Research On Immunoassay Methods For O,O-diethyl Organophosphorus Phosphorothionate Pesticides Residues

O,O-diethyl organophosphorus phosphorothionate pesticides are widely used in both agriculture and domesticity.In recent years,organophosphorus pesticides have played a more important role in the promotion of agricultural output and the control of different vectors.The development for a quick detection method is obviously necessary.At present,the methods of choice for pesticide analysis are instrumental methods,such as gas chromatography with mass spectrometry and liquid chromatography with mass spectrometry,have been applied successfully for detection for organophosphorus pesticides residues with great sensitivity and reliability as well.But these techniques are disadvantaged with their high costs and labor requirements,and the complicated procedure for sample preparation and inconvenience in the field should also be concerned.This research we have showed here is absolutely an easy,fast,low cost and large batch detection method.In this paper,seven organophosphorus haptens were synthesized,with NMR spectrum confirmed.Organophosphorus antigens were prepared with biomass MS spectrum confirmed.Polyclonal antisera were gotten and a fast and specific detection research were developed for four kind of representative organophosphorus pesticides residues.The contents are as below:1.Hapten synthesis.Seven haptens,which were Hapten 1,Hapten 2,Hapten 3,Hapten 4,Hapten 5,Hapten 6,Hapten 7,were designed as the chemical structure of kinds of organophosphorus pesticides and synthesized with different spacer arms in various ways,like direct method,nucleophilic substitution,carboxyl protection etc,with NMR spectrum confirmed.2.Antigen preparation.With the 7 haptens we synthesized and 3 haptens we purchased,10 immune antigens,which were Hapten 1-BSA,Hapten 2-BSA,Hapten 3-BSA,Hapten 4-BSA,Hapten 5-BSA,Hapten 6-BSA,Hapten 7-BSA,Hapten 8-BSA,Hapten 9-BSA,Hapten 10-BSA,were conjugated with BSA and 10 coating antigens,which were Hapten 1-OVA,Hapten 2-OVA,Hapten 3-OVA,Hapten 4-OVA,Hapten 5-OVA,Hapten 6-OVA,Hapten 7-OVA,Hapten 8-OVA,Hapten 9-OVA,Hapten 10-OVA,were conjugated,all confirmed by biomass MS and UV spectrum.3.Immunological analysis.Immune antigens were injected into mice and antiserum were gotten.The antiserum and coating antigens with the best titer were chosen by checkerboard test,which were Hapten 4-OVA(1:4000)and Sera 4(1:8000),Hapten 4-OVA(1:8000)and Sera 6(1:16000),Hapten 4-OVA(1:8000)and Sera 4(1:32000),Hapten 4-OVA(1:8000)and Sera 6(1:32000).Indirect competitive ELISA were performed to test the inhibition for the standard organophosphorus pesticides with the antiserum and coating antigens we chose and four organophosphorus pesticides which were phosalone,quinalphos,coumaphos,parathion,were with the best effects,and the standard curves were drawn and the IC50 were made up to 186ppb,506ppb,1409ppb,42596ppb,which all reached excellent standards.