抗肠道病毒71型单克隆抗体的制备及鉴定

Preparation and Identification of Monoclonal Antibody against Enterovirus 71

目的制备肠道病毒71型(EV71)单克隆抗体,并进行初步鉴定。方法将RD细胞和Vero细胞培养的EV71原液通过氯化铯超速离心纯化,分别作为免疫抗原和检测抗原,制备单克隆抗体。通过Westernblot分析、间接免疫荧光试验和ELISA法鉴定单抗的特异性。采用间接ELISA法测定单抗的酶标效价,微量细胞培养中和试验测定单抗的中和效价。结果获得1株可稳定分泌抗EV71单克隆抗体的杂交瘤细胞株,其分泌的单抗可与EV71特异性结合,酶标效价为1∶204800,中和效价为1∶28。结论已成功筛选出1株分泌抗EV71的单抗细胞株,为建立EV71疫苗抗原含量测定方法奠定了基础。

Objective To prepare and preliminarily identify the monoclonal antibody（McAb）against enterovirus 71（EV71）. Methods The bulks of EV71 cultured in RD and Vero cells were purified by cesium chloride ultracentrifugation and used as immunizing and detection antigens respectively for preparation of McAb. The prepared McAb was identified for specificity by Western blot, indirect IFA and ELISA, and determined for titer by ELISA and micro-cell culture neutralization test. Results A hybridoma cell strain stably secreting McAb against EV71 was obtained, and the secreted McAb showed specific binding to VP1 protein of EV71. The titers of McAb determined by ELISA and micro-cell culture neutralization test were 1：204 800 and 1：28 respectively. Conclusion A hybridoma cell strains secreting McAb against EV71 was successfully screened, which laid a foundation of developing a method for determination of antigen content in EV71 vaccine.