Studies On Immunochemistry For Analysis Of Cypermethrin And Flumethrin

Two haptens Cyp-Hp₁ and Flu-Hp₂ for cypermethrin and flumethrin were synthesized. The haptens were covalently conjugated to carrier proteins BSA and OVA by the method of modified active ester to prepare artificial antigens. After the conjugates were confirmed by UV analysis, Cyp-Hp₁-BSA and Flu-Hp₂-BSA were used as immunogens to immunize New Zealand white rabbits and the specific antiserum to the related antigens were obtained. The middle titers for anti-Cyp-Hp₁-BSA and anti-Flu-Hp₂-BSA serum were 1.6×104 determined by the indirect ELISA, and the titers were 1/16 measured by the method of agar double immunodiffusion. The antibodies were separated from antiserum by salting out method with 35% saturated ammonium sulfate(SAS) and freeze-dried in a high vacuum. The Cyp-Hp₁ and Flu-Hp₂ were conjugated with HRP and the resultant Cyp-Hp₁-HRP and Flu-Hp₂-HRP maintained both immunological activity and enzyme activity.The effects of pH value, ionic intension and organic solvent on the affinity of cypermethrin to antibody for antibody coated (enzyme-tagged-hapten, E-H) direct competitive ELISA were evaluated. The results showed that the optimal pH of coating buffer of antibody is 7.2, the affinity of cypermethrin to antibody was stronger in the medium of pH7.0 phosphate buffer (PB). In a certain range, the affinity of cypermethrin to antibody could be improved if the ionic intension in buffer was increased. Acetone and acetonitrile had an obvious influence on E-H direct competitive ELISA if their concentration is more than 5% in reaction medium.The direct competitive ELISA of cypermethrin has been developed successfully, and the ELISA conditions were selected by phalanx test, The antibody of 6μg/mL was used to coat microplate and the Cyp-Hp₁-HRP was diluted 64000 times. Under optimized conditions, the standard ELISA inhibition curve for the detection of cypermethrin was established and the regression equation was I=0.1919LogC+0.5618,R² = 0.9913,the linear concentrations of detection was ranged from 100μg/mL to 0.01μg/mL, the half-maximal inhibition(IC50)was 0.461μg/mL and IC20 was 12ng/mL with the relative standard deviation (RSD) of 17.26%(n=5).The standard cypermethrin was added into greengrocery at the levels of 0.5mg/kg or 5mg/kg. The spiked sample was extracted by acetone, determined by ELISA. The recovery was 80.29%～89.20% with RSD of 4.35%(n=5) at the spiked level of 0.5 mg/kg. And the recovery was 89.68%～120.78% with the RSD of 11.88%(n=5) at the spiked level of 5 mg/kg. It meets to the requirements of Cyp residue analysis.The direct competitive ELISA of flumethrin has also been developed successfully, and the ELISA conditions were selected by phalanx test. The antibody of 6μg/mL was used to coat microplate and the Flu-Hp₂-HRP was diluted 384000 times. The standard ELISA inhibition curve for the detection of flumethrin was established and the regression equation was I=0.1998LogC+0.6988,R²=0.9996,the linear concentrations of detection is ranged from 10μg/mL to 0.01μg/mL, the half-maximal inhibition(IC50)was 0.101μg/mL and IC20 was 3ng/mL with the RSD of 15.42%(n=5).