Study On Immunoassays For The Detection Of Zearalenone Based On Monoclonal Antibody

In this paper, a monoclonal antibody specifically recognised zearaleone was prepared.Enzyme-linked immuno sorbent assay and immunochromatographic strip analysis method based on up-conversion nanoparticle (UCNPs) were developed for the detection of zearaleone residue in corn and oat.Balb/c mouse was Immuned with immunogen of ZEN-KLH, which was prepared according to carbodiimide method. Three hybridoma cells named 2E8D3E4D6?2C7G9F3D5?6E11F9E6C4 were made after cell fusion and cell screening. Isotyping of the McAb was performed with use of an isotyping kit, where the isotype was identified as IgG1 along with kappa light chain.After optimized, the developed Indirect-competitive ELISA(ic-ELISA)was performed as follows: the optimal amount of coating antigen was 0.01 ?g/well,the antibody was diluted 40000-fold, 0.5% skim milk powder and pH 7.4 0.01 M PBS were used as blocking solution and standard dilution buffer, respectively. The half maximal inhibitory concentration (IC50) was 0.13±0.02 pg/L and the detection limit (IC15) was 0.02±0.01 ?g/L.The developed direct-competitive ELISA (dc-ELISA) was performed as follows: the optimal amount of antibody was 0.05 ?g/well, antigen-HRP was diluted 20000-fold,blocking solution and standard dilution buffer were the same as above. The half maximal inhibitory concentration (IC50) was 0.35±0.02 ?g/L and the detection limit (IC15) was 0.09±0.01 ?g/L. An ic-ELISA was developed for the detection of zearalenone in corn and oat samples. The detection limit (IC15) was 2.4 ?g/kg and 4.0 ?g/kg for corn and oat,respectively. The recovery rate was 82.8% to 109.2% and the coefficient variation was 3.3% to 15.4%. The method was confirmed by liquid chromatography tandem mass spectrometer and they were in good correlation.UCNPs with good fluorescent property and uniform morphology were synthesized by the solvothermal technology and the ligand exchange was then successfully modified.Immunochromatographic strip analysis method based UCNPs was developed for the detection of zearaleone residue in corn and oat. The optimized conditions were as follows:the connection ratio between antibody and UCNPs by activated ester was 0.3 to 5 mg, the dilution of the second antibody and the coating antigen were all 20-fold, the adding amount of ab-UCNPs conjugate was 5 ?L, the adding amount of working liquid was 20?L with the phosphate buffer. The detection limit was 2 ?g/L in standard buffer and 8,16 ?g/kg in corn and oat samples.The method was simple operation with high sensitivity, which can be used for the detection of zearalenone in corn and oat samples.