The Research On Enzyme-Linked Immunosorbent Assay For Haloxyfop-P-Methyl And Fluazifop-P-Butyl

The enzyme-linked immunosorbent assays were widely used in pesticide residue analysis, the indirect competitive enzyme-linked immunosorbent assays (ic-ELISA) for the herbicide haloxyfop-P-methyl and fluazifop-P-butyl were developed in the study. The work includes the synthesized of haptens and antigen, obtaining polyclonal antibody, and established ELISA, and at last, the recoveries of haloxyfop-P-methyl and fluazifop-P-butyl were analysed.Two haptens (GHs and GHs) of haloxyfop-P-methyl and two haptens (JHs and JHs) of fluazifop-P-butyl with different spacer-arm length were synthesized which were analyzed by MS,'H NMR. The haptens were conjugated with the carrier proteins bovine serum albumin (BSA) by Active ester method (AEM) and ovalbumin (OVA) by Mixed anhydride reaction (MAR), respectively. UV spectra show that these conjugates were obtained successfully. The polyclonal antibody were obtained by immunized the New Zealand rabbits. The most sensitive combinations of antibody/coating antigen were selected by homologous and heterologous assays. The effects of the organic solvent, ionic strength and pH of solution were evaluated, the optimum organic solvent was selected to be 20% methanol, the ionic strength of the assay phosphate buffer was selected to be 0. 3mol L-1 of Na+, meanwhile pH 7.5 was selected to be optimal, where the IC50 value was the lowest.Two kinds of indirect competitive immunosorbent assays (ic-ELISA) have been established for haloxyfop-P-methyl and fluazifop-P-butyl at the optimum assay conditions. The standard curve for haloxyfop-P-methyl was obtained by B/Bo (B=ODx-ODmin, Bo=ODmax-ODmin) versus LogC (C is the concentration of haloxyfop-P-methyl).%B/B0=-29.7651ogC+9.0044 (R2=0.9927), the assay showed that the IC50 was 0.0419 mg/L, and the LOD (IC10) was 0.00190 mg/L, the working range was assigned to 0.0019-0.926 mg/L for haloxyfop-P-methyl. And the standard curve for fluazifop-P-butyl was as followed: %B/B0=-20.506 logC+44.724 (R2=0.9969), with IC50 was 0.553 mg/L, and the LOD (IC10) was 0.00620 mg/L, the working range was assigned to 0.00620-49.358mg/L mg/L For the ELISAs, the Cross-Reactivities with other structurally related compounds were all low, which was negligible.The water, soil, cucumber, cabbage, tomato, potato with haloxyfop-P-methyl were analyzed according to the method, the recoveries were all in the range of 87.43～111.80%, and the coefficients of variation were 0.11～12.56%. The recoveries of fluazifop-P-butyl were obtained from water and soil samples were in the range of 86.80～103.41%, and the coefficients of variation were 3.96～12.32%. These results indicated that the ELISAs could be a convenient and supplemental analytical tool for monitoring fluazifop-P-butyl.The results showed that the antibodies for haloxyfop-P-methyl and fluazifop-P-butyl could be used for residues analysis and the immunoassay provide a fast, sensitive, cost-effective and selective method for the detection of herbicide residue.