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1.
D S Vallari B W Jett H J Alter L T Mimms R Holzman J W Shih 《Journal of clinical microbiology》1992,30(3):552-556
Serological markers for hepatitis C virus (HCV) infection were measured in serial samples from 14 posttransfusion chronic non-A, non-B hepatitis patients by a semiquantitative dot blot immunoassay. The assay detected antibodies to HCV by use of recombinant proteins that represent putative HCV capsid (core), nonstructural protein 3 (NS3) (33c), and NS4 (c100) epitopes. Seroconversion to anti-HCV antibodies (anti-HCV) was detected in all patients. The average time to active antibody production detected by any of the recombinant proteins was 13.8 (range, 3.6 to 22.0) weeks posttransfusion or 4.6 (range, -4.5 to 13.4) weeks after the first biochemical marker of illness. Anti-HCV were detected earliest by the core antigen in most cases; however, the patterns of anti-HCV responses varied significantly among individuals. Overall, the addition of the core and NS3 antigens to the assay enabled the detection of the antibody response 4 to 5 weeks earlier than did the addition of the c100 antigen, the sole antigen used in current screening tests in the United States. Passively transferred antibodies were detected by at least one antigen in early posttransfusion samples from 12 patients and decayed below detectable levels for all antigens in only 2 patients. Antibodies to all three gene products were evident in the last sample from all five patients monitored for greater than 3 years from transfusion indicating the persistence of antibodies in patients with chronic illness. Our data show that the period following the onset of hepatitis during which anti-HCV are not detected by current screening assays can be greatly shortened by the detection of anti-HCV responses by a combination of core, NS3, and c100 antigens. 相似文献
2.
It has been demonstrated that the immunodominant region of the HCV core protein and the hepatitis B surface antigen (HBsAg) have high degree of reactivity. In order to construct a chimeric protein that carries HCV and HBV epitopes and possesses immunogenicity to both HCV and HBV, four epitopes derived from residues aa2-21 (epitope C1), aa22-40 (epitope C2) of the core protein, residues aa315-328 (epitope E) of E1 protein of HCV, and residues aa124-147 (epitope S) of HBsAg were chosen to be displayed in a conformation-specific manner on the outer surface of the Flock House virus capsid protein and expressed in E. coli cells. The reactivity of these epitopes with antisera from hepatitis C and hepatitis B patients and induction of immune response in guinea pigs were determined. The results showed that when displayed in this system, the chimeric protein carrying only epitope S could react with anti-HBsAg positive human sera, elicit an anti-HBsAg response in guinea pigs. The chimeric protein carrying epitopes C1, C2 and E could react with antibodies to different HCV genotypes, elicit an anti-HCV response in guinea pigs. The chimeric protein carrying epitopes C1, C2, E, and S could react with antibodies against HCV and HBV, elicit anti-HCV and anti-HBsAg responses in guinea pigs. The results suggested that these epitopes displayed in this form could be considered for development of epitope-based vaccines against HCV/HBV infections. 相似文献
3.
HCV感染后NS5区抗体的动态观察与检测意义 总被引:5,自引:2,他引:5
本文报道用HCVNS5区两段抗原性较好的合成肽研制的ELISA试剂盒及UBIHCVNS5区抗体检测ELISA试剂盒观察14例HCV感染者NS5区抗体的动态变化,证实NS5区抗体如同NS4区抗体一样比C及NS3区抗体出现晚。NS5区抗体总体检出率近似于NS4区抗体;1.55%的血清为单独NS5区抗体阳性;存在NS5区抗体与其他区抗体滴度有互补作用的标本等提示NS5区抗体仍有一定的诊断价值。采用UBINS5区抗体检测试剂盒发现,95.65%(22/23)UBI试剂盒诊断为NS5区抗体阳性的标本中含HCVRNA,6/14HCv感染者用UBI试剂盒检出的抗体出现在ALT再次异常升高或剧烈升高(高于参考值的3倍以上)前后,4/14的感染者抗体出现于ALT首次升高前后(其余4/14的感染者未检出抗NS5抗体),因此UBI抗HCVNS5抗体诊断试剂盒检测的抗体似与疾病的活动有关。 相似文献
4.
Colette Jolivet-Reynaud Pascal Dalbon Florence Viola Stphane Yvon Glauricia Paranhos-Baccala Nadia Piga Laurence Bridon Mary Anne Trabaud Nicole Battail Genevive Sibai Michel Jolivet 《Journal of medical virology》1998,56(4):300-309
Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1–120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1–120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1–120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2–45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20–24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29–33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58–65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7–17, 34–39, and 73–86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton × 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected. J. Med. Virol. 56:300–309, 1998 . © 1998 Wiley-Liss, Inc. 相似文献
5.
Simultaneous detection of hepatitis C virus (HCV) core antigen and anti-HCV antibodies improves the early detection of HCV infection
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Laperche S Le Marrec N Girault A Bouchardeau F Servant-Delmas A Maniez-Montreuil M Gallian P Levayer T Morel P Simon N 《Journal of clinical microbiology》2005,43(8):3877-3883
To evaluate whether a new enzyme immunoassay developed for the simultaneous detection of hepatitis C virus (HCV) core antigen (Ag) and anti-HCV antibodies (anti-HCV Ab) (Monolisa HCV Ag/Ab ULTRA; Bio-Rad) could improve the early detection of HCV infection, we compared its sensitivity to that of anti-HCV, HCV core Ag, and HCV RNA assays. The populations studied included 12 blood donor samples positive for HCV RNA and HCV core Ag but negative for anti-HCV antibodies and 23 hemodialysis patients who developed anti-HCV Ab (seroconversion) during the follow-up. From these 23 individuals, 83 samples sequentially collected prior to seroconversion and 108 samples collected after seroconversion were tested. Six of 12 blood donations were positive by the HCV Ag/Ab assay. In the hemodialysis cohort, the 24 HCV RNA-negative samples were negative by the HCV Ag/Ab assay and 23 of the 59 HCV RNA-positive samples (39%) were positive. The HCV Ag/Ab assay detected HCV infection on average 21.6 days before the most sensitive antibody assay. The HCV Ag/Ab assay did not detect HCV infection as early as the HCV RNA assay (mean delay, 30.3 days) or HCV Ag assay (mean delays, 27.9, and 16.3 days by the HCV core Ag quantification assay and the HCV Ag blood screening assay, respectively). This new assay provides a notable improvement for the early detection of HCV infection during the so-called window period compared with anti-HCV Ab assays and could be a useful alternative to HCV RNA detection or HCV core Ag assays for diagnosis or blood screening when nucleic acid technologies or HCV core Ag detection are not implemented. 相似文献
6.
Gutierrez JA Klepper AL Garber J Walewski JL Bateman K Khaitova V Syder A Tscherne DM Gauthier A Jefferson D Rice CM Schiano TD Branch AD 《Journal of virological methods》2011,171(1):169-175
Many anti-HCV antibodies are available, but more are needed for research and clinical applications. This study examines whether ascitic fluid from cirrhotic patients could be a source of reagent-grade antibodies. Ascitic fluid from 29 HCV patients was screened by ELISA for anti-HCV antibodies against three viral proteins: core, NS4B, and NS5A. Significant patient-to-patient variability in anti-HCV antibody titers was observed. Total ascitic fluid IgG purified by Protein-A chromatography reacted with HCV proteins in immunoblots, cell extracts, and replicon-expressing cells. Affinity-purification using synthetic peptides as bait allowed the preparation of cross-genotypic antibodies directed against pre-selected regions of HCV core, NS4B, and NS5A proteins. The performance of the polyclonal antibodies was comparable to that of monoclonal antibodies. Anti-NS4B antibody preparations reacted with genotype 1a, 1b, and 2a NS4B proteins in immunoblots and allowed NS4B to be localized in replicon-expressing cells. Ascitic fluid is an abundant source of human polyclonal cross-genotypic antibodies that can be used as an alternative to blood. This study shows the utility of selectively purifying human polyclonal antibodies from ascitic fluid. Affinity purification allows antibodies to be selected that are comparable to monoclonal antibodies in their ability to react with targeted regions of viral proteins. 相似文献
7.
8.
Hepatitis C virus (HCV) has as yet no practical culture system so any antigen or antibody studies must be carried out using recombinant antigens. In this study, HCV core sequence was amplified by PCR, inserted into pRSET, and expressed in E. coli. The resultant core protein was purified using nickel affinity chromatography which bound the six histidine tag attached to the N-terminus of the protein. After elution in imidazole buffer, the core protein was used to immunise Balb/c mice and monoclonal antibodies against HCV core were raised. Six monoclonals were examined in a variety of assays. All of them recognised the p27 kDa protein which they were raised against and 2D2 and 3D7 recognised the core component of an HCV Recombinant Immunoblot Assay (RIBA). None of the antibodies recognised the linear peptides in an Innolia HCV assay. 2D2 showed cytoplasmic granular staining in 1–5% of cells in frozen sections of HCV infected livers. Cross-competition assays between themselves and human anti-HCV core positive sera divided the antibodies into two main groups (I and II), with a sub-division of group I into a and b. Group I antibodies were unable to be inhibited by human anti-HCV sera whereas group II antibodies were inhibited by these sera (up to 62%). Epitopes recognised by all the monoclonals were probably conformational with the group I epitope being located within the first 105 amino acids of the core sequence and the group II epitope between amino acids 105 and 160. © 1996 Wiley-Liss, Inc. 相似文献
9.
10.
Leary TP Gutierrez RA Muerhoff AS Birkenmeyer LG Desai SM Dawson GJ 《Journal of medical virology》2006,78(11):1436-1440
Hepatitis C virus (HCV) exposure in blood donors is determined serologically by the detection of anti-HCV antibodies in serum or plasma. However, a "window" period of 30-70 days after exposure exists where specific antibodies to HCV antigens are not detected. The use of nucleic acid testing for the detection of HCV RNA or antigen testing for the detection of HCV core protein have resulted in dramatic reductions in the pre-seroconversion window period. In this study, an automated HCV core antigen detection test was developed. This magnetic microparticle-based assay utilizes anti-HCV core monoclonal antibody to capture antigen present in human serum or plasma. Captured antigen is then detected using an anti-HCV core monoclonal antibody conjugated with a chemiluminescent compound. The specificity of this assay was established at 99% upon testing a population of normal volunteer blood donors. Sensitivity was determined by testing 16 commercially available HCV seroconversion panels representing genotypes 1a, 1b, 2b, and 3a. In each panel tested, HCV core antigen was detected prior to anti-HCV antibody, resulting in a reduction of the window period by greater than 23 days on average, and greater than 34 days on panels initially NAT negative. In addition, HCV core antigen was detected in >97% of HCV RNA positive/antibody negative specimens, exhibiting sensitivity nearly equivalent to nucleic acid testing in the pre-seroconversion window period for the panels examined. 相似文献
11.
目的 研究抗HCV阳性无病毒血症患者特异的体流及细胞免疫状态。方法 对15例抗HCV阳性无病毒血症患者,15例慢性HCV持续感染者及15例正常对照者进行研究。通过MTT、LDH释放法、酶联免疫等方法观察患者外周T细胞反应、自然杀伤(NK)细胞活性、细胞因子分泌及特异性体液免疫反应。结果 无病毒血症者T细胞增殖反应明显大于持续感染者及对照组,并且T细胞对核心区抗原增殖最大,NS3次之,对NS5则无明 相似文献
12.
Design of novel conformational and genotype-specific antigens for improving sensitivity of immunoassays for hepatitis C virus-specific antibodies 总被引:4,自引:0,他引:4
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Lin S Arcangel P Medina-Selby A Coit D Ng P Nguyen S McCoin C Gyenes A Hu C Tandeske L Phelps B Chien D 《Journal of clinical microbiology》2005,43(8):3917-3924
The current commercially licensed enzyme-linked immunosorbent assays (ELISAs) for hepatitis C virus (HCV) mainly use recombinant proteins containing linear epitopes. There is evidence, however, that conformational epitopes of HCV are more immunoreactive. Thus, we have designed an HCV antibody assay that employs a conformational protein, NS3NS4a PI (with functional protease and helicase activities), and a linear fusion protein, multiple-epitope fusion antigen 7.1 (MEFA 7.1) or MEFA 7.2. We have shown that NS3NS4a PI detects early-seroconversion conformation-sensitive antibodies better than c33c antigen. The correct conformation of NS3NS4a PI also cross-reacts with different genotype samples better than the c33c antigen. MEFA 7.1 and MEFA 7.2 incorporate all the major immunodominant and genotype-specific epitopes of HCV core, E1, E2 hypervariable region 1 (HVR1), E2 HVR1-plus-HVR2 consensus, NS3, NS4, and NS5. Since MEFA 7.1 is degraded by the active NS3NS4a PI protease, we designed a second MEFA 7.2 construct in which the six protease cleavage sites found in MEFA 7.1 were eliminated by amino acid mutation. We demonstrate here that MEFA 7.2 remains intact in the presence of NS3NS4a PI and preserves the epitopes present in MEFA 7.1. Compared to currently licensed assays, an ELISA incorporating a combination of the two antigens NS3NS4a PI and MEFA 7.1 or 7.2 demonstrates better serotype sensitivity and detects seroconversion earlier in many commercially available panels. We believe that an assay using NS3NS4a PI and MEFA 7.1 or 7.2 may have the potential to replace current HCV immunoassays for better sensitivity. 相似文献
13.
丙型肝炎病毒NS3蛋白单克隆杂交瘤细胞株的建立及初步鉴定 总被引:2,自引:1,他引:2
应用国产基因工程表达的丙型肝炎病毒(HCV)NS3区抗原免疫小鼠,然后取其脾细胞与小鼠骨髓瘤细胞系SP2/0融合,筛选出4株稳定分泌抗HCVNS3区蛋白单克隆抗体(McAb)的杂交瘤细胞株,分别命名为2B6,2F3,3D8,3D9,经初步研究表明这4株单抗与NS3抗原具有良好的反应性,与HCV核心区多肽及乙型肝炎病毒表面抗原和e抗原均无反应。抑制实验表明这4株抗体分别针对NS3抗原分子上的2个不同的抗原决定簇。 相似文献
14.
Human p85 glycoprotein bears three distinct epitopes defined by several monoclonal antibodies 总被引:4,自引:0,他引:4
M Letarte 《Molecular immunology》1986,23(6):639-644
A glycoprotein of apparent mol. wt 85,000 isolated from human cells of B lineage by affinity to 50B4-IgG immunoadsorbent was shown previously to express two spatially distinct epitopes identified with monoclonal antibodies 50B4 and 50E6 [Letarte et al. (1985) Molec. Immun. 22, 113-124]. It is now demonstrated that the p85 glycoprotein is homologous to the F10-44-2 antigen, defined initially as a T-lymphocyte-granulocyte-brain antigen [Dalchau et al. (1980) Eur. J. Immun. 10, 745-749], the A1G3 human medullary thymocyte antigen [Haynes et al. (1983) J. Immun. 131, 1195-1200] and the A3D8 antigen defined on erythrocytes [Telen et al. (1983) J. clin. Invest. 71, 1878-1886]. The 50B4 antigen purified either from a B lymphoblastoid cell line or a leukemic cell line, at a concn ranging from 0.25 to 2.0 micrograms protein/ml, could block the binding of either F10-44-2 and F-10-62-1 (a related antibody) or A1G3 and A3D8 antibodies to human leukemic cells. All of these antibodies immunoprecipitated, from the purified antigenic preparation, a single glycosylated polypeptide chain of apparent mol. wt 85,000. Competitive binding studies indicated that these antibodies define at least three epitopes. The F10-44-2 and A3D8 antibodies blocked the reactivity of monoclonal 50E6-IgG with human leukemic cells and thus bind to an epitope identical or spatially related to the 50E6 epitope. Antibody F10-62-1 competed for the binding of 50B4-IgG to leukemic cells and thus recognizes the 50B4-like epitope. The A1G3 antibody blocked 30% of the binding of 50E6-IgG but did not inhibit the binding of 50B4-IgG: it is thus reacting with a third epitope on the p85 glycoprotein distinct from but close to the 50E6 epitope. 相似文献
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16.
HCV核心抗原C33肽单克隆抗体的研制与初步鉴定 总被引:3,自引:2,他引:3
用HCV核心抗原(33肽)与鼠血清白蛋白交联后免疫BALB/c小鼠,用杂交瘤技术获得有实用价值的2株McAb。此两株McAb除与免疫抗原有较强的抗原-抗体反应外,与HCV核心抗原CP10也有很好的反应;但与HCVNS3、NS5及核心区CP9抗原无反应性。在竞争ELISA中,对抗HCV-IgG阳性血清有较好的抑制作用。将此两株McAb用于慢性丙型肝炎病人肝活检免疫组化研究,获得较为满意的结果。 相似文献
17.
Abdulmedzhidova AG Masalova OV Atanadze SN Ulanova TI Burkov AN Khudiakov IuE Fields H Kushch AA 《Voprosy virusologii》2002,47(1):21-25
Recombinant protein rNS3 imitating helicase region (1356-1459 amino acid residues) of hepatitis C virus (HCV) was expressed in E. coli cells and used for BALB/c mice immunization. Seven hybrydoma clones producing monoclonal antibodies (MAbs) to rHS3 were obtained. All MAbs reacted in ELISA with NS3 protein from Murex anti-HCV Version III and in immunoblotting from RIBA 3. These MAbs detect 5 individual epitopes, 4 of which were conformational and 1 discontinuous. All MAbs could compete for rNS3 binding with serum antibodies from patients with chronic hepatitis C, which suggests that these MAbs can recognize the natural HCV NS3 protein. 相似文献
18.
血清中丙型肝炎NS3抗原ELISA检测方法的建立和初步应用 总被引:1,自引:0,他引:1
目的 评价血清中丙型肝炎病毒(HCV)游离NS3抗原的酶联免疫吸附(ELISA)检测方法的特异性和灵敏度,初步探讨该方法在临床应用中的意义.方法 对77例正常人血清标本,173例抗-HCV阳性标本和3708例抗-HCV阴性的其他类型肝炎血清标本检测HCV游离NS3抗原;对部分HCV NS3抗原阳性标本进行验证,包括HCV RNA测定、中和试验和免疫斑点试验;对11例患者的25份系列血清标本进行了HCV游离NS3抗原、HCV RNA和HCV抗体的联合检测,并结合临床资料综合分析.结果 3708例抗-HCV阴性的其他类型肝炎血清标本中有48例为HCV NS3抗原阳性,其中3030例单纯乙型肝炎和445例其他类型肝炎血清标本中分别有44例和4例为HCV NS3抗原阳性;173例HCV抗体阳性标本中有42例为HCV NS3抗原阳性;77例正常人血清标本的HCV NS3抗原检测结果均为阴性;15例HCV NS3抗原阳性标本中有9例为HCV RNA阳性;23例HCV NS3抗原阳性标本的中和率和免疫斑点试验的阳性率分别为87.0%和69.6%;25份系列血清标本的检测结果显示其HCV NS3抗原的吸光度值与时间呈负相关,并有2例HCV NS3抗原阳性标本随着血清中HCV NS3抗原的吸光度值下降,其HCV抗体转阳.结论 血清中HCV游离NS3抗原的ELISA检测方法有较好的特异性和敏感度,在发展中国家应用此方法进行HCV感染的早期诊断有一定的临床意义和推广价值. 相似文献
19.
Fenwick F Bassendine MF Agarwal K Bevitt D Pumeechockchai W Burt AD Toms GL 《Journal of clinical pathology》2006,59(2):174-178
BACKGROUND: Patients with common variable immunodeficiency may exhibit rapidly progressive hepatitis when infected with hepatitis C virus (HCV), leading to cirrhosis and liver failure. Liver transplantation in these patients may result in a cholestatic form of HCV reinfection with exceptionally high virus loads. AIMS: To report an immunohistochemical investigation of the pretransplant and post-transplant liver of one such patient. METHODS/RESULTS: On immunohistochemical staining of frozen sections with anti-HCV core monoclonal antibody or fluorescein labelled human polyclonal anti-HCV IgG, no HCV antigens were demonstrated in the native cirrhotic liver removed at transplant, despite a viral load of 10(6.4) genomes/g. The transplanted liver, collected six weeks post-transplant, exhibited cholestatic recurrent hepatitis, had an HCV virus load of 10(10) genomes/g of liver, and revealed HCV antigen in the cytoplasm of most hepatocytes, with a pronounced periportal distribution. No virus antigen was demonstrable in other cell types. The core antigen was also detected in paraffin wax embedded, formaldehyde fixed tissue of this liver after high temperature antigen retrieval, but not in the native cirrhotic liver or a selection of HCV positive livers collected pretransplant from immunocompetent patients. Attempts to delineate the distribution of E1, NS3, and NS4 antigens were unsuccessful because monoclonal antibodies to these antigens produced "false positive" staining of foci of hepatocytes in the post-transplant livers of HCV seronegative patients with cholestasis. CONCLUSION: This case provided an opportunity to study the natural development of HCV during acute infection in the absence of an immune response, and may help to elucidate the pathogenesis of HCV recurrence in liver allografts. 相似文献
20.
HCV NS3蛋白单克隆抗体的制备及其识别区域的分析 总被引:2,自引:0,他引:2
目的 制备针对丙型肝炎病毒(HCV)非结构区NS3全长蛋白的单克隆抗体(MAb),并分析获得的单抗识别表位所在区域,为建立以NS3蛋白为靶位的抗HCV研究提供抗体工具。方法 用原核表达的HCV非结构区NS3全长蛋白作为免疫原,采用小鼠腹股沟皮下NC膜包埋法免疫小鼠,按常规杂交瘤细胞的制备方法,经细胞融合、克隆化制备抗NS3蛋白的MAb。用间接免疫荧光法和Westem blot鉴定其特异性。分别构建NS3丝氨酸蛋白酶(NS3蛋白的N末端1/3)编码基因的真核表达质粒pcDNA3.1(-)-ns3p、NS3解旋酶(NS3蛋白的C末端2/3)编码基因的真核表达质粒pcDNA3.1(-)-ns3A,将其瞬时转染COS-7细胞后,以获得的单克隆抗体作为一抗,通过免疫荧光分析获得单抗识别表位所在的区域。结果 获得了2株抗NS3蛋白的单克隆抗体,这2株MAbs均特异识别NS3蛋白,并确定了它们的结合区域。结论 获得了针对NS3蛋白的单克隆抗体,并对其单抗识别表位所在的区域进行了分析,为下一步进行以NS3蛋白为靶位的抗HCV研究奠定了良好的基础。 相似文献