Preliminary Study On Enzyme-linked Immunosorbent Assay For Multi-residue Detection Of Organic Phosphorus Pesticides

The existing multi-residue detections for pesticides are mainly based on the gas chromatography or the highly effective liquid chromatography and so on. Pre-processing and the opration of these methods are complex. Equipments are expensive, and the specialists are needed to operate them. These methods are not suitable for the massive samples and on-line rapid detection.The method based on ELISA is fast, keen, being suitable for trace substance's examination, meanwhile it needs low examination cost. The operation is simple, reliable. Preliminary study on ELISA which uses monoclonal antibodies and the immunity examination technology for multi-residue detection of organic phosphorus pesticides, may provide a technical support for development of ELISA kit.Methods: Coupling the organic phosphorus pesticides and the protein using the organic synthesis method, thus we can preparation the complete antigen. Immuning mice with complete antigen, and preparation monoclonal antibodies with aseptic technique technology, like cell culture, cloning and so on. To establish the ELISA method for the detection of one kind of organic phosphorus pesticides ,and check the performance index of the method. On these bases, we will do preliminary study on ELISA for multi-residue detection of organic phosphorus pesticides.The mainly results:1.Using chlorpyrifos and 3-mercaptopropionic acid as raw materials synthesis hapten ,then synthes complete antigen by carbodiimide .The results showed that the coupling ratio of chlorpyrifos and protein was 20:1, and we can know the experiment wass successful. The structure of methamidophos includes the amino, therefore we connected methamidophos and protein using EDC directly. But we found out this method did not work well from the following experiment.2.The titer of all mice immuned chlorpyrifos and several mice immuned methamidophos reached 2 104. These mice might be used in fusing experiment. Two cell strains which excreted chlorpyrifos monoclonal antibodies were obtained and named 2E4, 4B9. After purification, we obtained monoclonal antibodies and the concentration was 1.5mg/mL . The immunoglobulin class of McAb was IgG1. But positive cells which excreted methamidophos monoclonal antibodies were less and less , finally we had not obtained methamidophos monoclonal antibodies.3. Based on indirect competitive ELISA standard curve of chlorpyrifos was established, the cross reaction and the fortified recovery were tested and compared with GC.The chlorpyrifos concent ration (IC50) that inhibits 50% of antibody-antigen binding was 1.45μg/mL, the detection limit was 0.027μg/mL. Aparagus samples were detected with ELISA and GC after simple preprocession. The fortified recovery of ELISA was 82.6 %~98.96%. This result was verified by GC method.4.The results of experiment of multi-residue detection showed that there was no cross reaction between chlorpyrifos and parathion. And their indirect competitive ELISA standard curves unanimous. But the measure of detection for total multi-residue of these two kinds of pesticides need further improve.