Study On Enzyme-Linked Immunosorbent Assay Of Fumonisins Based On Monoclonal Antibody

A monoclonal antibody was prepared in this study,which could specifically recognize fumonisins.On the basis of this,three kinds of enzyme-linked immunoassays were established for the detection of fumonisins B1 residue in corn samples.The fumonisin B1(FB1)was conjugated to keyhole limpet hemocyanin(KLH)and ovalbumin(OVA)by glutaraldehyde,respectively.Then the prepared FBi-KLH was used as an immunogen for the immunization of Balb/c mice,and the prepared FB1-OVA was used as a coating antigen.Three hybridoma cell lines,1G1,1F8 and 2C4 were obtained after cell fusion and screening,and cell cloning,which can stably secrete monoclonal antibodies against FB1.The subtypes of antibody secreted by the three strains of cells were determined by the isotyping kit,where the isotype was identified as IgA along with kappa light chain.The colloidal gold nanoparticles(AuNPs)were modified by mercapto-undecanoic acid(MUA)on the surface thereof,and the HRP-goat anti-mouse IgA was conjugated by carbodiimide method.And the prepared AuNPs-HRP-goat anti-mouse IgA was used to establish colloidal gold indirect competitive ELISA.Three kinds of ELISA were established by selecting the monoclonal antibody secreted by the best cell line 1G1.After optimized,the developed indirect-competitive ELISA was performed as follows:the optimal amount of coating antigen was 0.1 μg/well,the antibody was diluted 8000-folds,the HRP-goat anti-mouse IgA was diluted 10000-fold,0.5%skim milk powder and pH 7.40.01 mol/L phosphate buffered saline(PBS)were used as blocking solution and standard dilution buffer,respectively.The half maximal inhibitory concentration(IC50)was 9.57 ±0.306 μg/L and the detection limit(IC15)was 2.34 ± 0.031 μg/L.The developed direct-competitive ELISA was performed as follows:the optimal amount of antibody was 0.1 μg/well,antigen-HRP was diluted 20000-fold,blocking solution and standard dilution buffer were the same as above.The half maximal inhibitoryconcentration(IC50)was 4.39 ±0.146 μg/L and the detection limit(IC 15)was 0.31 ± 0.007μg/L.The developed AuNPs-indirect-competitive ELISA was performed as follows:the optimal amount of coating antigen was 0.01 μg/well,the antibody was diluted 16000-fold,the AuNPs-HRP-goat anti-mouse IgA was diluted 10000-fold,0.5%skim milk powder and pH 7.4 0.01 mol/L phosphate buffered saline(PBS)were used as blocking solution and standard dilution buffer,respectively.The half maximal inhibitory concentration(IC50)was 0.93 ± 0.579 μg/L and the detection limit(IC15)was 0.078 ± 0.013 μg/L.Compared with the traditional indirect competitive ELISA,AuNPs-indirect competition ELISA is more sensitive,the sensitivity increased by about 10 times.The monoclonal antibody secreted by this cell line does not cross-react with other mycotoxins.The content of FBi in maize was determined by the established AuNPs-indirect competitive ELISA.The recovery ratio was 90.83%-95.83%and the coefficient of variation was 2.40%-11.42%.The method was confirmed by High Performance Liquid Chromatography Tandem Mass Spectrometer(HPLC-MS/MS).The linear correlation coefficient R2 between the two methods was 0.9988,and they were in good correlation.