Selection Of Hybridoma Cell Lines And Immunoassay For T-2Toxin

With the frequent occurrings of accident related with food safety, there has been much attention tofood safety issues caused by mycotoxins contamination for the last few years. Trichothecenes arechemically related mycotoxins that are produced by various fungal species, mainly of the genusFusarium and are divided into four subgroups (designated types A, B, C, D) based on their chemicalstructure. T-2toxin may contaminate a variety of cereal grains, such as wheat, maize, oats, barley andrice, especially in cold climate regions or during wet storage conditions. T-2toxin which are toxic toanimals and humans primarily because of their ability to inhibit DNA, RNA and protein synthesis andalso show immunosuppressive and cytotoxic effects both in vivo and in vitro is among the most potent(type A) trichothecenes. Therefore, mycotoxins are required to be detected in foods and feeds to keepgood foods quality and to reduce their negative impact on human and animal health.Analysis for mycotoxins in foods is a very important practice to ensure food quality and safety, andto eliminate or control the risk of consuming contaminated foods. Hence, many analytical methods forthe detection of these mycotoxins in food and feed have been described. However, most of them aretime-consuming and require sophisticated equipments. There is a need for rapid and inexpensive assaysfor screening mycotoxins in food and animal feed samples. Immunoassays based on monoclonalantibody were developed for on-site T-2detecting. No expensive instruments are needed as results arevisually evaluated. The main research contents are described as follows:1. A high-quality artificial antigen of T-2toxin was prepared. Rational design of the antigen is thebasis and premise to obtain the high-specific antibody. The methods proposed by Chu and Katsumi wereadopted for the preparation of T-2-protein conjugates: T-2toxin in pyridine reacted with glutaricanhydride in an oil bath and then was converted to T-2-HG. At the presence of EDC, DMF, NHS,T-2-HG was conjugated with BSA/OVA, and the result of ultraviolet scanning demonstrated that theT-2-protein conjugates was successfully synthesized. Preparation of T-2toxin antigen laid a foundationfor the next steps.2. A highly-sensitive anti-T-2toxin MAb with high specificity was prepared. High-quality antibodyis the most important key factor for the successful establishment of any immunological method. In orderto prepare high-quality antibodies anti-T-2toxin, a modified semisolid medium and a novel two-stepscreening method were adopted to select and breed the most excellent monoclonal hybridoma cell lines.By using the novel two-step screening method,5antibody secreting cell lines were obtained, which are4F12,2B5,2G7,1D7and4F3. Among them,2G7was most perfect with stable antidody production andhigh titer and sensitivity of the secreted antibody. The sensitivity (expressed by IC50) of2G7to T-2toxin was0.83ng/mL. The cross-reactivities of the antibody2G7to mycotoxin analogues, such as AFB1,DON, ZEN, OTA et al, were all less than1%. The antibody2G7showed high quality of sensitivity andspecificity.