Devlopment Of Immunoassays For Organophosphorus Pesticides Multi-residues Based On Phage Display

Organophosphorus pesticides?OPs?,a group of cholinesterase-inhibiting insecticides,have been widely used in agriculture because of their effective broad-spectrum insecticidal activity and relatively low stability under natural conditions.However,the extensive use of OPs led to contaminant residues in food and environment,and seriously harmful to the health of human being.Immunoassay is a rapid,sensitive,simple and cost-effective analytical tool for routine high throughput monitoring of pesticide residues samples.There are many kinds of OPs and they always used as a mixture of several kinds.Therefore,preparation of broad-specificity antibodies and development of immunoassays for OPs multi-resdues analysis are necessary.In the present study,a series of generic haptens of OPs were firstly designed,chemosynthesized and covalently attached to carrier proteins for using as immunogens or coating antigens.On the one side,BALB/C mice were immunized with immunogens,and two sensitive monoclonal antibodies?MAbs?with broad-specific for OPs were prepared.Then,immunoassays for analysis of OPs multi-residues were developed based on the MAbs with the optimum coating antigens.On the other side,a phage display scFv antibody library against OPs was prepared and a scFv antibody with broad-specific for OPs was selected.Then,a broad-specificity immunoassay for OPs based on the scFv antibody was developed.Furthermore,mimotopes against MAbs were selected from a random peptide phage display library.The selected mimotopes were expressed in prokaryotic expression system and used to developed broad-specificity immunoassays for OPs.The main results are in details.?1?Twenty-two kinds of haptens?Hapten 1^Hapten 22?were designed and chemosynthesized based on the common structures of OPs.The structures of the haptens were verified by ¹H-NMR.Hapten 1?O,O-diethyl O-?3-carboxyphenyl?phosphorothioate?and Hapten 2?O,O-dimethyl O-?3-carboxyphenyl?phosphorothioate?were covalently attached to BSA for use as immunogens by active ester method.All of the 22 haptens were covalenthly attaced to OVA for use as candidate coating antigens.?2?BALB/C mice were immunized with Hapten 1-BSA,after cell fusion,hybridoma selection,monocloning,ascites fluid preparation and purification,a monoclonal antibody?MAb3A7?with broad-specific for OPs was obtained.All of the coating antigens were tested for plate coating,and the highest sensitivity was obtaind by Hapten 22-OVA.Based on MAb3A7 and Hapten 22-OVA,a broad-specificity IC-ELISA for OPs was developed.Results of cross-reactivity and sensitivity study indicated that 15 O,O-diethyl OPs and 15O,O-dimethyl OPs could be detected by the developed method.With the optimum IC-ELISA,the IC₅₀0 values of the 30 OPs were determined as 23.1⁸99.9ng/m L,and the limits of detection?LOD?were determined as 5.7³19.2ng/m L.For the recovery study,the matrix effects of samples could be effectively eliminated by purified with dispersive solid-phase extraction?d-SPE?.The recoveries of OPs from the spiked samples were determined ranged from 89.4%to 131.5%,and the CV ranged from 3.5%to 15.7%.?3?BALB/C mice were immunized with Hapten 2-BSA,and MAb3C9 which showed broad-specific for OPs was obtained.The MAb was labeled with HRP to obtain MAb3C9-HRP.All of the coating antigens were tested for plate coating,and the highest sensitivity was obtaind by Hapten 5-OVA.Based on the MAb3C9-HRP and Hapten 5-OVA,a broad-specificity DC-ELISA was developed for analysis of OPs multi-residues.Results of cross-reactivity and sensitivity study indicated that the DC-ELISA showed class-specific for O,O-dimethyl OPs.With the optimum DC-ELISA,the IC₅₀0 and LOD values of 18O,O-dimethyl OPs were determined as 1.3²31.0ng/mL and 0.3⁵2.4ng/mL,respectively.For the analysis of spiked samples,the recoveries of OPs were determined ranged from 90.3%to112.8%,and the CV ranged from 3.5%to 14.1%.?4?Total RNA was extracted from spleen cells of the Hapten 1-BSA immunized BALB/C mouse,and cDNA was obtained by reverse transcription.The cDNA was used as template for the PCR amplification of murine antibody variable heavy?VH?and light?VL?chain genes.The VL and VH genes were assembled to acquire scFv gene fragments by SOE-PCR.The scFv gene fragments were inserted into pIT2-BAD phagemid and electroporated into E.coli TG1 to obtain the scFv antibody library?contain 5×10⁷ unique clones?.Then,the library was rescued by helper phage KM13 to obtain the phage displayed scFv antbibody library.After four rounds of panning,positive phages were enriched.Individual phage clones from the fourth round were rescued and screened by competitive phage-ELISA,and a scFv antibody?named sc Fv-5?with high sensitive and broad-specificity for OPs was obtained.The scFv antibody was fused with biotin accepted domain?BAD?and over-expressed as an inclusion body in E.coli BL21?DE3?.After refolding,biotinylation and purification,the biotinylated scFv antibody was obtained with yield of 66.7mg per liter cultures.Based on the biotinylated scFv-5-BAD and Hapten 22-OVA,a broad-specificity IC-ELISA for OPs was developed.The cross-reactivity study indicated that the ELISA could detect both O,O-diethyl OPs and O,O-dimethyl OPs.With the optimum IC-ELISA,the IC₅₀0 and LOD values of 30 OPs were determined as 19.4⁵15.2ng/m L and 4.1⁹0.7ng/m L,respectively.For the analysis of spiked samples,the recoveries of OPs were determined ranged from 88.9%to 117.6%,and the CV ranged from 3.8%to 16.7%.?5?To selected phage mimotopes of MAb3A7,three rounds of panning against MAb3A7were performed to enrich the positive phages from a commercial loop-constrained heptapeptide library.Individual phage clones from the third round were screened by a competitive phage-ELISA,and the best mimotope ME13?C-M-S-W-L-G-W-A-C?was obtained.ME13 was fused with GST and BAD,and over-expressed in E.coli BL21?DE3?.After biotinylation and purification,the biotinylated ME13-GST-BAD fusion protein was obtained.Based on the biotinylated ME13-GST-BAD and MAb3A7,a broad-specificity DC-ELISA for OPs was developed.With the optimum DC-ELISA,the IC₅₀0 and LOD values of 30 OPs were determined as 8.2⁴02.5ng/m L and 2.3¹27.7ng/m L,respectively.For the analysis of spiked samples,the recoveries of OPs were determined ranged from 91.3%to120.3%,and the CV ranged from 3.2%to 12.3%.?6?The loop-constrained heptapeptide library was preformed three rounds of panning against MAb3C9 to enrich the positive phage mimotopes.The individual clones from the third round were screened by a competitive phage-ELISA and the best mimotope ME20?C-T-G-T-T-P-F-Y-C?was obtained.The mimotope was over-expressed as ME20-GST-BAD fusion protein.After biotinylation and purification,the biotinylated ME20-GST-BAD fusion protein was obtained.Based on the biotinylated ME20-GST-BAD and MAb3C9,a broad-specificity DC-ELISA for OPs was developed.With the optimum DC-ELISA,the IC₅₀and LOD values of 18 O,O-dimethyl OPs were determined as 1.5²94.9ng/mL and0.3⁶3.2ng/m L,respectively.For the analysis of spiked samples,the recoveries of OPs were determined ranged from 92.4%to 121.5%,and the CV ranged from 2.9%to 13.6%.