乙型脑炎病毒SA14-14-2株和P3株单克隆抗体的制备及应用  

Preparation and application of monoclonal antibody against Japanese encephalitis virus SA14-14-2 and P3 strains

作　　者：周志军[1] 施金荣[1] 朱华松[1] 王平[1] 余模松[1] 

机构地区：[1]武汉生物制品研究所有限责任公司血液制剂室,武汉430060

出　　处：《中国生物制品学杂志》2012年第4期462-468,共7页Chinese Journal of Biologicals

摘　　要：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 and preliminarily apply the monoclonal antibody （McAb） against Japanese encephalitis （JEV） SA14-14-2 and P3 strains. Methods BALB mice were immunized with live attenuated JEV vaccine prepared with SA14-14-2 strain and P3 antigen respectively, and boosted crossly. The splenocytes of immunized mice were fused with SP2 / 0 cells, and positive hybridoma cells were screened by indirect ELISA. The clones negative for human serum albumin （HSA） but positive for JEV antibody were selected and subcloned for 2 times, based on which ascites was prepared and purified, and McAb and hybridoma were identified. Capture ELISA method was developed with McAbs against SA14-14-2 strain and used for determination of JEV antibody, while competitive inhibition ELISA and double antibody sandwich ELISA methods for determination of JEV antigen content. Results After 3 times of fusion, four hybridoma cell strains secreting McAb against SA14-14-2 strain and three hybridoma cell strains secreting MeAbs against P3 strain were obtained, but no hybridoma cell strains secreting McAbs with common antigenic determinants of SA and P3 strains were obtained. All the McAbs secreted by the seven hybridoma cell strains were IgG1 with high specificity but without neutralizing activity. In the order of relative affinity, the McAbs against SA14-14-2 strain were 3D1, 5E3, 6H3 and 4F12, all of which recognized the same SA14-14-2 antigenic epitopes; while the McAbs against P3 strain were 1C7, 5H12 and 3C4, all of which recognized the same P3 antigenic epitopes. The titers of McAbs prepared with the seven hybridoma cell strains 6 months after storage in liquid nitrogen and with those after continuous culttve for 3 months in vitro were stable. By using McAb 5E3 against SA14- 14-2 strain, the capture ELISA method for determination of JEV antibody as well as competitive inhibition ELISA method and double antibody sandwich ELISA methods for JEV antigen were developed. Conclusion The McAbs against SA14-14-2 and P3 strains of JEV were

关 键 词：乙型脑炎病毒 单克隆抗体 酶联免疫吸附测定 

分 类 号：R373.31[医药卫生—病原生物学] R392.33[医药卫生—基础医学]