Development of monoclonal antibody against Aflatoxin B1 and establishment of immunology quantitative ELISA

Objective The goal of this study was to synthesize artificial antigen of AFB1,to prepare monclonal antibody against AFB1.Methods Immunogen AFB1-BSA and coating antigen AFB1-OVA were synthesized using NHS by linking carrier proteins BSA and OVA to AFB1 and identified by ultraviolet scanning,SDS-PAGE.BALB/C mice were immunized with AFB1-BSA,The titre and sensitivity of polyclonal antibody was detected by indirect ELISA and blocking ELISA,so as to select the mouse used in cell fusion.AFB1mAb was prepared by hybridoma technology.The titer,affinity,sensitivity,specificity and subtype of the mAb were characterized.Massive AFB1mAb were induced from in vivo method.Results The results showed that the hapten AFB1 was successfully linked to carrier proteins by the UV scanning spectrum and SDS-PAGE electrophoresis.There hybridoma cell lines of 2H5-F6、2H5-C9、2H9-C3 were screened for specificity to AFB1,all the isotypes of the mAb were IgG1.The indirect ELISA titer of the mAb were 1∶ 2.0×102 ~1∶ 1.28×103 in supernatant,1∶ 1.28×106 of 2H5-F6 in ascites,and the affinity constant(Ka) was 2.65×1010 L/moL,the mAb of 2H5-F6 showed good sensitivity with IC50 of 2.58 ng/mL to AFB1.The rate of cross reaction of AFB1mAb with AFB2 was 1.61%,and there was no cross-reactivity to other compounds.Conclusion AFB1mAb of high-titer,sensitivity and specificity had been generated,it is possible to establish immunoassay of AFB1 residues in food.