Study On Enzyme Immuno-chemistry Of Organic Phosphorus Pesticides

The enzyme immuno-chemistry of pesticides including the preparation of the specific antibody for single compound and the "broad specific" antibodies for multi-residue analysis.Research on enzyme linked immunosorbent assay (ELISA) for multi-residue analysis of organic phosphorus (OP) pesticides was performed systematically for the first time in the first part. Most of the commonly used organic phosphorus pesticides share similar moiety in chemical structure. Three kinds of 'generic-OP' haptens were synthesized, which have the common moiety of the aromatic dimethoxyl phosphorothioate (MP, for parathion-methyl, fenitrothion et al.), the straight chain dimethoxyl phosphorothioate (SP, for dimethoate, malathion et al.), and the vinyl dimethoxyl phosphate (VP, for monocrotophos, phosphamidon et al.), respectively. Then the haptens were conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) by different methods.New Zealand white rabbits were immunized with the hapten-BSA comjugates, and the polyclonal antibodies (PAB) against the MP-BSA and SP-BSA were raised. The middle and final liters were 12800 and >50000 respectively for both of the antiserum. Using parathion-methyl and the SP hapten as the inhibitors, the indirect competitive ELISA was developed and optimized respectively.Using the optimized inhibition assays, the spacer arm and the other organic phosphorus analogues were used as inhibitors to test for the cross-reactivity. The results of these assays showed that, the greatest inhibition of the antiserum obtained from MP-BSA were recorded with parathion-methyl, fenitrothion and fenthion, the 50% inhibition were at 0.13 u g/mL, 0.50 u g/mL and 0.35 u g/mL, the limit of detection (I20) were 6.7, 20.9 and 12.1ng/mL, respectively. All the other OPs tested showed no cross reactivity. The fortified recovery tests of the three pesticides in cucumber were performed with the ELISA and GC method. The recoveries were 83.10%~93.46%, 80.45%~95.78%, 74.89%-86.78%; C.V% were 2.36%~ 10.36%, 5.14%~7.14% and 4.40%~6.34%, respectively. The results of the ELISA were verified by GC method.The greatest inhibition of the antiserum obtained from SP-BSA were recorded with acephate, dimethoate, parathion-methyl and malathion, the 50% inhibition were at 2.66, 3.31, 8.26 and 14.9 u g/mL, the limit of detection (I20) were 0.05 ,0.15, 0.19 and 0.53 u g/mL, respectively. The information showed that the generic polyclonal antibody (PAB) could recognize some of these chemical-similar-compounds.The immuno-chemistry of chlorpyrifos was studied in the second part. The hapten that has the similar structure with the chlorpyrifos was synthesized. Then it was linked to BSA or OVA through a 5-carbon spacer arm. The PAB that is specific to chlorpyrifos was raised against the hapten-BSA conjugate. The middle and final titer were 12800 and >50000, respectively. The indirect competitive ELISA for chlorpyrifos was developed and optimized, and the standard inhibition curve for chlorpyrifos was established, the 50% inhibition was at 76.8ng/mL, and the limit of detection was 5.2ng/mL. All the other OPs tested showed no inhibition except for chlorpyrifos-methyl (cross reactivity was 66.9%). The fortified recoveries of chlorpyrifos in soil, cucumber and river water were 73.81%~89.45%, 76.93%~ 102.8%, and 89.30%~ 103.2%, C.V% were 5.28%~9.83%, 7.62%~ 12.25% and 4.04%~5.18%, respectively. The results of the ELISA were verified by GC method.