Study Of Bispecific Monoclonal Antibody To Pesticide Carbofuran And Triazophos

One hapten of insecticide carbofuran 2,2-dimethyl-2,3-dihydrobenzofuranyl, 7-N-(3- carboxypropyl) carbamate (BFNB) and two haptens of pesticide triazophos O-ethyl, O-(1-phenyl-1H-1,2,4-triazol-3- yl) N-(3-carboxypropyl)phosphoramidothioate (THBu) and [O-ethyl, O-(1-phenyl-1H-1,2,4-triazol-3-yl) N-(5-carboxyamyl) phosphoramidothioate (THHe) were synthesized and then covalently coupled with carrier protein BSA and OVA, respectively. The mouse were immunized with artificial antigens, then mouse spleen lymphocytes were fused with myeloma cells by cross-tumour technique. The hybridoma cells were selected and cloned. The monoclonal antibodies were obtained with high titres, which of ascitic fluids up to 1×10^-6 by indirect ELISA. The tetradoma cell was selected based on the technique of lacking functional enzyme and the technique of microscopic operation, then the bi-specific monoclonal antibodyby were obtained with the characteristics of bi-specificity and high reactivity.The conditions of ELISA for those insecticides above were optimized as follows. Costar Immuno plate from corning corporation was selected as the coating plate in the experiment. The optimized coating buffer were PBS(0.05 M, pH8.0) and CBS( 0.05 M, pH9.6) for AC format(antibodies-coated) and CC format (conjugate-coated), respectively. The plate was incubated at 37°C for 2 hours and blocked by 2% defatted milk powder. The optimized buffers were 0.05M PBS (pH7.0) and 0.02M PBS (pH7.4) for carbofuran and triazophos, respectively. Based on the optimized ELISA conditions, the direct competitive ELISA (antibody coating format) were adopted for the determination of carbofuran and triazophos. The limits of detection(I₂₀) of the selected ELISA were 1.89 ng/mL and 0.15 ng/mL for carbofuran and triazophos, respectively. And the linear concentrations were ranged from 1.11 to 45.95 ng/mL and 0.07 to 3.90 ng/mL for carbofuran and triazophos, respectively. Different kinds of samples such as water and soil samples, vegetable and fruit samples were spiked with carbofuran and triazophos. The results showed that the recoveries of most of the samples ranged from 75% to 130%, except for rice and wheat powder samples. However, it was proved that the selected ELISA was reliable for detecting carbofuran and triazophos.In order to get the hybrid-hybridoma(tetradoma) cells, the hybridoma producing monoclonal antibody to carbofuran was selected by 8-N-Ag which make the cells lacking of hypoxanthibe- guanine phosphoribosyltransferase (HGPRT). And the hybridoma producing monoclonal antibody to triazophos was selected by 6-chloropurine which made the cells lacking of thymidine kinase(TK). Then the mouse hybrid hybridomas (tetradoma) was fused with the selected hybridomas and cloned by the technique of microscopic operation. One tetradoma line (12C1-2H12) secreted immunoglobulin manifesting parental and bi-specific binding characteristics was established. The bi-specific monoclone was produced by ascite and purified. Then the optimal conditions for the ELISA were established based on the optimization of buffer condition. From the result, the detection limits I₂₀ of the ELISA based on bi-specific monoclonal antibody were 4.46 and 0.36 ng/mL for carbofuran and triazophos, respectively. The value of I₂₀ was higher than that of the monoclonal antibody. The bi-specific monoclonal antibodies were obtained with high specificity to the analytes, and with very low cross-reactivity to carbofuran and triazophos related compounds. There were no obvious difference of cross-reactivity between the bi-McAb and McAb. It was testified that those bi-McAbs obtained was successful and could be used for detecting carbofuran and triazophos, respectively. The optimized ELISA based on Bi-McAb was applied in environmental samples, vegetable samples and fruit samples. From the results, the recoveries for water samples showed an abnormal tendency, especially from the sample of tap water, which had the highest tendency in tested water samples. The recoveries for soil, vegetable and fruit samples were ranged from 75% to 130%. Therefore, the Bi-McAb was suitable to detect the analyte residue in the soil samples, vegetable samples and fruit samples.In this study, the technique system for producing bi-specific monoclonal antibody based on hybrid-hybridoma and its ELISA method was esteblished successfully. And some of principle for bi-McAb to pesticide was discussed.