Study Of Multi-residue Immunoassay For Organophosphorus Pesticides

The general public and regulators pay more attention to the increasing problems of foodsafety and environmental issues caused by organophosphorus (OP) pesticides that are widelyused for a long term. Chromatographic analytical techniques, such as GC, HPLC, GC-MS andHPLC-MS, are commonly used for determining pesticides and their metabolites inenvironmental samples and food products. However, they are time-consuming due to thecomplicated sample preparation steps, and are operated by professional person andinconvenience for multitudinous samples screening and for use in the filed. Enzyme-linkedimmunosorbent assays (ELISA) for small molecules open up a new field for the multi-residuedetermination of OP pesticides. The development of a multi-residue immunoassay for OPpesticides using generic haptens is the mainly theme in this thesis.Most molecules of OP pesticides contain alkoxy phosphorothioate group, which can beused as a common structure to synthesize generic haptens. Sodium Ｏ,Ｏ-dimethyl (diethyl)phosphorothioate was used as material to react with chloroacetic acid and bromine propionicacid, respectively to synthesize six generic haptens named H1^H6with different linear spacerarms. All structures of target haptens were supported by NMR spectral data and mass spectra.The haptens were conjugated to bovine serum albumin (BSA) and to ovalbumin (OVA) usingthe methods of active ester and mixed-anhydride, respectively, to synthesize artificial antigens.The qualitative investigation for the coupling condition of artificial antigens was detected byUV absorbance spectrum. And conjugating ratios were analyzed by the method of2,4,6-trinitrobenzene sulfonic acid (TNBS).In order to obtain polyclonal antibody, artificial antigens named H2-BSA,H4-BSA,H5-BSA,H4-OVA,H6-BSA,H6-OVA as immunogens were injected to several BALB/c miceaccording to the immune protocol. Titration for antisera using coating antigen was given bynoncompetitive direct ELISA, and the results were256000:1,16000:1,16000:1,8000:1,128000:1,128000:1, respectively. To discuss the effects of different organic solvent, ionconcentration, pH on the activity of antisera with antigens, polyclonal antibody obtained byH6-OVA with higher titration and its coating antigen named H6-BSA working concentrationat5μg·mL^-1were used. The study showed that the phosphate buffer contained5%～10%acetone,10mmol·L^-1ion concentration, pH7.4was good for the activity of antisera. Based on competitive indirect ELISA, standard inhibition curves of H1, H2, H4and H6were established, respectively, using antisera H6-OVA at the optimum conditions. Inhibits50%of antibody-antigen binding (IC50) for H2was the lowest, which was34.43μg·mL^-1.Several OP pesticides were tested for cross-reactivity. It was noteworthy that the antibodyshowed no cross-reactivity to profenofos. It was also noteworthy that the antiserum showedhigher affinities to omethoate and chlorpyrifos, the IC50to them was12.59,8.91μg·mL^-1,respectively.The multi-residue immunoassay established in the thesis can detect several kinds of OPpesticides at the same time. Unfortunately, the immunoassay cannot be used to analyze realsamples since the sensitivity is much lower, which needs further improvement.