抗哈维氏弧菌脂多糖单克隆抗体的制备及其鉴定

Development and characterization of monoclonal antibody against Vibrio harveyi lipopolysaccharide

以哈维氏弧菌GYC1108-1细菌颗粒性抗原免疫8周龄雌性BALB/c小鼠,5次免疫后取其脾细胞与SP2/0-Ag-14骨髓瘤细胞融合。用间接酶联免疫吸附试验(ELISA)对杂交瘤进行筛选,阳性克隆经3次亚克隆后,获得一株针对GYC1108-1脂多糖(LPS)的特异性单克隆抗体,命名为2 F 9。该株单抗细胞培养上清ELISA效价为1∶1 600,腹水效价为6.4×104,交叉反应结果表明2 F 9除了与测试的多株哈维氏弧菌发生免疫识别外,还与溶藻弧菌、麦氏弧菌和副溶血弧菌代表株发生免疫交叉反应,但不与拟态弧菌、鳗弧菌、嗜水气单胞菌代表株等发生交叉反应。用温和高碘酸氧化法对LPS处理后,GYC1108-1的LPS与2 F 9的反应部分减弱,免疫印迹结果表明2 F9识别LPS的类脂A抗原。用抑制ELISA法半定量检测GYC1108-1培养上清的LPS,测得培养48 h LPS释放量不超过39μg/mL,大大低于使鱼类致死所需的LPS的剂量。

The 8-week-old female BALB/c mice were immunized with a pathogenic V. harveyi GYC1108-1, which was isolated from diseased great yellow croaker in Zhejiang Province in 2003. Spleen cells collected from immunized mice after the fifth immunization were fused with SP2/0-Ag-14 myeloma cells. Indirect Enzyme-Linked Immunosorbent Assay （ ELISA ） was used to screen hybridoma cells and limited dilution method was performed to subclone the positive clones. After three cycles of subcloning, a McAb against the lipopolysaccharide （LPS） of GYC1108 -1 was obtained, and was designated as 2F9. The obtained McAb 2F9 was proved to have high ELISA titers. The specificity test suggested that 2F9 reacted with V. harveyi, V. alginolyticus, V. parahaemolyticus, but did not react with V. mimicus, V. anguillarum, Aeromonas hydrophila and other isolates among 14 bacteria species tested. 2F9 could recognize GYC1108 -1 LPS, but the reaction was reduced after LPS oxidation by mild periodate acid. Immunoblotting after SDS-PAGE shows that 2F9 is specific for lipoid A of GYC1108 - 1. The LPS released into cultured medium in 48 hours was not more than 39 μg/mL semiquantitated by inhibition ELISA, which is by far less than the amount necessary for fish lethality.