| Organophosphorus pesticide is a kind of pesticide with the broad-spectrum insecticidal activity and relatively low stability,and has been widely used in agriculture.However,organophosphorus pesticides as a class of cholinesterase-inhibiting insecticides,its excessive and irrational utilization will harm to human health.Therefore,it is very essential to establish a simple,rapid and accurate method for detecting OPs residues in the environment and foods.Enzyme linked immunosorbent assay(ELISA)has been widely used in the detection of OPs residues,which has the advantages of low cost,high speed and detecting various samples at the same time.In our laboratory,we had obtained a broad-specificity monoclonal antibody(mAb)3A7 for OPs,but insufficient sensitivity still was a problem.In order to improve the sensitivity of detection,we used phage display technology screening the mimotope of organophosphorus pesticides and establishing a more sensitive immunoassay.The main results are as follows:(1)In this study,the broad-specificity mAb 3A7 for OPs as target,we used the mixtures of phage display random linear 7 peptide library,linear 12 peptide library and cyclic7 peptide library to select mimotope.The phages were successfully enriched by four rounds of biopanning,and the recovery increased from an initial 1.2×10-7 to 1.1×10-2.In the presence 250ng/m L of parathion,179 phage clones were selected for indirect competitive ELISA,and 91 phage positive clones had inhibition effect.After verification,14 phage clones were choosed for sequencing.Sequence results showed that we successfully screened five kinds of organophosphorus pesticides mimotope,including one kind of linear 7 peptide mimotope and four kinds of linear 12 peptide mimotope.(2)In a bid to develop P-ELISA with enhanced sensitivity,the best combination of antibody and phage antigen was choosed from 5 kinds of antibody/phage antigen on the basis of Amax/IC50 values.Phage peptide 21 was selected.By optimizing pH,ionic strength,concentration of methanol and skim milk powder,the optimal conditions of phage mimotopes was 2.5% methanol,0.4% skim milk,pH 6.5 and 5×PBS.(3)Under the optimal conditions,the developed P-ELISA showed high and uniform sensitivity for multi-OPs,particularly for eight O,O-diethyl OPs and six O,O-dimethyl OPs.The median inhibition concentration(IC50)for fourteen OPs ranged from 5.24 to 63.25ng/m L,and the average IC50 and the coefficient of variations(CVs)for the IC50 values of the fourteen OPs were 35.04ng/mL and 42.98%,respectively.Compared with the conventional ELISA,the sensitivity of P-ELISA was improved to some extent.The sensitivity for fenthion was improved more than 3 folds.The sensitivity for chlorpyrifos and pirimiphos-ethyl was improved 2 folds.The rest of 17 OPs was improved about 1 fold.(4)The spike recoveries were in the range of 82.03% to 103.63% for fenitrothion,parathion,fenthion and diazinon in cucumber,lettuce,apple and watermelon samples,and CVs were between 2.31% and 13.80%.These results declares that the establishment of P-ELISA is suitable for detecting organophosphorus pesticide residues in vegetables and fruits.In conclusion,phage has the advantages of non toxicity,easy amplification,high sensitivity and convenience.It can be used as a substitute for chemical antigen to establish a competitive ELISA analysis. |
PDF Full Text Request