Study On The Method Of ELISA For Determination Of Pyrethroid Pesticide Residues In Lycium Barbarum L.

Object The study is to research the rapid detection of residual pyrethroid pesticides in Lycium barbarum L.by ELISA.Method(1)3-Phenoxybenzoic acid(PBA)was used as the generic hapten,Immune antigen(PBA-BSA)and detection antigen(PBA-OVA)were prepared by conjugated bovine serum albumin(BSA)and chicken ovalbumin(OVA)with carbodiimide and carbonyl diimidazole method respectively.(2)Balb/c mice were immunized with the prepared immunizing antigen PBA-BSA.After four immunizations,the titers were the highest,and the cells with the strongest ability of PBA were taken.The spleen cells and myeloma cells were treated with polyethylene glycol(PEG 4000)were used to clone and isolate the positive cell lines by competitive inhibition ELISA and limited dilution method.The hybridoma cell lines secreting monoclonal antibodies against pyrethroids were obtained.The monoclonal antibody was prepared by ascites induction method and the titer and subtype of antibody titer and antibody titer and antibody subtype were determined after purification.(3)The effect of antibody specificity on pyrethroid pesticides in pesticide samples and Lycium barbarum L.samples was analyzed by ELISA and GC-MS/MS.The content of pyrethroid pesticide residues was compared and analyzed.Result(1)PBA-BSA and PBA-OVA conjugates were obtained by coupling PBA with carrier protein BSA with OVA by carbodiimide method and carbonyl diimidazole method respectively,A total of 10 mg of PBA-BSA and PBA-OVA conjugates at a concentration of 6.25 mg / mL were obtained.SDS-PAGE Gel electrophoresis showed that PBA-BSA and PBA-OVA antigen were successfully coupled.(2)Three strains of hybridoma cell lines secreting monoclonal antibodies against pyrethroids were obtained.They were 2E9-A5-F5,1G7-F6-D1 and 1G7-F6-G4 respectively.The titer of the antibody titer and subtype was 1?256000,and the antibody subtype was IgG 1.(3)The content of pyrethroid pesticide residues in Lycium barbarum L.was compared by ELISA and GC-MS / MS.The total residue of pyrethroids was 0.5809 mg / kg by ELISA.,and the results of GC-MS / MS GC-MS / MS method for the determination of pesticide residues in Lycium barbarum L.was 75753 mg / kg cypermethrin,0.0792 mg / kg fenvalerate,0.0854 mg / kg deltamethrin,The total amount of the three pesticide residues was 0.2399 mg / kg in Lycium barbarum L.by GC-MS / MS.The detection of the pyrethroid pesticide residues by monoclonal antibody ELISA was higher than that of the pyrethroid pesticide residues by GC-MS / MS.Monoclonal antibody ELISA was used to detect all pyrethroid pesticide with m-phenoxybenzoic acid structure,and only the designated pyrethroid pesticide residues were detected by GC-MS / MS.Conclusion As a broad spectrum of antibodies,the monoclonal antibody of Lycium barbarum L.It can detect all pyrethroid pesticides containing m-phenoxybenzoic acid in the nucleus,which lays the foundation for the rapid detection of these pesticides.