Development Of T-2 Toxin Immunological Rapid Detection Method

T-2 toxin is a kind of strong toxicity of mycotoxin,which poison people and animals by pollution crops and feed.At present,the instrument analysis is the most important method to test T-2 toxin.The instrument detection methods is sensitivity,but it is not suitable for on-site rapid detection because of the high technical requirements and it also limited its use scope.The immune analysis technology has some obvious advantages,such as quick speed,simple operation and so on,it has been widely used in the field of veterinary drug residues,pesticide residues and prohibited additives This study is proposed by T-2 toxin antigen structure analysis,preparation of artificial antigen;and by animal immunization,cell fusion and positive hybridoma screening to prepare monoclonal antibodies against T-2 toxin;and on the basis of this preparation was developed for rapid detection of T-2 toxin ELISA kit.The main contents and results of this study are as follows:1.Synthesis and identification of artificial antigen of T-2 toxinBased on the analysis of the chemical structure of T-2 toxin,the suitable carrier protein bovine serum albumin（BSA）and chicken ovalbumin（OVA）were selected according to the hydroxyl group of T-2 toxin.Then Artificial antigen T-2-BSA and T-2-OVA were synthesized by using N,N-carbonyl diimidazole method（CDI）.The results of UV and SDS-PAGE electrophoresis showed that the coupling protein absorption peak and carrier protein absorption peak did not overlap slightly offset,and the migration rate of the coupling protein is less than the carrier protein which means the artificial antigen were synthesized successfully.The results of animal immunization showed that the antiserum titer of immuned mice was more than 1:1× 104 and the half inhibitory concentration reached 8.32 ng/m L.The results showed that the prepared artificial antigen can not only has good immunogenicity but also stimulate the animal to create favourable specific antibody against T-2 toxin.2.Preparation and identification of monoclonal antibodies against T-2 toxinThe mice with high titer and high titer of T-2-BSA were selected as the fusion objects.we immuned mice with this antigen.Through cell fusion technology the mice splenocyte were fused with SP2/0 myeloma cells.The stable hybridoma cell lines were ultimately selected which By using ELISA screening system.After further expansion of positive hybridoma cells,hybridoma cells with high titer and high sensitivity were selected to be subcloned.Finally onehybridoma cells which could produce stable hybridoma cell lines of monoclonal antibodies,named 4F7,the cell supernatant titer wasabove 1:1× 10³.prepared ascites by inducing ascites in vivo and the ascites titer was as high as 1:6.4×105,the half inhibition of T-2 toxin(IC₅₀)could reach 2.28 ng/m L,the subtype of monoclonal antibody was IgG1 and the affinity constant of Ka is 1.49 × 1011 L/mol,the cross reaction rate of 4F7 with other mycotoxins was less than 0.01%.3.Preparation of T-2 toxin indirect competitive ELISA rapid detection kitBy using the checkerboard,we determine the concentration of coated antigen and the antibody concentration though indirect competitive ELISA;Through optimization of reaction time,we established and prepare the T-2 toxin indirect competitive ELISA rapid detection kit.based on the indirect competitive ELISA detection method When dilution the concentration of the coatingen to 1:4000 and the antibody to 1:32000,the standard curve of the T-2-kit was y=-0.2742x+0.5939,R²=0.9911,according to the equation figured out the IC₅₀ is 2.19 ng/mL and the sensitivity can reached 0.129 ng/m L,the recovery rate was between 83.8%⁹4.3%,the coefficient of variation was less than 15% and the cross reaction rate was less than 0.01%,and there was no quality problem in the storage of 4 °C for 180 d.