Development And Application Of Single-Analyte And Multi-Analyte Immunoassays For Organophosphorus Pesticides

O-Methyl-O-(4-nirtopheyl)-N-(2-carboxyethyl)-phosphoramidothionte (H1) was conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) by the active ester method. HI-OVA was used as coating antigen to scan hybridoma. Hl-BSA was used as immunogen to immunize BALB/c mice by intraperitoneal injection. Spleen cells of immunized mouse were fused with SP2/0murine myeloma cells by PEG. After several times of screening and subcloning, one hybridoma cell line (C8/D3) that stably produced monoclonal antibody (MAb) against organophosphorus (OP) pesticide was selected. The secreted MAb was IgG1, kappa type, and affinity constant (Kaff) reached2.3×107L/mol, which was a good material for development immoassays to detect OP pesticids.An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to detect the OP pesticides using the broad-selective antibody C8/D3. To develop a multi-analyte ELISA with increased sensitivity, the best antibody/coating antigen combination was selected from12antibody/hapten-OVA conjugations based on IC50values. IC50values of the novel ELISA system ranged from3.5to162.2ng mLT1for the eight OP pesticides. The selectivity of the ELISA was evaluated by measuring its cross-reactivity (CR) with23OP pesticides, and the results showed the ELISA had specific selectivity for the8OP pesticides (Parathion-methyl, Parathion, Fenitrothion, Cyanophos, EPN, Paraoxon-methyl, Paraoxon, Fenitrooxon). Spike recoveries were between78.9%and118.1%for the detection of single pesticide residues of parathion-methyl, parathion, and fenitrothion in Chinese cabbage, apple, and greengrocery. Moreover, ELISA results were validated by comparison with gas chromatography (GC) analyses. The results indicate that the ELISA established is a potentially useful tool for detecting OP pesticides in various samples.A rapid (less than10min), qualitative and quantitative immunochromatography assay (ICA) using colloidal gold-antibody3G7/F5was successfully developed and applied in determination of chlorpyrifos-methyl in water samples. The optimized pH value and ionic strength of working solution was selected as pH9.0and0.4M, respectively. The qualitative detection limit of chlorpyrifos-methyl was determined as0.6μg ml-1by using the naked eye observation. In the quantitative detection, the detection results of chlorpyrifos-methyl were scanned by a membrane strip reader, and a standard inhibition curve and calibration curve were established, and the regression equation y=-2.5229x+7.5951, R2=0.9889was obtained. IC50value and detection limit (IC10) were calculated to be1024.4ng/ml and137.9ng/mL, respectively. The cross-reactivities were less than1%with tested analog compounds and regarded as negligible. The recoveries obtained by standard chlorpyrifos-methyl addition to tap water samples were102.535%to107.584%. Therefore, the developed ICA test strip is very useful as a potential tool for rapid, quantitative or qualitative detection of chlorpyrifos-methyl in samples.Based on the gold-conjugated MAb C8/D3and an optimal antigen (H9-OVA), a simple and rapid (5min) immunochromatography assay (ICA) for detection of OP pesticides in different agricultural products was developed using a binding inhibition format on a membrane strip. On the optimized condition, IC50values of this ICA for the8OP pesticides were calculated to be from11.8to470.4ng mL-1under an optical density scanner. The selectivity of the ICA was evaluated by measuring its cross-reactivity (CR) with23OP pesticides, and the results showed the ICA had specific selectivity for the8OP pesticides (Parathion-methyl, Parathion, Fenitrothion, Cyanophos, EPN, Paraoxon-methyl, Paraoxon, Fenitrooxon). Spike recoveries were between70.6and131.9%for the detection of single pesticide residues of parathion-methyl, parathion, and fenitrothion in Chinese cabbage, apple, and greengrocery. Compared to enzyme linked immunosorbent assay (ELISA), the results of ICA were comparable with those of ELISA, and ICA has advantages over ELISA in test procedure, test time, matrix interference, on-site detection and so on. Therefore, the established ICA is very useful as a screening method for quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products.A phage-borne peptidomimetics enzyme-linked immunosorbent assay (P-ELISA) was developed to detect the organophosphorus (OP) pesticides by using4phage-borne peptides that isolated from a loop8peptide phage library. To develop a multi-analyte ELISA with increased sensitivity, the best antibody/phage antigen combination was selected from4antibody/phage antigen based on Amax/IC50values. IC50values of the novel ELISA system ranged from1.4to92.1ng mL-for the eight OP pesticides. The selectivity of the ELISA was evaluated by measuring its cross-reactivity (CR) with23OP pesticides, and the results showed the ELISA had specific selectivity for the8OP pesticides (Parathion-methyl, Parathion, Fenitrothion, Cyanophos, EPN, Paraoxon-methyl, Paraoxon, Fenitrooxon). Spike recoveries were between66.1%and101.6%for the detection of single pesticide residues of parathion-methyl, parathion, and fenitrothion in Chinese cabbage, apple, and greengrocery, all the coefficient of variations (CV) were less than16%. Moreover, P-ELISA results were validated by comparison with artificial coating antigen ELISA. The results indicate that phage-borne peptide can be used to accelerate development heterologous immunoassay, and have advantage on no toxicity, easy producing, and increased sensitivity.