| Organophosphorus pesticides are organic compounds containing phosphorus,and have been widely used in agricultural production to control pests and other plant pathogens in an effort to reduce yield losses and maintain high product quality.However,with their extensive use,harm to human,animal and the environment is more and more serious.In order to ensure safety and quality of foods,establishing rapid and sensitive methods for detection of Ops has attracted a lot of attention.The current methods for the detection of Ops residue are mainly instrument analysis,enzyme inhibition and immunochemical analytical method.Immunochemical analytical method,based on highly specific antigen-antibody interactions,would be desirable,offering several advantages compared to conventional techniques,i.e.,high selectivity,strong specificity,high throughput,low detecting cost,and it has been widely used in the detection of pesticide residues.However,because of its narrow spectrum and low sensitivity,immunochemical analytical technology is mainly applied to the detection of single pesticide residue,few study has been reported about the multi-residue detection of Ops.In this study,a new strategy was given to the immunogen of small molecule compounds and a dual-genetic hapten immunogen were prepared by conjugating two given genetic hapten of Ops to single protein molecule.After the dual-genetic hapten immunogen was immunized to rabbits or mice,broad-specificity polyclonal or monoclonal antibodies with high titer were obtained.Using the purified polyclonal or monoclonal antibody as the ligand,peptide mimetics of Ops genetic hapten were screened from phage display peptide libraries.It provides scientific basis for the establishment of rapid and sensitive heterologous ELISA for the multi-residue detection of pesticides.The main results and conclusions presented in this thesis are as follows:(1)Synthesis and identification of the generic haptenAccording to the general structure of phosphate OPs and thiophosphate OPs,generic hapten PM,PS,PO were synthesized,and the structure was identified by HNMR.(2)Synthesis of dual-genetic hapten immunogenThe two generic hapten(PM or PS)of methyl thiophosphate pesticides and two generic hapten(PO or DPA)of ethyl phosphate pesticides were conjugated to single protein molecule in turn to prepare dual-genetic hapten immunogens PM-DPA-BSA,PS-DPA-BSA,PM-PO-BSA ? PO-PS-BSA by diazotization method(containing amino hapten)or active ester method(containing carboxyl hapten).PM-OVA,DPA-OVA,PO-OVA,PS-OVA,DPA-PM-OVA,PS-DPA-OVA,PO-PS-OVA and DPA-PM-OVA were also prepared as coating antigens.(3)Preparation and screening of polyclonal antibody and monoclonal antibodyThe four dual-genetic hapten immunogen were immunized to rabbits to produce broad-specificity polyclonal antibodies PAb PMDPA,PAb PSDPA,PAb PMPO and PAb POPS.Four antibodies were purified by Protein G affinity chromatography and tested the sensitivity to pesticides by indirect competitive ELISA.Results showed that PAb PMPO and PAb POPS could recognize phosphate OPs and thiophosphate OPs well and were used for optimization of indirect competitive ELISA conditions.The four dual-genetic hapten immunogen were also immunized to BABL/c mice to produce monoclonal antibodies with the recognition of PO-OVA and PS-OVA.After detected by the indirect competitive ELISA,MAb POPS-4A7 was found to recognize parathion-methyl and paraoxon well.Therefore,4A7 was purified by Protein G affinity chromatography and used for optimization of indirect competitive ELISA conditions.(4)Establishment of multi-residues immunochemical analytical technology of OpsStandard curve were obtained by plotting absorbance against the analyte concentration.And then,IC50 values and IC20 values,cross-reactivity and analysis of spiked samples were determined.The IC50 value of PAb PMPO-2 for parathion-methyl was 0.819?g/mL with a detection limit of 0.034?g/mL(20%inhibition).The IC50 value of the assay for paraoxon was 0.307?g/mL with a detection limit of 0.009?g/mL.PAb PMPO-2 had the highest cross-reactivity with twelve Ops.Recoveries of parathion-methyl spiked samples ranged from 78.875%to 116.432%,and coefficient of variation from 1.328%to 7.976%,while recoveries of paraoxon spiked samples ranged from 77.432%to 103.651%,and coefficient of variation from 1.878%to 7.745%.The IC50 value of PAb POPS-1 for paraoxon was 0.353?g/mL with a detection limit of 0.011?g/mL(20%inhibition).The IC50 value of the assay for PS was 0.742?g/mL with a detection limit of 0.025?g/mL.PAb POPS-1 had the highest cross-reactivity with ten Ops.Recoveries of parathion-methyl spiked samples ranged from 81.2%to 105.5%,and coefficient of variation from 1.249%to 7.864%,while recoveries of paraoxon spiked samples ranged from 84.1%to 104.3%,and coefficient of variation from 1.403%to 7.389%.The IC50 value of MAb POPS-4A7 for paraoxon was 0.197?g/mL with a detection limit of 0.008?g/mL(20%inhibition).The IC50 value of the assay for PS was 0.184?g/mL with a detection limit of 0.007?g/mL.MAb POPS-4A7 had the highest cross-reactivity with sixteen Ops.Recoveries of paraoxon spiked samples ranged from 82.4%to 92.3%,and coefficient of variation from 1.468%to 7.045%.(5)Screening of peptide mimetics of generic hapten of organophosphorus pesticidesIn this study,with PAb PO-PS polyclonal antibody or MAb PO-PS monoclonal antibody as target molecule,reducing concentration of antibody,increasing the concentration of Tween-20,decreasing incubation time and concentration of competitive eluant,peptide mimetics of generic hapten were screened by phage display random cyclic seven peptide library and twelve peptide library.Results showed that,with PAb POPS-1 polyclonal antibody as target molecule,phage clone P12-8 was successfully screened from phage display random twelve peptide library,and the phage competitive ELISA inhibition curve was built.The IC50 value of PAb POPS-1 for paraoxon was 0.276?g/mL with a detection limit of 0.01?g/mL(20%inhibition).The IC50 value of the assay for PS was 0.209?g/mL with a detection limit of 0.009?g/mL.PAb POPS-1 had the highest cross-reactivity with fourteen Ops.Recoveries of paraoxon spiked samples ranged from 84.5%to 92.5%,and coefficient of variation from 1.382%to 6.826%.With MAb POPS-4A7 as target molecule,phage clone P12-2 was successfully screened from phage display random twelve peptide library,and the phage competitive ELISA inhibition curve was built.The IC50 value of MAb POPS-4A7 for paraoxon was 0.096?g/mL with a detection limit of 0.008?g/mL(20%inhibition).The IC50 value of the assay for PS was 0.062?g/mL with a detection limit of 0.006?g/mL.MAb POPS-4A7 had the highest cross-reactivity with eighteen Ops.Recoveries of paraoxon spiked samples ranged from 85.6%to 92.6%,and coefficient of variation from 1.634%to 6.653%. |
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