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目的制备乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14-2株(简称SA)和P3株单克隆抗体,并进行初步应用。方法分别用JEV SA株减毒活疫苗和P3抗原免疫BALB/c小鼠,交叉加强免疫1次,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,间接ELISA法筛选阳性杂交瘤细胞,选择人血清白蛋白(HSA)抗体阴性、JEV抗体阳性的克隆进行2次亚克隆,制备腹水并纯化,对单抗及杂交瘤细胞进行鉴定。用SA单抗建立捕获ELISA法检测JEV抗体,竞争抑制ELISA法和双抗体夹心ELISA法检测JEV抗原含量。结果经3次融合,共获得4株分泌抗JEV SA株单抗和3株分泌抗JEV P3株单抗的杂交瘤细胞株,未获得SA株和P3株共同决定簇的单抗;7株单抗均为IgG1型,特异性良好,但均无中和活性;SA株单抗的相对亲和力大小为:3D1>5E3>6H3>4F12,均能识别SA相同的抗原表位;P3株单抗的相对亲和力大小为:1C7>5H12>3C4,均能识别P3相同的抗原表位;7株杂交瘤细胞于液氮中放置6个月及体外连续培养3个月后,制备的腹水抗体效价保持稳定;用5E3株SA单抗建立了检测JEV抗体的捕获ELISA法及检测JEV抗原的竞争抑制ELISA法和双抗体夹心ELISA法。结论已制备了JEV SA株和P3株单克隆抗体;以SA株单抗建立的捕获ELISA法检测JEV抗体比间接ELISA法更简便,且敏感性更高,对于抗原浓度高的样品,双抗体夹心ELISA法检测比竞争抑制ELISA法更有优势。
Objective To prepare monoclonal antibodies against Japanese encephalitis virus (JEV) strain SA14-14-2 (SA) and P3 strain for preliminary application. Methods BALB / c mice were immunized with attenuated live vaccine of JEV strain SA and P3 antigen, respectively. The spleen cells of immunized mice were fused with SP2 / 0 myeloma cells and the positive hybridomas were screened by indirect ELISA . The clones that were negative for human serum albumin (HSA) antibody and positive for JEV antibody were selected and subcloned twice to prepare ascites and purified. The monoclonal antibodies and hybridoma cells were identified. Capture ELISA was used to detect JEV antibody with SA monoclonal antibody, competitive inhibition ELISA and double-antibody sandwich ELISA to detect JEV antigen. Results Four hybridomas secreting monoclonal antibody against JEV SA and three monoclonal antibodies against JEV P3 were obtained after three confluence, and monoclonal antibodies against the common determinant of SA and P3 were obtained. Anti-IgG1 type, good specificity, but no neutralization activity; SA monoclonal antibody relative affinity size: 3D1> 5E3> 6H3> 4F12, can identify the same epitope of SA; P3 monoclonal antibody The relative affinity was 1C7> 5H12> 3C4, which could identify the same antigenic epitope of P3. The seven hybridoma cells were kept in liquid nitrogen for 6 months and cultured in vitro for 3 months. Stable; 5E3 strain SA monoclonal antibody was established to detect the capture ELISA JEV anti-JEV antigen and competitive inhibition ELISA and double antibody sandwich ELISA method. Conclusion The monoclonal antibodies against JEV strain SA and P3 strain have been prepared. The detection of JEV antibodies by ELISA with SA monoclonal antibody is more convenient and sensitive than the indirect ELISA. For the samples with high antigen concentration, the double antibody sandwich ELISA assay is more advantageous than competitive inhibition ELISA.