Studies On Immunochemistry For Analysis Of Fenvalerate

Two haptens Fen-Hp₁ and Fen-Hp2 with different structure for fenvalerate were synthesized by different chemical pathes. 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 scanning, Fen-Hp₁-BSA , Fen-Hp2-BSA were used as immunogens to immunize New Zealand white rabbits and the specific antiserum to the related antigen were obtained. The middle titers for anti-Fen-Hp₁-BSA and anti-Fen-Hp₂-BSA serum were 3.2 ×10⁴ and 2.4 × 10⁴ respectively determined by the indirect ELISA, and the middle titers were 1/32 measured by the method of agar double immunodiffusion. The antibodies were separated from antiserum by salting out method with 35% saturated ammonium sulfate(S.A.S) and freeze-dried in a high vacuum. The Fen-Hpi was conjugated with HRP and the resultant Fen-Hp₁-HRP maintained both immunological activity and enzyme activity.The effects of pH value, ionic intension, tuwen 20 and organic solvent on the affinity of fenvalerate-antibody-1 for antibody coated (enzyme-tagged-hapten, E-H) direct competitive ELISA were evaluated. The results showed that the optimal coat pH of antibody is 6.4, the affinity of fenvalerate -antibody-1 was stronger in the medium of pH6.8, and it was weaker in the medium of basic condition. In a certain range the affinity of fenvalerate -antibody-1 could be improved if the ionic intension in buffer was increased. The sensitivity of ELISA for fenvalerate could be reduced by tuwen 20. 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 fenvalerate has been developed successfully, and the ELISA conditions were selected by phalanx test, which the antibody-1 of 8 μg/mL was used to coat microplate and the Fen-Hpi-HRP was diluted 12000 times. Under optimized conditions, the standard ELISA inhibition curve for the detection of fenvalerate was established and the regression equation was I = 67.22 + 21.62LogC( r = 0.9939), the linear concentrations of detection is ranged from 10.μg/mL to 0.001μg/mL,the half-maximal inhibition(IC5o)was 0.1598ug/mL and IC20 was 6.545ng/mL with the relative standard deviation (RSD) of 12.86%(n=6).The standard fenvalerate was added into greengrocery at the levels of 0.5mg Fen/kg or 5mg Fen/kg. The spiked sample was extracted by acetonitrile, purified by SEP-PAK Cis column, determined by ELISA. The recovery was 83.76% 109.5%, the RSD of repeated 5 times was 11.71% at the spiked level of 0.5 mg Fen/kg. The recovery was 93.64% 109.7%, the RSD of repeated 5 times was 7.25% at the spiked level of 5 mg Fen/kg. It reached the requirements of Fen residue analysis.The indirect competitive ELISA (IC-ELISA) was developed for some pyrethroids with the structure of 3-phenoxybenzyl methanol (PBM). Under optimized conditions, the standard inhibition curve of PBM was prepared, and its regression equation was I =49.21+ 14.71LogC(r = 0.9973), the linear concentrations of PBM is ranged from 10|ig/mL to 0.001 ng/mL, the half-maximal inhibition (IC50) was 1.132jig/mL and IC20 was 10.34ng/mL with the RSD of 5.93%(n=5). There is a high affinity of antibody-2 to permethrinN deltamethrinx lambdacyhalothrin, a lower affinity to cypermethrin, and a weak affinity to fenvalerate. The IC50 for these pyrethroids were 3.584jj.g/mLN 5.981 ng/mLN 9.786|ag/mL and 73.47jag/mL respectively. It indicated that the antibody-2 was practicable to select and detect the residues of these pyrethroids above.