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

ObjectiveThe goal of this study was to synthesize artificial antigen of AFB₁,to prepare monclonal antibody against AFB₁.MethodsImmunogen 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.AFB₁ mAb was prepared by hybridoma technology.The titer,affinity, sensitivity,specificity and subtype of the mAb were characterized.Massive AFB₁ mAb were induced from in vivo method.ResultsThe 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 show ed good sensitivity with IC₅₀ of 2.58 ng/mL to AFB₁.The rate of cross reaction of AFB₁ mAb with AFB2 was 1.61%, and there was no cross-reactivity to other compounds.ConclusionAFB₁ mAb of high-titer,sensitivity and specificity had been generated,it is possible to establish immunoassay of AFB₁ residues in food.