CHARACTERIZATION OF HCV AND ANTI-HCV

HCV 和抗 HCV 的特征

• 批准号: 3748305
• 负责人: Z P GUO
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3748305
• 关键词: Pan; antiviral antibody; cross immunity; enzyme linked immunosorbent assay; hepatitis C virus; human tissue; immunoglobulins; protein structure; recombinant proteins; tissue /cell culture; virus protein; western blottings

Although the lack of protective immunity against reinfection with HCV has been reported, our safety study in a chimpanzee infused with an HCV RNA positive intravenous immune globulin (IGIV) prepared from a c100-3 reactive plasma pool and other recent studies support the existence of neutralizing antibodies toward envelope proteins for HCV. To assess the presence of anti-E1 and anti-E2 in certain plasma and IGIV preparations, we partially purified recombinant E1 and E2 proteins produced in a baculovirus eukaryotic expression system. Cells infected with baculovirus were subjected to freeze-thawing, sonication, and solubilization with 8 M urea. After dialysis, the solution was applied to a GNA (a lectin)-agarose column to bind glycosylated proteins. The GNA bound proteins were eluted, analyzed on a gradient SDS-polyacrylamide gel, transferred to a nitrocellulose paper, and immune blotted. A monoclonal anti-E1 identified two prominent bands, 30 kd (E1) and 105 kd, while a monoclonal anti-E2 identified one major band, 70 kd and a faint band, 105 kd. Based on the construct of the recombinant clone, the 70 kd protein is an E2-polyhedrin fusion protein while the 105 kd protein may be an unprocessed E1-E2-polyhedrin, a complex between the two, or a multimer of E1. Thus, GNA bound proteins were used to set up ELISAs for detection of antibodies to E1 and E2 in hybridoma cell cultures, human sera or plasma, or immune globulin preparations. The specificity for our newly developed ELISA utilizing a peroxidase conjugated anti-mouse IgG as a second antibody to detect monoclonal antibodies can be demonstrated since neither a monoclonal anti-core nor a normal mouse serum reacted. A similar ELISA was also set up to detecting human anti-E1 and anti-E2 by using a peroxidase conjugated anti-human IgG. However, in both ELISA and immunoblot, we found that normal human sera had some cross reactivities unless they were further diluted. Although both assays remain to be further improved with respect to its specificity and sensitivity, our preliminary data indicated that the IGIV prepared solely from either anti-c100-3 reactive plasma or multiantigen anti-HCV reactive plasma had higher levels of antibodies to HCV envelop proteins than those IGIV prepared from multiantigen screened plasma.

尽管缺乏针对HCV再感染的保护性免疫， 据报道，我们在黑猩猩中进行的安全性研究注入了HCV RNA， 由c100 - 3制备的阳性静脉内免疫球蛋白（IGIV） 反应性血浆池和其他最近的研究支持了 针对HCV包膜蛋白的中和抗体。 评估 某些血浆和IGIV制剂中存在抗E1和抗E2， 我们部分纯化了重组E1和E2蛋白， 杆状病毒真核表达系统 感染的细胞 对杆状病毒进行冻融、超声处理， 用8M尿素溶解。 透析后，将溶液应用于 GNA（凝集素）-琼脂糖柱以结合糖基化蛋白。 的 洗脱GNA结合的蛋白质，在梯度SDS-聚丙烯酰胺上进行分析 凝胶，转移到硝酸纤维素纸上，并进行免疫印迹。 一 单克隆抗E1鉴定出两条显著的带，30 kd（E1）和105 kd， 而单克隆抗E_2只鉴定出一条70 kd的主带和一条微弱的 带，105 kd。 基于重组克隆的构建，70 kd蛋白是E2-多角体蛋白融合蛋白，而105 kd蛋白是E2-多角体蛋白融合蛋白。 可以是未处理的E1-E2-多面体蛋白，两者之间的复合物，或 E1的多聚体。因此，使用GNA结合的蛋白质来建立ELISA，以用于检测GNA结合的蛋白质。 人杂交瘤细胞培养物中E1和E2抗体的检测 血清或血浆或免疫球蛋白制剂。 我们的特异性 新开发的ELISA利用过氧化物酶结合的抗小鼠IgG 作为第二抗体检测单克隆抗体， 因为单克隆抗核心和正常小鼠血清都不反应。 建立了检测人源抗E1和抗E2的ELISA方法 通过使用过氧化物酶缀合的抗人IgG。 然而，在两种ELISA中， 和免疫印迹，我们发现正常人血清中有一些交叉， 除非它们被进一步稀释。 尽管两种检测方法 在其特异性方面仍有待进一步改进， 敏感性，我们的初步数据表明，IGIV准备单独 来自抗c100 - 3反应性血浆或多抗原抗HCV反应性血浆 血浆中抗HCV包膜蛋白抗体水平高于血浆中抗HCV包膜蛋白抗体水平。 从多抗原筛选血浆制备IGIV。