Investigation Of Immunoassays For T-2Toxin In Cereals And Feed

T-2toxin is a kind of compound of trichothecenes produced by Fusarium. T-2toxin is toxic, teratogenic, and carcinogenic. T-2toxin exists widely in nature, mainly contaminating rice, wheat, maize, other cereals and their products. In order to ensure safe tongue, many countries have established national standards for cereals and their products. Detection technologies can ensure the implementation of the standards. The main detection methods for mycotoxins are immune assay and instrument analysis. In this paper, two kinds of immune assays and one kind of instrument analysis for T-2toxin were established by using a highly sensitive monoclonal antibodies. The main contents include the following:1. Indirect competitive ELISA method for T-2toxin detection was established by using a highly sensitive monoclonal antibody which was produced in our lab. By optimizing experimental conditions including the concentration of coating antigen, antibody dilution, the reaction system pH, salt concentration, the closure, the methanol content, three kinds of standard curve were established. The sensitivity of the method for rice was2.79ng/mL, LOD was0.16ng/mL, the linear range was0.43-63.15ng/mL. The sensitivity of the method for corn was3.21ng/mL, LOD was0.18ng/mL, the linear range was0.46-66.82ng/mL. The sensitivity of the method for feed was5.23ng/mL, LOD was0.21ng/mL, the linear range was0.61-71.34ng/mL. The recoveries of the method were94.8%～112.3%. The results of actual samples detection showed that the coefficient of variation between2.8%～13.3%, indicating good reproducibility of this method.2. Time-resolved fluorescence immunochromatographic strips for T-2toxin were developed in this paper. A kind of fluorescent probe was prepared by using europium-marked polystyrene microspheres which was conjugated with monoclonal antibody. Europium has a good time-resolved fluorescence property. By optimizing the amount of EDC, the amount of antibody, nitrocellulose membrane, the blocking solution for sample pad, sustained release liquid, the amounts of antigen and antibody on the strip,the dilution for fluorescent probe and other factors, time resolved fluorescence immunochromatographic strips were prepared. According to different matrixs, standard curves of time-resolved fluorescence immunoassay assay for rice, corn, feed were established. The linear range of this method for rice and corn was0.125～200ng/mL, The correlation coefficient was above0.99, LOD was0.09ng/mL. The linear range of this method for feed was0.25～200ng/mL, The correlation coefficient was above0.98, LOD was0.17ng/mL. The recoveries of the method were between94.2%～111.0%. The results of actual samples detection showed that the coefficient of variation between2.31%～14.84%, indicating good reproducibility and stability of the method. The detection process can be completed within15minutes and the portable time-resolved fluorescence tester can show the test results directly. The method is simple, rapid, and can be used for on-site inspection.3. Immunoaffinity column for T-2toxin was prepared and immunoaffinity column clean-LC-MS/MS method was established for detecting T-2toxin in cereals and feed. The LOD of this method was0.05ng/mL, LOQ was0.17ng/mL, the linear range ranged from0.5ng/mL to500.0ng/mL, correlation coefficient was above0.9997, and the recoveries of the method were92.9%～109.7%.