腹泻性贝毒软海绵酸单克隆抗体的制备和酶联免疫检测方法的建立

Preparation and application of the monoclonal antibody against okadaic acid

采用细胞融合技术制备抗赤潮毒素腹泻性贝毒软海绵酸的单克隆抗体,应用此抗体发展建立了软海绵酸的间接竞争酶联免疫检测方法。采用碳二亚胺法,将半抗原与载体蛋白偶联,得到免疫抗原和包被抗原。免疫BALB/c小鼠,用免疫小鼠的脾细胞与骨髓瘤细胞融合,ELISA方法筛选阳性克隆,经多次克隆化,获得了4株能稳定分泌抗软海绵酸单克隆抗体的杂交瘤细胞株。通过小鼠体内诱生腹水方法获得单克隆抗体,建立分析检测软海绵酸的间接竞争酶联免疫方法。最低检出限为31.2ng/ml,批内平均变异系数8.1%,回收率为87%-112%。

An competitive indirect enzyme-linked immunosorbont assay was developed for measurement of okadaic acid （OA）, a marine biotoxin associated with red tide. OA was coupled to BSA and OVA by earbodiimide reaction. OA-BSA as immunogen were injected in BALB/c mice. Titres of the antisera against OA were determinated using OA-OVA as coating ligand by ELISA method. The spleen cells of immunized mice were fused with Sp2/O cells. After cloning, four hybridoma cell strains stably produced anti-OA monoclonal antibody were obtained, The monoclonal antibody were produced by the mouse ascites method. The competitive indirect ELISA for okadaic acid in shellfish was established. Under optimal condition, the detection limit of OA was 31.2ng/ml and the recovery was 87 % - 112 %, with a mean coefficient of variation of 8.1.