Preparation And Characterization Of Monoclonal Antibodies Against T-2 Toxin

T-2 toxin is the metabolite of fusarium species,and it is the most potent toxin of Trichothecenes,commonly found in crops and their food products.T-2 intoxication in human and other animals leads to acute poisoning and causes varied physiological and clinical disorders,and the symptoms include vomiting,diarrhea,fever,severe damage to the hematopoietic system and death.In 1973,the joint meeting of the FAO and WHO held in Geneva declared T-2 toxin as an important food contaminant of natural food crops.Therefore,the qualitative and quantitative analysis of T-2 toxin are critically important.T-2 is a small hapten and cannot induce immunogenic response in the target specimen.Therefore,the first step is the production of complete antigen by using the EDC conjugation method.In this method,EDC linked small T-2 hapten to the carrier proteins such as BSA and OVA.T-2-HS-BSA was used as an immune antigen in Balb/c mice,while T-2-HS-OVA was used as a detection antigen.After five times i.p.immunization of Balb/c mice,the highest titer reached 1:32000.An ELISA was used to detect the anti-T-2 serum antibody to determine the optimal working conditions.The optimized concentration of coating antigen of T-2-HS-OVA was 1.25 ?g/mL,and the dilution of HRP-IgG was 1:4000.Cell fusion between Sp2/0 myeloma cells and murine spleen cells was carried out by using 50%PEG-1450.Three anti-T-2 antibody hybridoma cell lines were obtained after vigorous screening,named 4B1(IgG2b),7C3(IgG2b),7A3(IgG2b).The chromosomes count assay showed hybridoma cells had 102 to 105 number of chromosomes each cell.4B1(IgG2b)was used for the production of ascites in Balb/c mice to obtain higher anti-T-2 titers.The higher specific affinity of purified antibody from ascites showed that affinity constants Kaff was 4.5×108 L/mol.The titer assay by ELISA showed that the titer was 1:8000 for the ascites and 1:4000 for McAb respectively.The McAb showed no cross-reactivity with other related mycotoxins.The high affinity McAb against T-2 toxin prepared in this study lay the foundation for assembling ELISA kits,and this McAb can be applied for colloid gold detecting kit for T-2 toxin.