The Research On Enzyme-Linked Immunosorbent Assay For Aryloxyphenoxypropionate Herbicides

For the research of immunorbent assay for pesticide multiresidue, two haptens (fenoxaprop-p and cyhalofop-p) for fenoxaprop-p-ethyl and cyhalofop-butyl were synthesized. Haptens were successively conjugated to BSA by active ester method. The conjugates ((fenoxaprop-p)m(cyhalofop-p)n-BSA) were confirmed by UV spectra, the coupling ratio of conjugates was around9.2:7.8:1(m:n:BSA). Fenoxaprop-p and cyhalofop-p were conjugated to OVA via mixed anhydride reaction, respectively. The conjugates were used as multi-determinant immunogen to immunize female New Zealand white rabbits and the specific antiserum to the related antigens were obtained. The antiserum had the high affinity and the affinity constants for fenoxaprop-p-ethyl and cyhalofop-butyl were5X109and7.5X109, respectively. The titers of the broad specificity antibodies for fenoxaprop-p-ethyl and cyhalofop-butyl were5.12×05and1.02×106, respectively.The optimal concentrations of coating antigen and antiserum were determined by a checkerboard titration assay. The effects of organic solvent, ionic intension and pH value on the affinity of fenoxaprop-p-ethyl and cyhalofop-butyl to antibody were evaluated. The results showed that30%methanol, pH of7.5and0.5mol/L Na+were determined to optimum assay conditions.Under optimum conditions, the standard competitive curves for fenoxaprop-p-ethyl and cyhalofop-butyl were obtained using%(B/Bo) versus log concentration of fenoxaprop-p-ethyl or cyhalofop-butyl. For fenoxaprop-p-ethyl, the equation y=-24.799x+31.816was obtained with R2=0.9975, an IC50value of0.185mg L-1, a LOD (IC10) of0.004mg L"1and a linear range of0.005mg L-1-5mg L"1. For cyhalofop-butyl, the equation y=-30.776x+8.422was obtained with R2=0.9956, an IC50value of0.045mg L-1, a LOD (IC10) of0.002mg L-1and a linear range of0.005mg L-1-1mg L-1. The antibody cross-reactivity of cyhalofop-butyl was411.1%and displayed no cross-reactivity with most of the APPs tested, except for metamifop (CR%=55.56%). At spiked different levels (0.01-5mg/L) recoveries of fenoxaprop-p-ethyl and cyhalofop-butyl in tap water, paddy water, soil, rice and soybean samples were86.86-114.52%and82.07-119.11%with CV of0.72%～9.32%and1.23%-7.75%, respectively. It meets to the requirements of residue analysis.Two indirect competitive enzyme-linked immunosorbent assays (ic-ELISA) were developed to detect mixture of cyhalofop-butyl and fenoxaprop-p-ethyl. Then the assays were applied to the analysis of the tap water, river water, soil and rice. The optimized ELISA detected with broad specificity antibodies showed the limit of detection (IC10) was0.017mg/L, the recoveries ranged from77.6%-118.2%and the relative standard deviation (RSD) were found to be1.5-7.0%. The optimized ELISA detected with mix antibodies showed the limit of detection (IC10) was0.039mg/L, the recoveries ranged from79.1%-118.1%and the relative standard deviation (RSD) were found to be1.5-8.9%.By the established and optimized ic-ELISA, the residue of aryloxyphenoxypropionates could be monitored. The method was satisfied with the requirement of the pesticide multiresidue analysis, so the present study laid the foundation for production of the ic-ELISA kits.