Immunological Methods Of T-2Toxin And Bisdesoxycydox

T-2toxin is one kind of trichothecene mycotoxins produced by various Fusarium molds with strong toxicity. It is widely distributed in nature, and main toxin pollutants of field crops and inventory grains. Due to its high toxicity, high food pollution rate, and the great harm to human health and animal husbandry, an efficient, rapid and sensitive detection method to monitor T-2toxin residues is very necessary. Although caydox has been used as a safe and efficient feed additive, considering its role through the food chain on human body, it may produce negative effects, so to establish a detection method of caydox residues in edible animal tissues is needed. As the residual marker of caydox, bisdesoxycyadox was studied in this research to explore veterinary drug residues in animal tissues. This paper aims to establish two kind of immunological analysis: immuno-chip assay for T-2toxin and enzyme-linked chemiluminescence assay (CL-ELISA) for bisdesoxycyadox.①After the synthesis of T-2-HS-BSA, we used spotting capillaries to print it onto agarose-modified glass slides, and established an indirect competitive immuno-chip assay to detect the T-2toxin residues in food crops, such as wheat. First, the optimization of various parameters (which influenced the reaction) were carried on, such as the spotting concentration, Mab working concentration, blocking materials and concentration, Cy5-IgG concentration. Then a standard curve was established, the detection range (IC2o-IC80) was0.005ng/mL-9.58ng/mL,50%inhibition concentration (IC50) was0.22ng/mL, and the limit of detection (LOD) was0.001ng/mL. Compared with the standard curve of ELISA, the detection range was0.27ng/mL-11.82ng/mL, IC50was1.77ng/mL, and LOD was0.14ng/mL, the immuno-chip assay showed higher sensitivity and wider detection range. The spiking experiment was detected by ELISA, immuno-chip and HPLC, the recovery rate were almost meet the requirements of analysis detection. The whole experiment process is simple, rapid, high sensitivity, high-throughput with small amounts of samples, suitable for toxicants residue analysis in food crops. ②By adopting luminol-H2O2system and using HRP-IgG, we established an indirect competitive enzyme-linked chemiluminescence immunoassay (CL-ELISA) to detect the bisdesoxycyadox (Cy4) residues in chicken chest muscle and liver tissues. First, we use Cy4polyclonal antibody to explore CL-ELISA method and established a standard curve, the detection range was0.017～10.34μg/mL, IC50was0.42μg/mL, and LOD was5.82ng/mL. Then we use Cy4monoclonal antibody and elaborately optimized various parameters (which influenced CL-ELISA reaction), such as the coating buffer constitutes and coating concentration, antibody working concentration, blocking materials, blocking concentration and blocking time, DMSO concentration in standard dilution buffer, competitve reaction time, HRP-IgG dilution ratio, and the reaction time between enzyme and substrate. Then we established a standard curve, the detection range was0.027ng/mL-67.67ng/mL, IC50was1.36ng/mL, and LOD (IC10) was0.007ng/mL. The result of cross-reaction with its structural analogs and metabolites indicated good specificity, and the recovery rate with variation coefficient were meet the requirements of analysis detection. In an actual residue study, the result obtained by CL-ELISA correlated well with those obtained by HPLC, and the residue levels of Cy4in treated chicken decreased with time and dropped rapidly. The whole experiment process is simple, rapid, high sensitivity with good specificity and wide detection range, suitable for veterinary drug residue analysis.