Preparation And Application Of Anti-Podophyllotoxin Monoclonal Antibody

The evergreen prostrate shrub Sabina vulgaris Ant. belonging to the genus Sabina ( family Cupressaceae) is widely distributed in the northwest of China.One type of mainly secondary metabolites of the plant, podophyllotoxin and its congeners exhibit pronounced biological activity mainly as strong antineoplastic drugs, and as insecticides.Therefore, a podophyllotoxin-based insecticide (0.75%, EC) from S. vulgris, was developed by R & D Center of Biorational Pesticide, Norwest A & F University. This dissertation focused particularly on studies on the preparation of monoclonal antibody and immunochemistry analysis of the podophyllotoxin. The results were described as bellow.1.To avoid antibody production to the linker, the succinoyl-podophyllotoxin (hapten) mimicking the analyte, podophyllotoxin, was synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) by mixed anhydride reaction (MAR) and active ester method (AEM). monoclonal antibodies raised against Hapten-BSA synthesized by MAR were screened and selected for the IC-ELISA. The coupling ratio of Hapten-BSA conjugatesis 45.8:1 and the coupling ratio of Hapten-OVA conjugate is 26.5:1.2.One hybridoma-cell line 6E5 secreting monoclonal antibodies against insecticide podophyllotoxin were produced by fusing mouse myeloma cells (sp2/0) with spleen cells from Balb/c mice immunized by the hapten-BSA. The production and characterization of hybridoma cell line secreting positive antibodies permanently are described. The effect of several physicochemical factors on the monoclonal antibodies for podophyllotoxin was studied and the results were used to optimize the assay system. when incorporated into a competitive indirect ELISA, the antibody provided a detection limit of 0.0326ngmL-1 and I50 6.97ng﹒ mL-1 of podophyllotoxin. Different cross-reactions with the closely related compounds including deoxypodophyllotoxin,β-Picropodophyllototoxin and Podophyllinic acid hydrazide were observed, with I50 value of 11.6%,11.6%,<0.0697%μg·mL-1, respectivity.The result indicated the antibody was practicable to determinate the concentration of podophyllotoxin.3.The IC-ELISA protocol was developed for screening podophyllotoxin on vegetables...