Study On Indirect Competitive Enzyme-Linked Immunosorbent Assay (IC-ELISA) For Toosendanin

Toosendanin is a good kind of botanical pesticide with high insecticidal activity isolated from M.toosendan et Z. and M.azedarach L.. Toosendanin-based botanical pesticides,such as 0.5% toosendanin pesticide-EC, have been used in agriculture pest control, therefore, it has very important significance to further research on the metabolic rule, environmental security, the mechanism of toosendanin and quality control, by establishing highly efficient, rapid and sensitive toosendanin-detection methods. This dissertation focused particularly on the synthesis of artificial antigens and preparation of polyclonal antibodies for toosendanin, and the establishment and confirmation of indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for toosendanin. Key results are as follows:1. Two haptens, toosendanin-succinil (TS) and toosendanin-glutaryl (TG) were synthesized successfully and characterized by HRMS, NMR and IR,then conjugated with carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) to form artificial immunogens (TS-BSA and TG-BSA) and coating antigens (TS-OVA and TG-OVA) by mixed-anhydride method(MA) and active ester method(AE) respectively. Also the artificial antigens were confirmed by ultraviolet spectroscopy and infrared spectroscopy.2. Two types of anti-toosendanin polyclonal antibodies (TS-Ab and TG-Ab) with high titers (1/64,000 and 1/320,000) were obtained by immuring New Zealand white rabbits with immunogens. Specific antibodies against toosendanin were produced after the first booster immunization assayed by indirect ELISA and indirect competitive ELISA, and with the increase of immunity times, also on the rise in antibodies.3. The IC-ELISA protocols were established based on antibodies TS-Ab and TG-Ab for detecting toosendanin in optimized conditions. The limit of detection (LOD) IC10 and IC50 for toosendanin was 0.0363μg/ml and 1.3266μg/ml detected by the combination (TS-Ab,TG-OVA), and its linear equation was y=0.8203x+4.8993(r=0.9917) in the linear range of 0.01-50μg/ml. The IC10 and IC50 for toosendanin was 0.1178μg/ml and 3.0323μg/ml detected by the combination (TG-Ab,TG-OVA), and its linear equation was y=0.9084x+4.5623(r=0.9858) in the linear range of 0.1-50μg/ml. The recovery determined by IC-ELISA based on antibody TS-Ab was in the range of 82.4-106.0% in tap water,and the results were confirmed by HPLC. The recovery determined by the same ELISA format was in the range of 66.0-79.3% in cabbage.