Preparation And Directed Evolution Of Broad-specific Scfv Antibody For Organophosphorus Pesticides Recognization

Organophosphorus pesticides(OPs)are the most widely used agri-chemicals for insect control in the crop production.However,their residues in food and environment will cause serious damage to human health and ecosystem.Due to the similar toxicity,and characteristics,as a family chemicals,the risk of cumulative toxicity remains when various of OPs coexistent.Thereby,it is urgency to develop a method for the simultaneous determination of family pesticides.At present,several methods have been employed for the detection of Ops residues,the immunoassays cause attention for its sensitivity,simpleness,and high-throughput.Mechanism study of antibody affinity has been profiled that for low molecule weigh compounds binding,the interaction mainly happened in the pocket or groove of the interface between VH and VL,so the size and structural stability of the Abs inevitably affects the stability of pocket,and further influence the combination between small molecules antigens(Ags)and Abs.For high molecular weigh compounds,such as protein,the combination mainly happened on the surface of the conjunction between VH and VL.The binding domain of Ab is composed by CDRs region and FR region,and the CDRs are believed to be responsible for Ag recognition,and the FR residues are considered as a scaffold for the CDRs,which also play an important role in Ags binding.In this study,the research object was small molecules compounds,the size of pocket has influence on the extensive adaptability feature of antibody,so we focused on the research of FRs of Abs.In this study,site-directed mutation have been conducted in FRs of scFv Abs,based on the qualified monoclonal antibody(hybridoma cell line 3D2B3),which can simultaneous recognize 8 Ops.Main results have been presented as follows:1?Variable region genes of antibody have been cloned and tertiary structure of scFv antibody has been modelled and docked with antigen.The results show that if turning Asparagine at H3 positon and Valine at H4 in FR1 region into glutamine and leucine could extend the size of binding pocket,and that would improve the activity of antibody,theoretically.2?Site-directed mutation have been conducted in FRs of sc Fv Abs,the scFv and scFv mutant [scFv(M)] were expressed in Ecoli system,using prokaryotic expression vector of pET-26b(+).The results show that all 8 OPs can be recognized by expressed scFv antibody before and after mutation,but affinity of Ab for several Ops binding has been improved after mutation.The IC50 ranged 1.25-20.701 ?g/mL for scFv,for scFv(M)IC50 ranged from 1.216-10.740 ?g/m L,repectively.Except disulfoton,the sensitivity of sc Fv(M)for others Ops were higher than scFv.Compared with Mab,the sensitivity of scFv for 8 Ops were all below parent mAb,whereas scFv(M)can specifictal recognize four pesticides(phosalone,phorate,terbufos,malathion)which with IC50 similar to parent mAb,even the sensitivity to malathion was improved 2 folders than mAb after mutation.3?An indirect competitive enzymelinked immunosorbent assay(ic-ELISA)has been established based on scFv(M).The recoveries and coefficient of variation values to spiked sample corn and wheat are 72% to 127% and 1.2% to 8.6%,and 79% to 139% and 1.3% to 11%,respectively.The resuLts reveal that the ic-ELISA could be used for OPs residues determination in cereal samples.4?scFv(M)has been expressed in eukaryotic expression system with vector Pcho1.0,and using Chinese hamster ovary(CHO)cells lines as host;The Western blot results imply that the scFv(M)antibody is successfully expressed with antigen binding activity,but the yield is very low,expression conditions still need further optimization.