新型HCV EIA诊断试剂盒的研制

丙型肝炎病毒（ＨＣＶ）基因组结构区核壳蛋白（Ｃ）区抗原、膜蛋白Ｅ１和Ｅ２区抗原，以及非结构区ＮＳ３－ＮＳ５区抗原的区段，已经在原核细胞中获得有效的表达。同时，相应区段中的优势抗原表位肽也经化学合成法大规模地制备。ＨＣＶ基因组上各区段抗原性的分析发现，由Ｃ区和ＮＳ３区分别编码的Ｃ抗原和Ｃ３３ｃ抗原是ＨＣＶ基因组上两个优势抗原区段。其相应的抗体出现早（感染后６周可检出抗Ｃ３３ｃ抗体），阳转率高（约９９％阳性检出率），特异性和重复性均优于其它区段抗原。以中国人ＨＣＶ的Ｃ３３ｃ重组蛋白和分支状合成肽ＭＡＰ－Ｃ－１９为复合抗原，研制了适合我国抗ＨＣＶ抗体检测的新型丙型肝炎病毒酶免疫测定（ＨＣＶＥＬＡ）诊断试剂盒。它同当代美国Ａｂｂｏｔｔ／ＵＢＩＨＣＶＥＬＡ诊断试剂的符合率约９８％，同加拿大ＹＥＳ公司ＨＣＶＥＩＡ诊断试剂的符合率约９７．８％，阳性检出率提高了约２％，３次重复性达１００％，表明其特异性、敏感性和重复性均达到了当代第二代ＪＣＶＥＬＡ诊断试剂的水平。我国人群中抗ＨＣＶ抗体的分布情况为：正常人群的检出率１％－２％；外科类住院病人检出率约２８．８％；肝炎患者抗ＨＣＶ阳性率为３４．４％，慢活肝、肝硬化和重症肝炎患者

DEVELOPMENT OF A NEW TYPE HCV EIA DIAGNOSTIC

ffectuve antigenic domains of structural C ,E1-E2 and nonstructural NS3-NS5 regionsthroughout the HCV genome, derived from Chinese HCV carriers,were cloned and expressed in E.coli cell,and many immunodominant epitope peptides corresponding to these regions were synthesized by a chemically synthetic method .These recombinant and synthetic-derivedantigens were then used to capture and measure reactive antibodies curculating in the different individuals.We studied their antigeniucities and found that nucleocapsid (C),NS3 and NS5 polypeptides contain nearly all immunodominant epitopes ,their antibodies come out early (NS3 and NS5 polypeptide for 6 weeks ,C polypeptide for 9 weeks after transfusion ),and are specific and broadly reactive. The C33c recombinant protein corresponding to NS3 region ofHCV is a better antigen than the others .Purified C33c recombinant protein combined with a branched peptide MAP-C-19 representing immunodominant epitopes on the nucleocapsid re-gion was used to develop a New Type HCV EIA Diagnostic Kit for the detection of an-ti-HCV antibodies.Its specificity , sensitivity and reproducibility were all in keeping with the in -dexes of the national reference sera .The conformity rate between our kit and the Abbott/UBIHCV EIA ,the 2nd generation diagnostic kit ,was 99.3%,and our kit identification rate for positive anti -HCV was (2%) more than those of Abbott and YES kits.We have found that the great diversity of HCV genome in different area would affect the establishment of the diagmostic tools.The positive rate of anti-HCV antibodies among different groupe in China,obtained by application of our kit for serodiagnosis ,was 1.4% in normal population,1.2%in vounteer donors ,24%in paid donors ,28.8% in in-patients relative to surgery and 34.4%inhepatitis cases .These results showed that injection,vaccination , acupuncture and surgical instru-ments play a great role in HCV infection (85% of the total cases)