Synthesis Of Two Artifical Antigens And Production Of Polyclonal Antibodies Against Cantharidin

Cantharidin is a kind of monoterpenes compound produced by blister beetle, which has been widely studied as insect pesticide because it is poison and feed-refusal to many target insects. However, its target in insect pests is still not clear Thus, this experiment deals with the synthesis the haptens of Cantharidin and their artificial antigens, then immunize New Zealand rabbits to get polyclonal antibodies against cantharidin. This will provide a basic approach for identifying the effect target and understanding the toxic mechanism of cantharidin to insect pests through enzyme immunoassay. The main results are as follows:1. The hapten N-ethyloiccantharidimide(BG)and N-ethoxylcantharidimide(BY)were synthesized, then BYS synthsis by mixed anhydride method. The hapten was characterized by MS, NMR, IR, TLC and melting point measuring methods. All the results showed that the haptens had synthesized successfully.2. To get the immunogens and coating antigens, the hapten was coupled to carrier proteins BSA and OVA by means of mixed anhydride reaction and Carbodiimide method respectively. These artificial antigens, BG-HS-BSA, BYS-HS-BSA, BG-TY-BSA, BYS-TY-BSA, BG-HS-OVA, BYS-HS-OVA, BG-TY-OVA and BYS-TY-OVA were characterized and verified by IR and MS. The results showed that the artificial antigen have synthesized successfully.3. Polyclonal antibodies were obtained by immunizing rabbits with immunogens of BYS-HS-BSA, BYS-TY-BSA, BG-HS-BSA and BG-TY-BSA. The titers of the polyclonal antibodies BYS-HS-Ab, BYS-TY-Ab, BG-TY-Ab and BG-HS-Ab obtained were 8000, 16000, 128000 and 300 respectively, so BG-TY-Ab was used for following experiment.4. To establish enzyme immunoassay of Cantharidin. Taken the combination of BG-TY-Ab and BG-TY-OVA, its detection limit for N-ethyloiccantharidimide was 3.156μg/mL, IC50 was 215μg/mL and the liner range was from 3.125μg/mL to 625μg/mL, R2=0.9818. The results showed that the polyclonal antibody produced by N-ethyloiccantharidimide can easily detect N-ethyloiccantharidimide.Artificial antigens for cantharidin were obtained through the methods of mixed anhydride reaction and Carbodiimide respectively in this study. It can be used for detecting cantharidin derivatives. This experiment helps establishing enzyme-linked immunosorbent methods for cantharidin and its other derivatiovers.