复合免疫制备3种有机磷农药单克隆抗体的技术研究

Preparation of organophosphorus pesticide monoclonal antibodies against multiple antigens

以α-氨基丁酸和农药中间体为原料合成了甲基对硫磷、杀螟硫磷和甲基毒死蜱3种有机磷农药人工半抗原(H1、H2和H3);通过活泼酯法将H1、H2和H3分别交联于牛血清白蛋白(BSA)和卵清蛋白(OVA)上制备免疫原和包被原。以等体积混合的H1-BSA、H2-BSA和H3-BSA复合免疫BALB/c小鼠,采用常规杂交瘤技术制备单克隆抗体获4株抗上述复合抗原的单克隆抗体,其中甲基对硫磷1株(H9/A3),杀螟硫磷2株(H9/B3、C8/H6),甲基毒死蜱1株(G7/F5)。所获4株抗体的类型及亚类均为IgG1,轻链均为κ链,间接ELISA法测定腹水效价均高达1×106。间接竞争ELISA法测定H9/A3、C8/H6和G7/F5细胞株的抑制中浓度(I50)分别为107.0、40.4和75.2μg.L-1,且与类似物无交叉反应。本研究建立了一种可在较短的研制周期内,通过单次细胞融合获得多种不同农药的单克隆抗体的有效方法。

Hapten of parathion-methyl, fenitrothion, chlorpyrifos-methyl was synthesized by active ester method, and termed as H1, H2, H3, respectively. Immunogen and coating antigen were prepared by conjugating （ H1, H2, H3 ） with carried protein bovine serum albumin （BSA） and ovalbumin （OVA）, respectively. Four hybridoma cell lines which secreted monoclonal antibody （McAb） against the multiple immunogen （mix equally with H1-BSA, H2-BSA and H3-BSA） were obtained. Among them, one hybridoma cell line against parathion-methyl （H9/A3）, two against fenitrothion （H9/B3, C8/H6） and one against chlorpyrifos-methyl （G7/F5） was obtained, respectively. Isotype and subclass of all monoclonal cells were classified as IgG1. The light chain of the McAb was indentified to be K. These titres of ascites fluids were up to 1 × 10^6 by indirect ELISA. The inhibition rate of 50% of McAbs secreted by hybridoma cell lines （ H9/A3, C8/H6 and G7/F5） by competitive indirect ELISA were 107.0, 40.4, 75.2 μg ·L^-1 respectively, while the McAbs had no cross-reaction to analogues. This work provided a new strategy for production of different monoclonal antibodies at the same time.