Production And Characterization Of Peptide-mimetics, Human Domain Antibodies For Organophosphorus Pesticide Antigen Epitope Mimicking And Recognization

Organophosphates pesticides have been wildly used for the pests control in agricultural production. Due to its good characteristics of broad spectrum and high efficiency toxic to insects, more than 50% of total agrichemicals used in China have been occupied by organophosphates pesticides.. In another hand, the high toxicity and environmental effect and accumulative toxic risk of organophosphates pesticides have been concerned word widely. Pesticide residue in food becomes the biggest issue for human health and food safety in china, especially for organophosphates pesticide residue in food.In recent years, with the continuous development of recombinant engineering technology, such as phage display technology, peptides, anti-idiotypic antibody based human friendly, less toxic rapid test methods have been intensity studied for the determination of small molecules, such as pesticides. According to reports, anti-idiotypic antibody based method for pesticide detection has been established, and phage born anti-idiotypic antibody for toxin detection also reported, which shows remarkable affinity. However, few study focus on the human friendly methods development for the accumulative toxicity detection of organophosphates pesticide. Thereby, the rapid test method is urgently needed for the identification of accumulative toxic risk, which also requires characterization of being friendly to human and environment.In this study, phage displayed peptide library and human domain antibody library have been used for polypeptide and anti-idiotypic antibody screening, using BPB as hepten, which presenting the general structure of organophosphates pesticides. Thereafter, peptide based ELISA method has been developed for the determination of BPB and organophosphates pesticides. The main results are as following.1. Production and identification of monoclonal antibody and antigen.Previously achieved two monoclonal antibody cell lines, which can recognize 8-15 organophosphates pesticides have been recovered and scale-up cultured and ready for ascetic fluid generation. Through caprylic acid-ammonium sulfate precipitation and protein G column purification, the antibody has been purified out of the ascetic fluid then identified by SDS-PAGE and ic-ELISA. The results confirm that the purified antibody possesses good specificity and affinity. Antigen materials are BPB coupled amination magnetic beads and coupled antigen BPB-BSA.2. Antigen epitope mimicking peptides screening by phage displayed 12 peptides library. This study completed the antigen BPB-BSA screening, BPB-BSA monoclonal antibody screening and BPB-KLH monoclonal antibody screening. In BPB-KLH monoclonal antibody screening, five positive strains clones are successfully identified and selected. The results reveal that the amino acid of selected peptide is HPAPDHSSQSHQ, which can mimic the antigen epitope of hapten. Near N terminal part, the polypeptides(amino acid start from 3 to 10) has good antigenicity and hydrophilicity, and surface accessibility, which also can form β-pleated sheets and loops.3. Human domain antibodies for organophosphorus pesticide antigen epitope mimicking and recognization. Using BPB coupled amination magnetic beads to screen single domain antibodies against organophosphorus pesticide, Using the monoclonal antibody against BPB-KLH(keyhole limpet hemocyanin) to screen anti-idiotypic.The experiments obtained the phages which can recognize target, but the competition effects are negative, we need to do further research.4. Polypeptides based Ic-ELISA method development for the determination of organophosphates pesticide. An indirect competitive ELISA method for the determination of BPB and organophosphates pesticides has been established using Polypeptide as immobilized antigen. The results show that the IC50 and the Limit of detection of method for BPB detection are 1.776μg/ml and 0.358μg/ml, respectively. The cross-reaction for the organophosphates pesticides are Parathion: 58.74%; coumaphos: 44.93%; disulfoton:52.03%; phoxim: 43.98%; quinalphos: 36.82%; triazophos: 4.87%.