| Neonicotinoids insecticides and organophosphorous pesticides have been widely used for controlling the pest. Neonicotinoids insecticides are mainly used to control the pests of sucking mouthparts on the rice, corn and vegetables. Organophosphorous pesticides use to control the pests on the apple, vegetables and other crops. Due to the improper use of organophosphorous pesticides and neonicotinoids insecticides, they have seriously jeopardized the environmental quality and human health. Hence, it was imperative to developed rapid and sensitive assays for two type of pesticides residue. The enzyme-linked immunosorbent assay (ELISA) plays a significant role in screening multi-pesticides residues because of its simpler and more suitable for monitoring a huge number of samples than traditional methods. Three ELISAs have been developed for the determination multi-residues,1) broad-specificity antibody which prepared by multi-haptens antigen could be applied to analyzing pesticides;2) different antibodies were applied to analyze pesticides;3) two antibodies were mixed to detect pesticides simultaneously.For broad-specificity antibody strategy, haptens P1and I1were successively conjugated to BSA in turns by active ester method. 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 optimal concentrations of coating antigen and antibody were determined by a checkerboard titration assay. The effects of organic solvent, ionic intension and pH value on the affinity of parathion and imidacloprid to antibody were evaluated. Under optimum conditions, the standard competitive curves for parathion and imidacloprid were obtained using%(B/Bo) versus log concentration of parathion and imidacloprid. For parathion, the linear equation was y-19.785x＋+25.300was obtained with an IC5o value of0.056mg/L, LOD (IC10)=0.0005mg/L and a linear range of0.0017-1.85mg/L. For imidacloprid, the linear equation was y=-25.418x+55.847was obtained with an IC50value of1.7mg/L, IC10of0.0045mg/L and a linear range of0.11-25.70mg/L. The antibody cross-reactivity of imidaclothiz was23.9%and displayed no cross-reactivity with most of organophosphorous pesticides and neonicotinoids insecticides. Recoveries of parathion and imidacloprid were84.37-116.89%with RSD of0.37-7.16%in tap water, river water, and soil and cabbage samples at0.002-1mg/L spiked, which meets to the requirements of residue analysis.For the second method, the anti-parathion antibody (Pab-P) and anti-imidaclothiz (Pab-I) were coated at different wells in one micro-well plate, and then dc-ELISA had been developed. The optimal concentrations of coating antibody and enzyme tracer were determined by a checkerboard titration assay. The effects of organic solvent, ionic intension and pH value on the affinity of parathion and imidaclothiz to antibody were evaluated. Under optimum conditions, the standard competitive curves for parathion, parathion-methyl, imidaclothiz and imidacloprid were obtained. For parathion, the linear equation was y=-19.895x+25.291was obtained with an IC50value of0.057mg/L, IC1o=0.0005mg/L and a linear range of0.0018-1.84mg/L. For parathion, the linear equation was y=-19.895x+25.291was obtained with an IC50value of0.057mg/L, and a linear range of0.0018-1.84mg/L with a LOD of0.0006mg/L. For parathion-methyl, the linear equation was Y-23.006x+32.283, the linear range of parathion-methyl was from0.00843to3.419mg/L, with the average IC50value of0.1698mg/L, IC10value of0.0006mg/L. For imidaclothiz, the linear equation was Y=-20.587x+23.562, the working range was from1.82to1489mg/L with the IC50value was0.05248mg/L and a LOD of0.00062mg/L For imidacloprid, the linear equation was Y=-19.776x+24.784, the working range was from0.0016to1.745mg/L, with the IC50value was0.0531mg/L and a LOD of0.0005mg/L. The antibodydis played negligible cross-reactivity with most of organophosphorous pesticides and neonicotinoids insecticides. The veracity of the method was evaluated by spiked recoveries study, the recoveries of parathion, parathion-methyl, imidaclothiz and imidacloprid in river water, soil and cabbage samples were82.54%to116.29%with RSD of1.59-8.09%at0.005-0.5mg/L spiked. It fulfilled the requirements of residue analysis.For the third method which use mixed different antibodies (anti-imidaclothiz antibody and anti-thiacloprid antibody) for screening pesticides, the concentrations of coating antibodies and enzyme tracers were optimized by three different ECL-ELISA formats. Under the optimum conditions, multi-enzyme tracers ECL-ELISA was used for the analysis of imidaclothiz and thiacloprid residue mixtures. The standard curve was Y=-33.609x-13.252, R2=0.988, with a LOD (IC,0) of0.0018mg/L and a linear range of0.0036-0.275mg/L. Recoveries of imidaclothiz and thiacloprid in tomato, cabbage and rice samples were83.7-109%with RSD of2.0-5.8%at0.04-0.25mg/L spiked. Meanwhile, the results showed that the multi-enzyme tracers ECL-ELISA could be applicable for accurately analyzing real tomato samples, and the results correlated well with high-performance liquid chromatography (HPLC), with the correlation coefficient of0.996. Therefore, the new strategy for developing immunoassay was suitable for simultaneous quantitative detection of imidaclothiz and thiacloprid residues in agricultural samples.By the established ELISA, the residue of pesticides could be monitored in environmental and agricultural samples. Those methods were saisified with demand for rapid and economical screening programs and met with the requirement of the multi-residue of pesticide analysis, so the present research laid the foundation for development of the ELISA kits. |
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