Preparation Of Parathion Monoclonal Antibody And Preliminary Study On Electrochemical Immunosensor

China is one of the biggest countries for production of pesticides and the second largest country for consumption of pesticides, which results in the serious pesticide pollution. Although some high-toxicity pesticides such as methamidophos and parathion have been banned to use, there are also some hazards caused by the illegal use of these kinds of pesticiedes.Since there are some shortages with the traditional pesticide detection method such as complex pre-processing and expensive instrument, it's of great importance to establish a new fast method to detect the pesticide residue. Electrochemical immunosensor, as a fast, cost-effective detection instrument has become an important studying point recently. It can be widely used in pharma, food analysis, industrial production as well as environmental detection. In this study, we chose parathion and methyl-parathion as target pesticide, prepared parathion monoclonal antibody and established a method to detect methyl-parathion by electrochemical immunosensor.The main contents are as follows:1. Synthesis and identification of artificial antigens for parathionMolecular weight of parathion is very small and it can not elicit an antibody response alone and must be attached to a carrier protein to form an artificial antigen first. Half antigen, amino-parathion was synthesized through reduction reaction.Then bovine serum albumin (BSA) and ovalbumin (OVA) were conjugated to the hapten through diazotization reaction. The half atigen was determined by H-NMR and the artificial immunizing antigen and coating antigen were determined by P-NMR. The results showed that the artificial antigens were synthesized successfully. Conjugating ratios were calculated through UV specturm.The results were as follows: immunizing antigen was 1∶32 and coating antigen was 1∶25.2. Preparation, purification, determination and application of parathion monoclonal antibodyBalb/c mice were immunized by intraperitoneal injection of immunizing antigen. Antiserum was determined by indirect ELISA four weeks later, and the mouse that produced polyclonal antibodies of the highest titer was used for cell fusion to prepare monoclonal antibody. Spleen cells of immunized mouse were fused with SP2/0 murine myeloma cells by 50%PEG 4000. After several times of screening by indirect ELISA and indirect competitive ELISA and cloning by limited dilution, positive hybridoma cells were acquired. All the Balb/c mice produced polyclonal antibodies for parathion.The mouse D2 had the highest titer of 1:100000.Ten positive hybridoma cells were obtained.Culture supernatants of hybridoma cells did not react with carrier proteins of BSA and OVA, but reacted with artificial coating antigen. Results of indirect competitive ELISA showed parathion could combine with antibodies of hybridoma cells culture supernatants, so the cells were monoclonal cells secreting anti-parathion McAbs which were satisfied with our anticipation. The fusion rate and positive rate were 90% and 3.7% respectively. Isotyping of monoclonal antibody determined by ELISA showed that cells belong to IgG1 type, the affinity is 1.6×105L/mol. Mouse ascites antibody was purified with caprylic acid ammonium sulphate precipitation method. Indirect competitive ELISA for parathion was established. The titer of parathion monoclonal antibody in ascites was 1:100000. The optimum ELISA conditions were as follows: concentration of coating antigen was 10μg/mL and antibody concentration was 0.2μg/mL. The half-maximize inhibition concentration (IC50) of inhibition standard curve for parathion was 0.188μg/mL, the linear range and the detection limit were 0.071μg/mL-0.4951μg/mL and 0.05μg/mL respectively.3. Electrochemical immunosensorMercaptopropionic acid was self assembled to the Au electrode through the Au-S covalent bond. Then, antibody against methyl parathion is immobilized by means of covalent coupling with the activation of 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). The change of electrode potential is recorded to determinate the amount of methyl parathion. The detection limit of the immunosensor is 0.085μg/mL, the linear range is 0.5-15μg/mL. The immunosensor can be regenerated and repeatedly used.