Screening And Identification Of ScFv Antibody Against Pesticide Of Thiopgosphates And The Optimizing Of Elisa Conditions

Currently, with the improvement of people’s attention in pesticide residues, thepesticide residue analysis of agricultural and sideline products is becoming more andmore significantly. Especially in the real-time detection of the spot, geneticallyengineered antibody have broad application prospects in the aspects of pesticideantibody preparation and detection technology development, because it has high fluxscreening, short testing cycle, high efficiency, strong specificity and broad affinityselection range etc. In this study, the enzyme immunoassay of organophosphoruspesticide residues was optimized, using single chain antibody fragment (ScFv).Firstly, the builted ScFv primary antibody library against the pesticides ofthiophosphates was gathered and biopanned. After selecting48monoclonal strains inthe last antibody library, the monoclonal strains which have high affinity werescreened through the method of ELISA. Utilized this specific phages to infectlogarithmic phase of the E. coli HB2151, soluble antibody can be made after IPTGinducing expression. Expressed part was chosing by indirect ELISA, which usedHRP/Anti-E Tag as secondary antibody, then the protein in this expressed part wasused during subsequent experiments.After purifying the large number of expressed soluble antibody with the method ofammonium sulfate fractional precipitate, via qualification of SDS-PAGE,the proteinwhich be precipitated by60%ammonium sulfate is about30kD. Via detection ofWestern blotting, the protein can combine with HRP/Anti-E Tag, therefore, it sure thatthe protein is recombinant antibodies which could express the nature of resisting thethiosulfuric acid pesticides.A recombinant antibody of organophosphorus pesticide with a titer of1:25600and affinity constant of6.2×107had been obtained by immunizing mice. By indirect and direct ELISA, the optimum coating temperature and time is37℃,2h; theoptimum working concentration of coating antigen, antibody is10μg/mL and1:6,400;the LOD is2.88μg/mL. The recombinant ScFv antibody exhibited cross-reactivitywith chlorpyrifos was339.6%, whereas reaction with phoxim, profenofos, triazophos,malathion was61.6%，69.5%，16.7%，52.0%.