| Organophosphate pesticides, which have been considered as the most commonly used pesticides until now, include the most popular dichlorvos (DDVP) and phosmet(PMP).It is immpossible to ensure no residue in all foods for the wide application of DDVP and PMP. Up to now, almost all the countries and regions all over the world have regulated the maximum limit for DDVP and PMP in foods as the mandatory standard. Recently, the main detection methods are gas chromatography that has disadvantages of the relatively complicated procedure for sample preparation and expensive instrument and enzyme inhibition method, which has a relatively high false positive.Therefore, immunological methods have been become a hot research topic due to their rapid, sensitive, easy to handle and low-cost merits.In this paper, Mannich reaction adopted for constructing the complete antigen (DDVP-cBSA and PMP-cBSA) to couple DDVP, PMP and cationized bovine serum albumin (cBSA). cBSA, DDVP-cBSA and PMP-cBSA had been identified and analyzed by ultraviolet-visble (UV-Vis) spectroscopy and fourier transform infrared (FT-IR) spectroscopy. The development, titers and sensitivitis of anti-DDVP and PMP antisera produced by both antigens were obtained by injecting DDVP-cBSA and PMP-cBSA. Then the hybridoma cells, which were capable of continuously secreting the monoclonal antibodies (McAbs) against DDVP, had been screened out by fusion and clone of cells, which were from mouse splenic cells immunized by DDVP-cBSA and the murine Sp2/0 myeloma cells, respectively, and McAbs had been studied on the aspects of titer and sensitivity. The main results were followed.1. DDVP-cBSA and PMP-cBSA complete antigen construction and its spectroscopy analysisDDVP and PMP have low molecule substance with 190 and 317 molecule weight, and hapten, which has reactionogenicity without immunogenicity. DDVP and PMP must couple with carrier protein to form complete antigen in order to stimulate animals to secrect antibodies.In Mannich method, cBSA was firstly prepared, then was coupled with DDVP and PMP. Conjugates DDVP-cBSA and PMP-cBSA with the molar ratio of 40:1 and 1.5:1 were obatained.UV-Vis and FT-IR were applied to analyze cBSA, DDVP-cBSA and PMP-cBSA. In UV-Vis, the maximum absorbent wavelenght (λmax) of cBSA was slight blue shift compared with that of BSA, and there was an absorbent peak at 278 nm in DDVP-cBSA, which was the superposition peak of the characteristic absorbent peak of DDVP and BSA. The absorbent peak at 278 nm in PMP-cBSA was the superposition of the characteristic absorbent peak of PMP and BSA. In FT-IR, the strong bands at 1650 cm-1 of protein and the weak bands at 1286～1258 cm-1 and 1050～950 cm-1 of DDVP appeared in DDVP-cBSA. At the same time, the strong bands at 1650 cm-1 of protein and the weak bands at 833～790 cm-1 and 663～645 cm-1 of PMP appeared in PMP-cBSA.In conclusion, the conjugation between DDVP, PMP and cBSA did occur.2. Anti-DDVP and PMP polyclonal antibodies preparation and their characters analysisBALB/c mice were immunized by DDVP-cBSA and PMP-cBSA. The results showed that both of two complete antigens could induce mice to produce antibodies after 3rd～4th immunization. The antibody against DDVP had the maximum titer of 1.5×104 and The antibody against PMP had the maximum titer of 1.6×104 by ELISA. The two kinds of antisera had a sensitivitie of 473 ng/mL and 482 ng/mL.3. Anti-DDVP monoclonal antibodies preparation and its characters analysisThe splenic cells from BALB/c mice immunized by DDVP-cBSA were fused with the murine Sp2/0 myeloma cells. As a result, the fusion ratio of the cells was up to 3.5 %, in which the positive fusion ratio of anti-DDVP McAb was 10 %. One hybridoma cell lines :1D3 , which was capable of continually secreting high and sensitivity McAb against DDVP, was screened out by the limited dilution method and produced antibodies with the sensitivity of 800 ng/mL.
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