risk of somaclonal variation. Micropropagation effectiveness reached 3.5-5.7 per passage, which implies potential production of up to 6600 plant clones within a maximum 5 months. Multiplication and rooting rates were statistically homogenous for most transgenic and control plants. For most lines, more than 90 % of clones obtained via in vitro micropropagation had HBsAg content as high as reference plants directly developed from seeds. Clones were also several times more uniform in HBsAg expression. Variation coefficients of HBsAg content did not exceed 10 % for approximately 40-85 % of clones, or reached a maximum 20 % for 90 % of all clones. Tissue culture did not affect total and leaf biomass yields. Seed production for clones was decreased insignificantly and did not impact progeny condition. Micropropagation facilitates a substantial increase in the production of lettuce plants with high and considerably equalised HBsAg contents. This, together with the previously reported optimisation of plant tissue processing and its long-term stability, constitutes a successive step in manufacturing of a standardised anti-HBV oral vaccine of reliable efficacy.

  7. Herpes simplex virus antigen direct detection in standard virus transport medium by Du Pont Herpchek enzyme-linked immunosorbent assay.

    PubMed Central

    Verano, L; Michalski, F J

    1990-01-01

    A commercial 5-h direct herpes simplex virus (HSV) antigen detection enzyme immunoassay kit (Du Pont Herpchek) was compared with a cell culture isolation system by using primary rabbit kidney and MRC-5 cells with 779 clinical specimens received in virus transport medium and with stock tissue culture preparations of HSV types 1 and 2. In the first study of 422 specimens from symptomatic patients, Herpchek detected 110 of 111 HSV-positive specimens (26.3% of all specimens), with a sensitivity of 99% and a specificity of 100%. In the second study of 357 specimens primarily from asymptomatic pregnant women, however, Herpchek detected 70 of 119 HSV-positive specimens (33% of all specimens), with a sensitivity of 58.8% and a specificity of 99.5%. Stock virus dilution experiments showed that Herpchek was 10 to 100 times less sensitive than culture. Herpchek was found to be an acceptable test for symptomatic patients, but for asymptomatic patients shedding a low titer of HSV it was not as sensitive and cell culture of Herpchek-negative specimens is recommended for such cases. PMID:2174903

  8. Herpes simplex virus antigen direct detection in standard virus transport medium by Du Pont Herpchek enzyme-linked immunosorbent assay.

    PubMed

    Verano, L; Michalski, F J

    1990-11-01

    A commercial 5-h direct herpes simplex virus (HSV) antigen detection enzyme immunoassay kit (Du Pont Herpchek) was compared with a cell culture isolation system by using primary rabbit kidney and MRC-5 cells with 779 clinical specimens received in virus transport medium and with stock tissue culture preparations of HSV types 1 and 2. In the first study of 422 specimens from symptomatic patients, Herpchek detected 110 of 111 HSV-positive specimens (26.3% of all specimens), with a sensitivity of 99% and a specificity of 100%. In the second study of 357 specimens primarily from asymptomatic pregnant women, however, Herpchek detected 70 of 119 HSV-positive specimens (33% of all specimens), with a sensitivity of 58.8% and a specificity of 99.5%. Stock virus dilution experiments showed that Herpchek was 10 to 100 times less sensitive than culture. Herpchek was found to be an acceptable test for symptomatic patients, but for asymptomatic patients shedding a low titer of HSV it was not as sensitive and cell culture of Herpchek-negative specimens is recommended for such cases.

  9. HIV incidence in the Estonian population in 2013 determined using the HIV-1 limiting antigen avidity assay.

    PubMed

    Soodla, P; Simmons, R; Huik, K; Pauskar, M; Jõgeda, E-L; Rajasaar, H; Kallaste, E; Maimets, M; Avi, R; Murphy, G; Porter, K; Lutsar, I

    2017-08-01

    Estonia has one the highest number of new HIV diagnoses in the European Union, mainly among injecting drug users and heterosexuals. Little is known of HIV incidence, which is crucial for limiting the epidemic. Using a recent HIV infection testing algorithm (RITA) assay, we aimed to estimate HIV incidence in 2013. All individuals aged ≥18 years newly-diagnosed with HIV in Estonia January- December 2013, except blood donors and those undergoing antenatal screening, were included. Demographic and clinical data were obtained from the Estonian Health Board and the Estonian HIV-positive patient database. Serum samples were tested for recent infection using the LAg-avidity EIA assay. HIV incidence was estimated based on previously published methods. Of 69,115 tested subjects, 286 (0.41%) were newly-diagnosed with HIV with median age of 33 years (IQR: 28-42) and 65% male. Self-reported routes of HIV transmission were mostly heterosexual contact (n = 157, 53%) and injecting drug use (n = 62, 21%); 64 (22%) were with unknown risk group. Eighty two (36%) were assigned recent, resulting in estimated HIV incidence of 0.06%, corresponding to 642 new infections in 2013 among the non-screened population. Incidence was highest (1.48%) among people who inject drugs. These high HIV incidence estimates in Estonia call for urgent action of renewed targeted public health promotion and HIV testing campaigns. © 2017 British HIV Association.

  10. 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.

  11. Use of an Interferon Gamma Release Assay (IGRA) to test T-cell responsiveness to soluble Leishmania infantum antigen in whole blood of dogs from endemic areas.

    PubMed

    Zribi, Lilia; El-Goulli, Amel F; Ben-Abid, Meriem; Gharbi, Mohamed; Ben-Sghaier, Ines; Boufaden, Imed; Aoun, Karim; Bouratbine, Aïda

    2017-11-15

    Interferon-Gamma (IFN-γ) Release Assays (IGRAs) are easy tests that allow rapid screening of primed memory T-cells immunity in response to antigen. The aim of this study was to use IGRA to assess IFN-γ release in response to Soluble Leishmania infantum antigen (SLA) in whole blood of dogs living in endemic area of visceral leishmaniasis and to interpret IGRA results according to clinical examination, specific anti-Leishmania humoral response and presence of L. infantum DNA in blood. The study was carried out on 56 dogs living in greater Tunis area. Physical examination, quantitative serology and PCR on blood were used to characterize dogs' status in relation to Leishmania infection and disease. IGRA consisted on testing by ELISA for IFN-γ-secretion in whole blood after a 20-h challenge with SLA. PBS and Phytohemagglutinin (PHA) stimulations were used as controls. Four groups of dogs were characterized: 31 were negative by both serology and PCR, two had doubtful serology, 10 presented no to mild clinical signs but low antibodies levels and 13 were affected by Canine Leishmaniasis (CanL). In seronegative dogs, IGRA was little contributory in 4 puppies (age <6months) and 5 old dogs (median age=72months, IQR: 45-84 months) that didn't respond to PHA stimulation, IGRA was negative in 19 and positive in three animals with lymph node enlargement. In dogs with doubtful serology, IGRA was positive in one dog and negative in the other. In infected dogs with no to mild clinical signs, one dog exhibited high level of IFN-γ in absence of antigenic stimulation and all the other were positive by IGRA. CanL dogs showed variable IGRA results. Negative IGRAs (n=4) were shown in animals with the highest parasitic burden whereas positive IGRAs (n=5) were shown in dogs with negative PCR or low parasitic load. The 4 remaining dogs either didn't respond to PHA (n=2) or showed non-specific secretion in PBS tube (n=2). The results of this study showed that IGRA is a useful new tool

  12. Detection of Recent HIV-1 Infection Using a New Limiting-Antigen Avidity Assay: Potential for HIV-1 Incidence Estimates and Avidity Maturation Studies

    PubMed Central

    Duong, Yen T.; Qiu, Maofeng; De, Anindya K.; Jackson, Keisha; Dobbs, Trudy; Kim, Andrea A.; Nkengasong, John N.; Parekh, Bharat S.

    2012-01-01

    Background Accurate and reliable laboratory methods are needed for estimation of HIV-1 incidence to identify the high-risk populations and target and monitor prevention efforts. We previously described a single-well limiting-antigen avidity enzyme immunoassay (LAg-Avidity EIA) to detect recent HIV-1 infection. Methods We describe here further optimization and characterization of LAg-Avidity EIA, comparing it to the BED assay and a two-well avidity-index (AI) EIA. Specimen sets included longitudinal sera (n = 393), collected from 89 seroconverting individuals from 4 cohorts representing 4 HIV-1 subtypes, and sera from AIDS patients (n = 488) with or without TB co-infections from 3 different cohorts. Ninety seven HIV-1 positive specimens were purchased commercially. The BED assay, LAg-Avidity EIA, AI-EIA and HIV serology were performed, as needed. Results Monitoring quality control specimens indicated high reproducibility of the LAg-Avidity EIA with coefficient of variation of <10% in the dynamic range. The LAg-Avidity EIA has an overall mean duration of recency (ω) of 141 days (95% CI 119–160) at normalized optical density (ODn) cutoff of 1.0, with similar ω in different HIV-1 subtypes and populations (132 to 143 days). Antibody avidity kinetics were similar among individuals and subtypes by both the LAg-Avidity EIA and AI-EIA compared to the HIV-IgG levels measured by the BED assay. The false recent rate among individuals with AIDS was 0.2% with the LAg-Avidity EIA, compared to 2.9% with the BED assay. Western blot profiles of specimens with increasing avidity confirm accurate detection of recent HIV-1 infections. Conclusions These data demonstrate that the LAg-Avidity EIA is a promising assay with consistent ω in different populations and subtypes. The assay should be very useful for 1) estimating HIV-1 incidence in cross-sectional specimens as part of HIV surveillance, 2) identifying risk factors for recent infections, 3) measuring impact of

  13. Evaluation of a dengue NS1 antigen detection assay sensitivity and specificity for the diagnosis of acute dengue virus infection.

    PubMed

    Hermann, Laura L; Thaisomboonsuk, Butsaya; Poolpanichupatam, Yongyuth; Jarman, Richard G; Kalayanarooj, Siripen; Nisalak, Ananda; Yoon, In-Kyu; Fernandez, Stefan

    2014-10-01

    Currently, no dengue NS1 detection kit has regulatory approval for the diagnosis of acute dengue fever. Here we report the sensitivity and specificity of the InBios DEN Detect NS1 ELISA using a panel of well characterized human acute fever serum specimens. The InBios DENV Detect NS1 ELISA was tested using a panel composed of 334 serum specimens collected from acute febrile patients seeking care in a Bangkok hospital in 2010 and 2011. Of these patients, 314 were found to have acute dengue by either RT-PCR and/or anti-dengue IgM/IgG ELISA. Alongside the InBios NS1 ELISA kit, we compared the performance characteristics of the BioRad Platelia NS1 antigen kit. The InBios NS1 ELISA Ag kit had a higher overall sensitivity (86% vs 72.8%) but equal specificity (100%) compared to the BioRad Platelia kit. The serological status of the patient significantly influenced the outcome. In primary infections, the InBios NS1 kit demonstrated a higher sensitivity (98.8%) than in secondary infections (83.5%). We found significant variation in the sensitivity of the InBios NS1 ELISA kit depending on the serotype of the dengue virus and also found decreasing sensitivity the longer after the onset of illness, showing 100% sensitivity early during illness, but dropping below 50% by Day 7. The InBios NS1 ELISA kit demonstrated high accuracy when compared to the initial clinical diagnosis with greater than 85% agreement when patients were clinically diagnosed with dengue illness. Results presented here suggest the accurate detection of circulating dengue NS1 by the InBios DENV Detect NS1 ELISA can provide clinicians with a useful tool for diagnosis of early dengue infections.

  14. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. A double antibody sandwich enzyme-linked immunosorbent assay for detection of soft-shelled turtle iridovirus antigens.

    PubMed

    Zhang, M; Yang, J X; Lin, X M; Zhu, C H; He, J Q; Liu, H; Lin, T L

    2010-08-01

    A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detection of the soft-shelled turtle iridovirus (STIV) was developed using a specific monoclonal antibody (mAb) against STIV and anti-STIV rabbit serum. Using DAS-ELISA, the detection limit of STIV was found to be 10(3)PFU/ml. The positive rate of 15 STIV samples was 100%, while the positive rate of 100 other aquatic virus samples was 0%. These data show that DAS-ELISA is highly specific and sensitive for the detection of STIV. In clinical tests, 128 samples isolated from pond-reared turtles were subjected to DAS-ELISA and PCR. The overall agreement between the results obtained by DAS-ELISA and PCR was 98.4%. The results indicate that the DAS-ELISA method could be used for diagnosing diseases caused by STIV. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  16. 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.

  17. The testing of antibodies raised against poultry red mite antigens in an in vitro feeding assay; preliminary screen for vaccine candidates.

    PubMed

    Wright, Harry W; Bartley, Kathryn; Nisbet, Alasdair J; McDevitt, Regina M; Sparks, Nickolas H C; Brocklehurst, Sarah; Huntley, John F

    2009-06-01

    Dermanyssus gallinae (De Geer), the poultry red mite, is a blood-feeding ectoparasite that infests many bird species. We have used an in vitro feeding assay to allow the identification of protective D. gallinae antigens that may have potential as vaccine candidates. Homogenised mites were extracted sequentially with PBS, Tween 20, Triton X100 and urea giving four protein fractions. Five experimental groups of Lohmann Brown hens were used to generate antibodies; four groups were injected with one of each of the protein fractions in QuilA adjuvant and a control group was injected with adjuvant only. Booster injections were administered 2 and 4 weeks after initial immunisation. Eggs were collected throughout the experiment and soluble IgY antibodies were extracted from a pool of egg yolks collected at week six post-injection. Western blots, performed using post vaccination antibodies from test and control groups, revealed a strong antibody response against a range of injected proteins. Fresh chicken blood, supplemented with antibodies raised against these protein fractions, was fed to mites in an in vitro feeding assay in order to determine whether the antibodies had an anti-mite effect. Although there was variability in the numbers of feeding mites, it was found that the strongest anti-mite effect was seen with the PBS protein fraction, which had a cumulative average mortality of 34.8% 14 days after feeding compared with 27.3% for the control group (P = 0.043).

  18. 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.

  19. 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.

  20. Liver transplantation from hepatitis B surface antigen positive donors: a safe way to expand the donor pool.

    PubMed

    Loggi, Elisabetta; Micco, Lorenzo; Ercolani, Giorgio; Cucchetti, Alessandro; Bihl, Florian K; Grazi, Gian Luca; Gitto, Stefano; Bontadini, Andrea; Bernardi, Mauro; Grossi, Paolo; Costa, Alessandro Nanni; Pinna, Antonio Daniele; Brander, Christian; Andreone, Pietro

    2012-03-01

    The main limitation of orthotopic liver transplantation (OLT) is the scarcity of available donor organs. A possibility to increase the organ pool is to use grafts from hepatitis B virus surface antigen (HBsAg) positive donors, but few data are currently available in this setting. We assessed the clinical, serovirological, and immunological outcomes of liver transplant from HBsAg positive donors in a single centre study. From 2005 to 2009 10 patients underwent OLT from HBsAg positive donors, for HBV-related disease (n=6) or HBV-unrelated disease (n=4). The median follow-up was 42 months (range 12-60). All recipients were HBcAb positive and were given antiviral prophylaxis. Patients transplanted for HBV-related disease never cleared HBsAg. Two HBsAg negative patients never tested positive for HBsAg, whereas the others experienced an HBsAg appearance, followed by spontaneous production of anti-HBs, allowing HBsAg clearance. No patient ever had any sign of HBV hepatitis. HBV replication was effectively controlled by antiviral therapy. The immunologic sub-study showed that a most robust anti-HBV specific T cell response was associated with the control of HBV infection. OLT from HBsAg positive donors seems to be a safe procedure in the era of highly effective antiviral therapy. Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  3. Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets: I. Evaluation of the GTI QuikID assay and analysis of antibody patterns.

    PubMed

    Zachary, A A; Delaney, N L; Lucas, D P; Leffell, M S

    2001-03-01

    The development of solid phase immunoassays using solubilized HLA molecules as targets has provided a means of detecting HLA-specific antibodies that overcomes many of the shortcomings of lymphocyte based assays. We have evaluated a commercially available assay, the GTI QuikID (QID), that uses solubilized class I molecules from 40 subjects selected for their HLA phenotype, to characterize HLA-specific antibodies. We tested 595 sera from 319 subjects and compared the results obtained with QID to those obtained with cytotoxicity (CYT) and with GTI QuikScreen (QS) as well as to historic data. The correlation of QID with CYT (r = 0.54) was comparable to that between QID and QS (r = 0.60). The majority of disparities between QS and QID were apparent false negatives with QID that could be overcome by analyzing QID data at lower cutoff values. In contrast, most of the disparities between QID and CYT were false negatives in CYT due to the relatively low sensitivity of that assay. As expected, the ELISA was more sensitive (97%) than CYT (78%) but had a somewhat lower specificity (87% vs. 92%) due, most likely, to selection of sera that excluded most sera that were known to be nonspecific by CYT. Determination of antibody specificity could be achieved quickly by manual analysis of the QID data because of the way the data are presented by the manufacturer's software. Interestingly, the frequencies of different antibodies detected by ELISA differed from those detected by CYT with ELISA identifying more sera containing antibodies to both A and B locus antigens.

  4. [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.

  5. Optimal conditions for elution of hepatitis B antigen after absorption onto colloidal silica.

    PubMed Central

    Pillot, J; Goueffon, S; Keros, R G

    1976-01-01

    Hepatitis B surface antigen (HBSAg) adsorbed from sera onto colloidal silica could be completely eluted through the use of 0.25% sodium deoxycholate in 0.01 M borax, pH 9.3, at 56 degrees C. The HBSAg recovered in the eluate represented 100% of that present in the original serum, and it was contaminated by only trace amounts of serum proteins (in decreasing amounts: beta-lipoprotein, immunoglobulin G, albumin). This preliminary step greatly facilitates purification of large amounts of HBSAg and provides small volumes of highly concentrated material for subsequent purification by density gradient centrifugation. PMID:9423

  6. A potent hepatitis B surface antigen response in subjects with inactive hepatitis B surface antigen carrier treated with pegylated-interferon alpha.

    PubMed

    Cao, Zhenhuan; Liu, Yali; Ma, Lina; Lu, Junfeng; Jin, Yi; Ren, Shan; He, Zhimin; Shen, Chengli; Chen, Xinyue

    2017-10-01

    Hepatitis B surface antigen (HBsAg) clearance represents a clinical cure, although the clearance rate is extremely low. The aim of this study was to evaluate the feasibility and safety profiles of pegylated-interferon α-2a (PEG-IFNα-2a) as a therapeutic option for inactive HBsAg carriers. There were 144 inactive HBsAg carriers enrolled and divided into a therapeutic group (102 subjects) and a control group (42 subjects). PEG-IFNα-2a and PEG-IFNα-2a combined with adefovir dipivoxil were used for treatment group subjects with hepatitis B virus DNA <20 IU/mL and 20 IU/mL ≤ hepatitis B virus DNA < 2,000 IU/mL, respectively. Total therapy duration was no more than 96 weeks. HBsAg clearance and seroconversion rates at therapeutic weeks 48 and 96 were used to evaluate the therapeutic efficacy. Per protocol analysis showed that the HBsAg clearance rate and seroconversion rate in the treatment group were 29.8% and 20.2% at week 48 and increased to 44.7% and 38.3% at week 96, respectively. However, the HBsAg clearance rate in the control group was 2.4% at weeks 48 and 96, and no subject achieved seroconversion. The quantitative HBsAg levels and changes during the early period of treatment (at week 12 and week 24) as well as the alanine aminotransferase elevation at week 12 were strong predictors of HBsAg clearance. The adverse events were similar to those with treatment for chronic hepatitis B patients. High rates of HBsAg clearance and seroconversion could be achieved by PEG-IFNα-2a-based treatments and the treatments were relatively safe for inactive HBsAg carriers. (Hepatology 2017;66:1058-1066). © 2017 by the American Association for the Study of Liver Diseases.

  7. 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

  8. Rapid mycobacterial liquid culture-screening method for Mycobacterium avium complex based on secreted antigen-capture enzyme-linked immunosorbent assay.

    PubMed

    Shin, Sung Jae; Anklam, Kelly; Manning, Elizabeth J B; Collins, Michael T

    2009-05-01

    Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer's instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1

  9. Evaluation of a hepatitis C virus (HCV) antigen assay for routine HCV screening among men who have sex with men infected with HIV.

    PubMed

    Vanhommerig, Joost W; van de Laar, Thijs J W; Koot, Maarten; van Rooijen, Martijn S; Schinkel, Janke; Speksnijder, Arjen G C L; Prins, Maria; de Vries, Henry J; Bruisten, Sylvia M

    2015-03-01

    For detection of early HCV infection and reinfection, commercial HCV-RNA tests are available. However, these tests are relatively time-consuming and expensive. A commercially available test that may supplement current screening methods, targets the HCV core protein. During five waves of anonymous surveys at the Amsterdam STI clinic between 2009-2012, all HIV-infected MSM (N=439) were tested for HCV-antibodies (AxSYM HCV 3.0, Abbott), and HCV-RNA (TMA Versant, Siemens). To evaluate the potential value of the ARCHITECT HCV antigen (HCV-Ag) assay (Abbott), all HCV-RNA-positive sera (N=31) were tested with this assay, as well as two HIV-infected HCV-RNA-negative controls. In addition, all included samples were tested for alanine aminotransferase (ALT). Among 439 HIV-infected MSM, 31 (7.1%) tested positive for HCV-RNA; the HCV-Ag assay showed concordant positive results for 31/31 (100%). A substantial number of MSM, i.e., 5/31 (16.1%), had detectable HCV-RNA but were HCV-seronegative at the time of screening and were presumed to have been recently infected. Concordant HCV-RNA-negative results were obtained in 57/60 control-samples. Specificity was 95.0% (95% CI: 86.1-99.0). The detection limit was between 3.0 and 3.7 Log10 IU/mL, irrespective of HCV genotype/subtype. ALT concentrations were elevated (i.e.,>40 U/L) in 9/31 (29.0%) HCV-RNA positive MSM, including 1/5 (20.0%) MSM with recent HCV-infection. The HCV-Ag assay proved a valuable screening tool for detection of active HCV infection among HIV-infected MSM with and without anti-HCV. Adding ALT to current screening methods would improve case finding marginally. We therefore recommend implementation of routine HCV-Ag screening for populations at risk for HCV-(re)infection. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. 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.

  11. PRISM hepatitis B surface antigen detection of hepatits B virus minipool nucleic acid testing yield samples.

    PubMed

    Linauts, Sandy; Saldanha, John; Strong, D Michael

    2008-07-01

    Hepatitis B virus (HBV) residual risk has been estimated at 1:63,000-1:205,000 and introduction of more sensitive serological tests and nucleic acid testing (NAT) would reduce that risk. Sensitivity of the recently licensed Abbott PRISM hepatitis B surface antigen (HBsAg) CLIA and minipool (MP) HBV NAT has been described as comparable and thus the need for HBV NAT has not been compelling. In this study, eight samples identified as yield samples with MP HBV NAT were tested using the PRISM test. Seven samples were identified using the Roche COBAS AmpliScreen HBV test and one additional sample was obtained from the clinical trial for the Roche cobas TaqScreen MPX test. Each of these samples was reactive by MP HBV NAT and nonreactive for HBsAg using one of three licensed enzyme immunoassay (EIA) tests. After licensure of the PRISM HBsAg, aliquots were tested with this assay, and DNA quantitation and genotyping were repeated where sample volume permitted. Three samples (2000, 2300, and 61,000 copies/mL) produced reactive results with PRISM. Four samples with viral loads less than 300 copies per mL produced nonreactive results. One sample, originally quantitated at 37,000 copies per mL (but 3850 copies/mL in repeat testing) was also nonreactive by PRISM. Genotyping of this sample indicated a type C genotype with no mutations. Adding serological sensitivity of PRISM CLIA reduced the NAT yield from the original 1: 385,555 to 1:610,488. However, MP HBV NAT still provides additional sensitivity over CLIA, even for a donation with a viral load of almost 4000 copies per mL.

  12. Low occurrence of HBsAg but high frequency of transient occult HBV infection in vaccinated and HBIG-administered infants born to HBsAg positive mothers.

    PubMed

    Zhou, Shan; Li, Tingting; Allain, Jean-Pierre; Zhou, Bin; Zhang, Yuming; Zhong, Mei; Fu, Yongshui; Li, Chengyao

    2017-05-23

    The status of chronic and occult HBV infection (OBI) in neonatal hepatitis B vaccine and immunoglobulin (HBIG) vaccinated infants born to HBsAg+ mothers was investigated at a major hospital in China. Seventy-seven and 15 blood samples were collected in first or second follow-up detection from the vaccinated babies aged 3-36 months born to 43 HBsAg+ or plus 25 HBeAg+ mothers. HBV infection was analyzed between the paired baby and mother by serology and DNA analysis. Among 77 children born to 68 HBsAg+ mothers, 3.9% (3/77) were HBsAg+, and 36.4% (28/77) were HBV DNA+/HBsAg- (OBIs) by a single PCR, respectively. Thirteen of 28 HBV DNA+/HBsAg- samples were conformed by two PCRs or S sequence, which accounted for 16.9% (13/77) of children. Three HBsAg+ and six OBIs were genotyped in consistent with their mother's HBV strains. Of 77 babies' blood samples, anti-HBs reactivity varied slightly according to age groups, while passively transmitted anti-HBc reactivity declined from 100% high reactivity at age 3-5 months to mostly negative at age ≥12 months. Babies with apparent OBI had higher levels of anti-HBc and lower levels of anti-HBs than those without OBI but all eight OBI babies with second follow-up samples became HBV DNA negative beyond 1 year of age. The vaccinated infants born to HBsAg+ mothers presented the low rate of HBsAg occurrence as vaccination failure and high frequency of viral persistence in the form of transient OBIs since no evidence of active HBV infection occurred beyond 1 year of age. © 2017 Wiley Periodicals, Inc.

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

    PubMed

    Stief, Thomas W

    2007-01-01

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

  14. Construction of Human Immunoglobulin Combinatorial Library and Screening of Phage Antibodies to Hepatitis B Surface Antigen.

    PubMed

    Wang, Xue; Wang, Hai-Tao; Chen, Wan-Rong; Xu, Jing

    1997-01-01

    Human immunoglobulin combinatorial library was generated by using phage surface-display expression system, and phage antibodies (Fab fragments) to hepatitis B surface antigen (HbsAg) were screened from it. The products by half-nested PCR using signal peptide sequences as primers were superior in quality and quantity to those by PCR with conserved sequences in the 5'-end variable regions as primers. After three round of selections by biopanning, the ratio of positive clone was 69%. The inhibition assay showed the phage antibodies to be specifically anti-HbsAg. The V(H) genes were derived from V(H) I and V(H) III, while V(L)s belonged to V(lambda) II and V(lambda) I as shown by DNA sequencing.

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

    PubMed

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

    2016-08-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. Copyright © 2016 Adungo et al.

  16. 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

  17. 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.

  18. 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. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  19. Development of a Recombinant Cell-Based Indirect Immunofluorescence Assay for the Determination of Autoantibodies against Soluble Liver Antigen in Autoimmune Hepatitis

    PubMed Central

    Radzimski, Christiane; Probst, Christian; Teegen, Bianca; Rentzsch, Kristin; Blöcker, Inga Madeleine; Dähnrich, Cornelia; Schlumberger, Wolfgang; Stöcker, Winfried; Bogdanos, Dimitrios P.; Komorowski, Lars

    2013-01-01

    Autoantibodies against soluble liver antigen (SLA) are specific markers for autoimmune hepatitis (AIH) type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA), detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA) to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors). Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases. PMID:23401700

  20. Development of a recombinant cell-based indirect immunofluorescence assay for the determination of autoantibodies against soluble liver antigen in autoimmune hepatitis.

    PubMed

    Radzimski, Christiane; Probst, Christian; Teegen, Bianca; Rentzsch, Kristin; Blöcker, Inga Madeleine; Dähnrich, Cornelia; Schlumberger, Wolfgang; Stöcker, Winfried; Bogdanos, Dimitrios P; Komorowski, Lars

    2013-01-01

    Autoantibodies against soluble liver antigen (SLA) are specific markers for autoimmune hepatitis (AIH) type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA), detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA) to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors). Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases.

  1. [Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Salmonella Typhi lipopolysaccharide O and capsular polysaccharide Vi antigens in persons from outbreak of typhoid fever].

    PubMed

    Rastawicki, Waldemar; Kałużewski, Stanisław

    2015-01-01

    The laboratory diagnosis of typhoid fever is dependent upon either isolation of S. Typhi from a clinical sample or the detection of raised titers of serum antibodies in the Widal test or the passive hemagglutination assay (PHA). In this study we evaluated the usefulness of ELISA for detection of antibodies to S. Typhi lipopolysaccharide O and capsular polysaccharide Vi antigens in the sera of persons from outbreak of typhoid fever. Fifteen serum samples from patients with laboratory confirmed typhoid fever and 140 sera from persons suspected for contact with typhoid fever patients from outbreak in 1974/75 in Poland were tested by ELISA. Additionally, as the control group, we tested 115 sera from blood donors for the presence of S. Typhi anti-LPS and anti-Vi antibodies. Anti-LPS and anti-Vi antibodies were detected in 80% and 53.3% of sera obtained from patients with laboratory confirmed typhoid fever, respectively. The high percentages of positive results in ELISA were also noted in the group of persons suspected for contact with typhoid fever patients (51.4% and 45%) but not in the group of blood donors (7.8% and 6.1%, respectively). The ELISA could be a useful tool for the serological diagnosis of typhoid fever in patients who have clinical symptoms but are culture negative, especially during massive outbreaks of typhoid fever.

  2. 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.

  3. 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.

  4. 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

  5. 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.

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

    PubMed

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

    2016-01-01

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

  7. Seroprevalence of antibodies specific for gram-negative core antigens in chickens on the basis of an Escherichia coli J5 enzyme-linked immunosorbent assay.

    PubMed

    Ruble, Randall P; Wakenell, Patricia S; Cullor, James S

    2002-01-01

    Antibodies directed toward gram-negative core antigens (GNCAs) have been demonstrated in many mammalian species but to date are unexamined in any avian species. An enzyme-linked immunosorbent assay with phenol-killed whole cell Escherichia coli J5 was used to assess the presence of serum antibodies directed toward GNCAs in chickens. The first experiment consisted of collecting blood samples from randomly selected hens at egg laying ranches in northern California. The ages ranged from several days of age to 77 wk of age. Birds were classified into age groups (hatchling [1 day-4 wk], pullet [4-18 wk], pullet cycle [18-60 wk], and postmolt [>60 wk]) and husbandry style for titer comparison. The geometric mean titer (GMT) for all adult hens regardless of age was 2147. The geometric mean titers were 220, 5691, 2304, and 1776 for hatchlings, pullets, pullet cycle hens, and postmolt hens, respectively. The age group titer trends were similar to those of humans rather than those of farm animals in that the highest titers occurred during "adolescence" (pullets) and titers decreased slightly with maturity. The GMTs were 2870 for hens housed intensively and 1872 for those housed extensively. The second experiment looked at the progression of GNCA titers within individual birds over a 1-yr period. Individual titers increased slightly throughout the study time of the second experiment.

  8. 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

  9. 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

  10. 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

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

    PubMed Central

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

    1985-01-01

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

  12. 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.

  13. 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

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

    PubMed

    Pár, A; Mihály, I; Kömives, K

    1994-09-25

    An one-step enzyme-linked immunoabsorbent method, named as HBsAg Uniform II has been described for the detection of serum HBsAg, and a comparison was made with a widely used ELISA technique HBsAg Hepanostika test, to evaluate sensitivity, specificity and reproducibility of the method. A total of 531 serum samples from patients with l