抗狂犬病病毒抗体的制备及酶联免疫法的建立

Preparation of Antibody against Rabies Virus and Development of ELISA Method for Determination of Rabies Virus Antigen

目的 制备具有中和活性的抗狂犬病病毒（Rabies virus，RV）抗体，并建立检测RV抗原的ELISA法。方法 以RV全病毒免疫2只新西兰家兔，制备抗RV多克隆抗体。以RV全病毒免疫BALB／c小鼠，取脾细胞与小鼠骨髓瘤细胞SP2／0融合，建立稳定分泌抗RV单克隆抗体的杂交瘤细胞株。以小鼠中和试验（MNT）检测抗体的中和活性。以ELISA双抗体夹心法和ELISA竞争法检测RV抗原。结果 2只家兔的多克隆抗体ELISA效价分别为1：6．0×10^3和1：1．2×10^4，纯化的兔抗RVIgG中和活性分别为46．3和29．2IU／ml。获得了9株稳定分泌抗RV的单克隆抗体杂交瘤细胞株，属于IgG1或IgG2b亚型，腹水的抗体ELISA效价为1：1．0×10^5～1：1．0×10^7。单克隆抗体3E5、4C2和4F8具有中和活性，Western blot分析提示，单克隆抗体4C2是针对RV糖蛋白线性表位的抗体。以单克隆抗体4C2作为捕捉抗体，免多克隆抗体作为检测抗体，建立了ELISA双抗体夹心法，检测RV抗原。同时建立了另一种ELISA竞争法，加入固定工作浓度的单克隆抗体4C2，与RV病毒液孵育，以ELISA间接法检测RV病毒抗原。结论 所获得的狂犬病毒多克隆和单克隆抗体具有中和活性，可在ELISA中用于检测RV抗原。

Objective To prepare the antibody against rabies virus（RV） ,with neutralizing activity,and develop an ELISA method for the determination of RV antigen. Methods The polyclonal antibody（PcAb） against RV was prepared by immunizing New Zealand rabbits with RV strain. The hybridoma cell strain stably secreting the monoclonal antibody（McAb） against RV was established by fusing murine myeloma SP2/0 cells with the splenocytes of BALB/c mice immunized with RV strain. The neutralizing activity of antibody was determined by murine neutralization test. The RV antigen was determined by double antibody sandwich ELISA and competitive ELISA. Results The ELISA titers of PcAbs of two rabbits were 1 ： 6. 0 × 10^3 and 1 ： 1.2 × 10^4, and the neutralizing activities of purified rabbit IgG against RV were 46.3 and 29.2 IU/ml respectively. Nine hybridoma cell strains stably secreting McAb to RV,of IgG1 or IgG2b subtype,were established. The ELISA titers of McAbs secreted by the strains in ascites were 1：1 ×10^5-1：1 × 10^7. McAbs 3E5, 4C2 and 4F8 showed neutralizing activities. Western blot proved that McAb 4C2 recognized the linear epitope of RV glycoprotein. A double antibody sandwich ELISA for RV antigen was developed using McAb 4C2 as capture antibody and rabbit PcAb as detection antibody. Meanwhile,a competitive ELISA was developed by incubating McAb 4C2 at a constant concentration with RV then determining RV antigen by indirect ELISA. Conclusion Both the prepared PcAb and McAb showed neutralizing activities and could be used for the determination of RV antibody by ELISA.