CHARACTERIZATION OF HCV AND ANTI-HCV

尽管缺乏对丙型肝炎病毒再次感染的保护性免疫力 据报道，我们对一只注射了丙型肝炎病毒RNA的黑猩猩进行了安全性研究 C100-3制备阳性静脉免疫球蛋白(IGIV) 反应血浆池和其他最近的研究支持存在 针对丙型肝炎病毒包膜蛋白的中和抗体。要评估 某些血浆和免疫球蛋白制剂中存在抗-E1和抗-E2， 我们对重组的E1和E2蛋白进行了部分纯化 杆状病毒真核表达系统。感染病毒的细胞 对杆状病毒进行冻融、超声波处理和 用8M尿素增溶。透析结束后，应用该溶液 与GNA(一种凝集素)-琼脂糖柱结合糖基化蛋白。这个 GNA结合蛋白被洗脱，在梯度SDS-聚丙烯酰胺上分析 凝胶，转移到硝酸纤维素纸上，免疫印迹。一个 单抗E1鉴定出两条明显的条带，分别为30kd(E1)和105kd。 而抗E_2的单抗鉴定出一条主带，70kd，一条微弱的 波段，105kd。基于重组克隆的构建，70 KD蛋白是一种E2-多角体蛋白融合蛋白，而105kd蛋白 可以是未经处理的E1-E2多角体、两者之间的络合物或 E1的多聚体。因此，GNA结合蛋白被用来建立ELISA 人杂交瘤细胞培养中抗E1和E2抗体的检测 血清或血浆，或免疫球蛋白制剂。我们的专业性 应用过氧化物酶偶联抗鼠免疫球蛋白建立新的酶联免疫吸附试验 作为第二抗体可用于检测单抗 因为抗核心的单抗和正常的小鼠血清都不反应。 建立了一种类似的检测人源性抗-E1和抗-E2的酶联免疫吸附试验 通过使用过氧化物酶偶联的抗人免疫球蛋白。然而，在两种酶联免疫吸附试验中 免疫印迹，我们发现正常人血清中有一些交叉 反应性，除非它们被进一步稀释。虽然这两种检测方法 在其特殊性方面仍有待进一步完善， 敏感性，我们的初步数据表明，IGIV单独准备 从抗C100-3反应性血浆或多抗原抗丙型肝炎病毒反应性 血浆中抗丙型肝炎病毒包膜蛋白抗体水平高于 多抗原筛选血浆制备免疫球蛋白IV。

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.