Immunoassay Methods Development For The Determination Of Organophosphate Pesticides Based On Monoclonal Antibody And Single-chain Variable Fragment Antibody

Using Agri-chemicals for the pest control has been a critical strategy of the integrated pest management?IPM?in the agriculture production,including biological pesticide and chemical pesticide using.As a high efficiency and broad-spectrum chemicals,organophosphate insecticides?OP?have been used for pest control world wildely.In China,organophosphate insecticides is the largest chemicals has been used in agriculture production system,and also occupy half of total yield of agri-chemicals producted in china per year.Due to the intensive using of pesticide,the food safety issue releated to pesticide residue became serious problem to human health,especially for the potential cumulative risk of family chemicals.Therefore,It is urgent to develop rapid detection method with multiple detection capability,for immunoassay,broad specific antibody generation is the key.In this study,a chemical presenting common structure of organophosphorus pesticides was designed as hapten,after mice immunization,hybridoma cell selection,monoclonal antibody was produced and characterized with specific recognization for OPs,thereafter a single-chain variable fregment?scFv?was produced by assembling variable region gene sequences together.The main results as follows:First,hapten design and antigen synthesis:Compound with carboxyl?BPB?was synthesized and conjugated with carrier protein?bovine serum albumin BSA?for artificial antigens preparation.Synthesized chemical was conformed and dentified by mass spectrometry and ultraviolet spectrophotometry for the complete antigens identification,the result showed that:Haptens and the complete antigen was successfully achieved,and the coupling ratio of the carrier protein and hapten was 1:25.Second,monoclonal antibodies generation:Balb/c mice were immunized with the complete antigen BPB-BSA several times and serum titer was monitored,thereafter,spleen cells of mice were removed and fused with SP2/0 myeloma cells to prepare hybridoma cells.Hybridoma cell line BPB-BSA-4A5 which secrets the monoclonal antibody stably was selected.After cellular cleavage and proliferation,the monoclonal antibody was prepared by injecting the cell strain to mice^?s enterocoelia in vivo,and the properties of antibody were characterised with indirect competition ELISA.The results showed that the subtype of the obtained monoclonal antibody was Ig G1 type,and eight different organophosphorus pesticides could be recogonized by this Mab.The corresponding IC₅₀ of each pesticide were:0.612?g/ml for coumaphos,0.492?g/ml for parathion,1.65?g/ml for triazophos,7.23?g/ml for phorate,13.86?g/ml for disulfoton,11.08?g/ml for quinalphos,14.39?g/ml for diazinon,19.53?g/ml for phoxim,respectively.Third,variable region genes extraction and single-chain variable fragment scFv expression:Total RNA of hybridoma cell strain No.BPB-BSA-4A5 was extracted and reversly transcripted to c DNA for further PCR conduction.The heavy and light variable region genes V_H and V_L were cloned and amplified,and the heavy and light variable region genes were assembled by SOE-PCR,and ligated it to the cloning vector PMD19-T,followed by digesting of restriction enzyme NcoI and NotI,then assembled scFv was ligated with the expression vector PET26-B by T4 DNA ligase.Finally,it was transformed into competent cells E.coli BL21?DE3?to express scFv antibody.Four,characterization of scFv antibody:The expressed scFv antibody was purified to be95%more by Ni-NTA affinity column and its molecular weight was conformed to be 27 kDa.Eight organophosphorus pesticides can be recognized by expressed scFv antibody,and their corresponding IC₅₀ values were:Coumaphos 2.03?g/ml,parathion 3.51?g/ml,triazophos 6.20?g/ml,phorate 7.35?g/ml,disulfoton 8.98?g/ml,quinalphos 15.24?g/ml,diazinon 19.36?g/ml,and phoxim 20.01?g/ml,respectively;Comparing with monoclonal antibodies,the detection spectrum of single chain variable fragment is the same but its detection sensitivity is reduced.