稳定分泌抗羊种布鲁菌脂多糖单克隆抗体细胞株的筛选与应用

研制抗羊种布鲁菌脂多糖抗原的单克隆抗体,并应用其建立检测布病的双夹心ELISA方法。本研究采用热酚水法提纯羊种布鲁菌(16M菌株)的脂多糖抗原,并经SDS-PAGE鉴定。用脂多糖和灭活的羊种布鲁菌16M作为免疫抗原,交替免疫6～8周龄BALB/c雌鼠,第1次免疫用羊种布鲁菌标准菌16M全菌加等量弗氏完全佐剂;第2次免疫用脂多糖加等量弗氏不完全佐剂。将脂多糖作为包被抗原建立间接ELISA方法,筛选针对抗羊种布鲁菌(16M菌株)脂多糖的单克隆抗体杂交瘤细胞株。筛选出3株能稳定分泌抗羊种布鲁菌脂多糖单克隆抗体的杂交瘤细胞株,分别命名为5H3、6B8和3H7,细胞培养上清的ELISA效价在1∶1 000～1∶5 000,小鼠腹水单克隆抗体ELISA效价在1∶10 000～1∶160 000;抗体亚类鉴定表明:5H3、6B8属于IgM亚类,3H7属于IgG3亚类;特异性试验结果显示:3株杂交瘤细胞分泌的抗体不与大肠杆菌O157裂解抗原、鸡白痢沙门氏菌裂解抗原、鸭源鸡杆菌脂多糖抗原以及福氏志贺菌裂解抗原反应,仅与灭活的羊种布鲁菌(16M)发生反应。布鲁菌虎红平板凝集试验和试管凝集试验检测结果显示,获得的单抗可与标准检测抗原形成明显的颗粒凝集物和伞状凝集物。利用所建立的单克隆抗体细胞株,建立了一种检测布鲁菌的双夹心间接ELISA方法,并进行了特异性和敏感性检测。对模拟样品和临床样品进行检测,准确性均很高。

Development and Primary Application of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against LPS Antigen of B.Melitensis

The aim of this study was to prepare the specific monoclonal antibody(McAb) against LPS of B.melitensis,and to develop a sandwich ELISA for detecting B.melitensis.The LPS from B.melitensis strain 16M was purified by using hot phenol extraction method,and identified by SDS-PAGE.Six to eight-week-old female BALB/c mice were immunized with inactivated thalli of B.melitensis(strain 16M) in Freund’s complete adjuvant and LPS in Freund’s incomplete adjuvant successively.Three hybridoma cell lines secreting McAb against LPS were obtained and named as strains 5H3,6B8 and 3H7,respectively.The ELISA titers of cell cultures supernatant and ascites fluids of all three hybridoma cell lines ranged from 1∶1 000 to 1∶5 000,and 1∶10 000 to 1∶160 000,respectively.Immunoglobulin subclass tests differentiated corresponding mcAbs from the three hybridoma cell lines as IgG3(3H7) and IgM(5H3 and 6B8).The specificity assay showed that mAbs from 6B8,5H3 and 3H7 had non-cross-reactivity with antigens from Colibacillus O157,Salmonella pullorum,Flexneri Shigella and LPS of Gallibacterium anatis,and only reacted with the inactivated thalli of B.melitensis(strain 16M).Rose-Bengal plate and tube agglutination assays indicated that the acquired mAbs could react with standard antigen of B.melitensis,which further demonstrated that the mAbs from 6B8,5H3 and 3H7 had strong specificity.A sandwich ELISA for detecting B.melitensis was developed by using the mcAbs produced from hybridoma cell lines.Simulative samples and clinical samples were tested with the developed sandwich ELISA.Results showed that this ELISA had very high accuracy.Specific hybridoma cell lines secreting monoclonal antibodies against LPS Antigen of B.Melitensis were successfully developed,and provide a basis for establishing rapid diagnostic method of Brucella Melitensis.