Development Of Immunoassay Techniques For Ivermectin

Ivermectin is a semi-synthetic macrolide-compound which is the reduction derivative of avermectin. And it is active against helminthes and arthropods. It has good effect to kill so many pests on vegetables and melon crops. Because of its tow residue in the vegetables and low solubility in water, it is difficult for apparatus to detect ivermectin in high level. Immunoassay is an important tool for analysis and detection of pesticide and its derivatives. Compared to the chromatographic analytical and techniques, it has some advantages in high sensitivity, high specificity and fast efficiency. In this study, development of immunoassay techniques for monoclonal antibody for ivermectin was studied. The hapten of ivermectin was designed and it was conjugated with the carrier protein to synthesize the artificial antigens. CI-ELISA to detect ivermectin were established with corresponding monoclonal antibody and the results were described as below:1. Hapten synthesis and conjugation of the artificial antigens. The 5-o-succinoyl ivermectin was obtained afer the reaction of ivermectin with succinic anhydride in anhydrous pyridine, and then the hapten were bound to BSA or OVA by the carbodiimide activation.2. Monoclonal antibody for ivermectin was produced. Six Balb/c mice were immunized with IVM-BSA. And the titers of antiserum for mouse A2 is 1:128,000 which is the highest: Splenocytes from mouse A2 were fused with SP2/0 myelomas and the fusion frequency was 78%. One hybridoma-cell line 4B6 secreting monoclonal antibodies against ivermectin was produced by screening. The antibody was identified as IgG.. The titers of monoclonal antibody from mice ascites has reached 1:512,000.3. The indirect competitive ELISA to detect ivermectin based on monoclonal antibody was developed. The titers of IVM-OVA is 1:800 and the monoclonal antibody is 1:40000. And 10% methanol-PBS as the basic working buffer for ELISA was selected after optimizing. IVM-OVA-based CI-ELISA conducted with the monoclonal antibody, the linear range for ivermectin was between 0.146-431.949 ng/ml. The lower limit of detection (I₁₀) for ivermectin was 0.039ng/ml and the half-maximal inhibition (I₅₀) was 7.947ng/ml. The cross reactivities for avermectin B₁ was 51% and for milbemycin oxime was less than 0.2%. In the cabbages, the recovery of ivermectin was in the range of 90%～105% with the coefficient of variation was 2.1%～12.6%.The results showed that the monoclonal antibody for ivermectin could be used for residue analysis. And the antibody for ivermectin can be produced permantly by the hybridoma cells. And the hybridoma cells could be used as the foundation material to construct the phage display library for avermectins. In the later test, mRNA could be prepared from the hybridoma cells and amplified by RT-PCR. Then the variable region gene of the antibody could be purified and linked by PCR. Then the antibody library of avermectins would be generated by phage display technology. It is significient in national immunoassay detection for pesticides.