特异性O1群稻叶型霍乱弧菌单克隆抗体的制备、筛选及识别抗原表位的分析

Preparation and characterization of monoclonal antibodies against Vibrio cholerae O1 Serotype Inaba and analysis on targeting antigenic epitopes of the monoclone antibodies

目的制备并筛选出高特异性高活性的抗O1群稻叶型霍乱弧菌(Vibrio cholerae O1 Serotype Inaba)单克隆抗体(monoclonal antibody,McAb),为霍乱的预防诊断提供有力的抗体工具。方法应用杂交瘤技术,通过灭活的O1群稻叶型霍乱弧菌免疫的BALB/c小鼠的脾细胞与SP2/0细胞融合制备特异性McAb,以间接ELISA法对所需的杂交瘤细胞株进行了特异性及排除性筛选,对筛选出的特异性McAb进行了包括亚型鉴定,效价、亲和常数的测定以及特异性的鉴定与分析,并通过单克隆抗体的位点相加实验、与O1群稻叶型霍乱弧菌脂多糖(LPS)的间接ELISA和Western blot实验对McAb识别的抗原表位进行了初步分析。结果融合了386株能分泌抗O1群稻叶型霍乱弧菌McAb的杂交瘤细胞株,通过用O1群小川型、O139霍乱弧菌及9种非霍乱弧菌的细菌进行的筛选,最后得到4株能稳定分泌特异性的针对该McAb的细胞株,其分泌的抗体亚类分别为2株IgG1,1株IgG2a,1株IgG2b;腹水效价均达10-6;亲和常数达108以上。间接ELISA法及Western blot证实所获的McAb可与O1群稻叶型霍乱弧菌发生特异性反应。ELISA相加实验结果显示4株McAb识别不同的抗原表位,与LPS的反应表明其中2株针对O1群稻叶型霍乱弧菌脂多糖,2株针对非脂多糖抗原位点。结论获得霍乱弧菌O1群稻叶型特异性McAb,初步定位单克隆抗体识别表位所在区域。

Objective To prepare and select monoclonal antibodies （McAbs） with high specificity against Vibrio cholera O1 Serotype Inaba and to provide a potent medical tool for the prevention and diagnosis of cholera, Methods BALB/c mice were immunized with Vibrio cholerae Ol Serotype Inaba, Hybridoma cell lines secreting monoclonal antibodies against Vibrio cholerae O1 Serotype Inaba were established after cell fusion of mouse splenic cells and SP2/0. Both of these McAbs were characterized for their antibody titre in the culture supernatant as well as ascites, class, sub-class and affinity analysis, The antigen specificity and strain cross reactivity for McAb were evaluated and confirmed by indirect enzyme-linked immunosorbent assay （ELISA） and Western blot. The antigen epitope identifying of McAb were observed by ELISA additive assay, indirect ELISA with LPS of cholerae O1 Serotype Inaba and Western blot. Results Totally 386 positive monoclonal cell lines were produced ; four of them showing high specificity for Vibrio cholerae O1 Serotype Inaba. As to the immunoglobulin subclass of McAb, two were IgG,, others were IgG2a and IgG2b, respectively. Indirect proof from ELISA and Western blot showed that the McAbs reacted specifically with Vibrio cholerae O1 Serotype Inaba.Results from ELISA additive assay indicated that these McAbs recognized different epitopes. The reaction with LPS indicated that two McAbs were targeted to the LPS of cholerae O1 Serotype Inaba, while others were targeted to the non-LPS sites of cholerae O1 Serotype Inaba. Conclusion Four highly specific McAbs against Vibrio cholerae O1 Serotype Inaba were obtained successfully. The antigen epitope by combined experimental site was located initially. It provides a fast and exact tool on the early diagnosis and prevention of Vibrio cholerae O1 Serotype Inaba.