| Two haptens, pretilachlor-3-mercaptopropionic acid (PMPA) and acetochlor-3-mercaptopropionic acid (APMA) were synthesized, and then covalently coupled with carrier protein BSA via active ester method or OVA by mixed anhydride reaction, respectively. The polyclonal antibodies were obtained by immunized New Zealand rabbits against PMPA-BSA and APMA-BSA, and the titres of the solutions of the corresponding antibodies protein (1000 mg/L) were 1.02×106 and 2.56×105, respectively.The optimal concentrations of coating antigen and antiserum were determined by a checkerboard titration assay. Then the influence of several assay conditions such as organic solvent, ionic strength and pH of solution were evaluated. The optimum parameters of ELISA procedure for pretilachlor were determined as follows:concentration of coating antigen PMPA-OVA and antiserum was 0.5 mg/L and 1 mg/L, content of methanol was 10%, pH was 7.5, and ionic strength was 0.14 mol/L. And for acetochlor, the ionic strength of 0.4 mol/L was selected as optimum to keep an acceptable signal-to-sensitivity ratio, meanwhile the optimum pH was 7.5.Under optimum conditions, calibration curves of the optimal assays were constructed by Logit (B/Bo) and the logarithm of both herbicides concentration as the two influencing factors. It was observed that the immunoassay had a linear relationship from 0.001 to 1.000 mg/L between Logit (B/Bo) and the logarithm of the pretilachlor concentration. Within this range, a regression equation was obtained (y=-0.591x-0.8539, R2= 0.9909). The method was shown to have an IC50 of 0.0415 mg/L and a linear working range from 0.001 to 1.000 mg/L. And the immunoassay for acetochlor was shown to have a linear relationship from 0.025 to 5.000 mg/L between Logit (B/Bo) and the logarithm of the analyte concentration. Within this range, a regression equation was obtained (y=-0.7421x-0.2996, R2= 0.9902). The method was shown to have an IC50 of 0.3718 mg/L and a linear working range from 0.025 to 5.000 mg/L. For both ELISAs, the cross-reactivities with other structurally related compounds were all low, which was negligible.The spiked recoveries and the relative standard deviation (RSD) were calculated to evaluate the accuracy and precision of the ELISAs. Water samples were diluted 10 times with PBS to reduce the matrix effect prior to an analysis. At different spiked levels (0.05-5 mg/L), the recoveries of pretilachlor in water were in the range of 77.0-115.2% and the RSD were found to be 2.3-9.4%. And at different spiked levels (0.1,0.5 and 1 mg/kg), the recoveries and RSD in soil were in the range of 79.1-92.3% and 4.0-8.4%, respetively. For acetochlor, at different spiked levels (0.1-10 mg/L), the recoveries in different water samples were in the range of 73.2-117.6% and the RSD were found to be 3.2-6.4%. And at different spiked levels (0.1,0.5 and 1 mg/kg) in soil, corn and soybean, the recoveries and RSD were in the range of 77.3-98.6% and 1.7-9.2%, respetively. These data indicated that the matrix interference of water samples can be minimized by diluting the samples 10 times with PBS. The accuracy and precision of the methods well matched requirements of residue analysis. These results suggested that ELISA could be a convenient and supplemental analytical tool for determinations of pretilachlor and acetochlor, and the method provides an alternative valuable analytical method for monitoring the both herbicides residue in agricultural and environmental samples. |
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