Development Of Enzyme-linked Immunosorbent Assay For The Detection Of Aflatoxin B₁

In this work,the polyclonal antibodies and monoclonal antibodies against AFB1(Aflatoxin B1)were prepared sucessfuly and ELISAs for the detection of AFB1 using these antibodies were established respectively.New Zealand white rabbits were immunized with the immunogen(AFB1-BSA)to obtain polyclonal antibodies against AFB1.By optimizing the working conditions of icELISA,the optimal amount of coating antigen was 0.05 ?g/well,the antibody was diluted to 1:30000,0.5%skim milk powder as blocking solution and pH 7.4 0.01M PBS as assay buffer to establish icELISA of AFB1.The assay sensitivity(IC50)is 1.21±0.08 ng/mL and the limit of detection(IC15)is 0.0610.01 ng/mL.By optimizing the working conditions of dcELISA,the developed dcELISA was performed as follows:the concentration of antibody was 0.1?g/well,the solution of antigen-HRP was 1:200000.0.5%skim milk powder was choosed as blocking solution and pH 7.4 0.01 M PBS as assay buffer.The sensitivity(IC50)of this method was 0.31±0.01 ng/mL,the limit of detection(IC15)was 0.04±0.001 ng/mL.Detected the specificity of the polyclonal antibodies against AFB1 by icELISA and dc ELISA,the cross-reactivity rates of the polyclonal antibody with AFG1 were19.56 and 18.29%respectively,and it was less with other analogues of AFT(Aflatoxin).Immunized mices with immunogen AFB1-BSA,after a fusion of the immunized mice spleen cells and SP2/0 myeloma cells,cells screened and subcloned,a strain of cells which can produce anti-AFB1 monoclonal antibody was obtained.Ascites was produced by mice vivo culcure and purified to obtain antibody.The subtype of the antibody was identified by antibody subtype identification kit:the heavy chain was IgG1,and the light chain was Lambda.By optimizing the working conditions of icELISA,the optimal amount of coating antigen was 0.05 ?g/well,the antibody was diluted to 1:15000,0.5%skim milk powder as blocking solution and pH 7.4 0.01M PBS as assay buffer to establish icELISA of AFB1.The assay sensitivity(IC50)is 4.5 5±0.13 ng/mL and the limit of detection(IC15)is 0.7410.15 ng/mL.After optimized,the developed dcELISA was performed as follows:the optimal amount of antibody was 0.02?g/well,antigen-HRP was diluted to 1:100000,0.5%skim milk powder was choosed and pH 7.4 0.01 M PBS as sample diluent.The sensitivity of the method was 1.07±10.01 ng/mL,the limit of detection was 0.13±0.01ng/mL.Detected by icELISA and dcELISA,the cross-reactivity rates of the monoclonal antibody with AFG1 were 23.90%and 26.10%,with AFM1 were 26.32%and 25.36%,and it was less with other analogues of AFT.