Preparation And Application Of Specific Antibodies Against T-2 Toxin And Aflatoxin

T-2 toxin and aflatoxin both are the toxic secondary metabolites of fungi, which distributed in nature widely and become a major pollutants of agricultural commodities, such as peanuts, corns, soybeans and rice, etc, yield and quality of agricultural products be seriously affected. They are not only able to cause immune toxicity and cytotoxicity to humans and livestock, but also a strong carcinogenic effect. Aflatoxin is considered to be one of the strongest carcinogen ever discovered. Many countries in the world have set up strict regulations towards mycotoxins in agro-products, and take it as one of the means of trade barriers, due to its harmful to humans and animals. With the continuous improvement of living standards, People give more attention to food safety issues, food safety issues related to social stability and harmony. How to break barriers to international trade, reduce economic losses, provide people safe and secure food becomes a serious problem. The study and the establishment of mycotoxins immunoassay technology is an important means to solve this problem.Antibody is the core of immunoassay technique, the specificity and sensitivity of antibody directly determines the quality of the immunoassay technology. Conventional immunoassay techniques are mostly based on monoclonal antibodies and polyclonal antibodies. Hybridoma cells can be survival in vitro and passaged. As long as the mutations do not occur, the cell line will be produce high specificity and high uniformity antibody continuously. However, the sensitivity and specificity of the current T-2 monoclonal antibodies were not good enough to meet the detection needs, so the development of the higher sensitivity of T-2 monoclonal antibody is imperative.In recent years, nanobodies gradually into the spotlight, it resistant to organic solvents, it was expected for its high temperature, acid and alkali resistance and many other advantages. Detection technology which based on nanobodies have emerged, and creating a new research field. With increasing demand for nanobodies, how to achieve mass production of nanobody will become a vital part of the nanobody technological development.To solve above urgent problems, main research contents and novelties of this work are as follows:T-2 toxin was reacted with succinic anhydride to prepare T-2-hemisuccinate(T-2HS). Then, T-2HS was conjugated to bovine serum bovine serum albumin(BSA) and ovalbumin(OVA) to obtain the conjugates T-2HS-BSA and T-2HS-OVA. As immunogen, T-2HS-BSA was intradermally injected in Balb/c mince. Then, spleen cells and myeloma cells were fused. A modified semisolid medium was adopted to culture cells. Monoclonal hybridoma cell lines were selected by a novel two-step screening method, which could secrete anti-T-2 toxin antibody. Indirect competitive ELISA was used to evaluate the specificity of the obtained antibody. Four positive hybridoma cell lines were obtained, named 1D6, 2A8, 2C5 and 4G3. The antibody from 2C5 has the best sensitivity(expressed by IC50), which was 0.13 ng/mL. This antibody was specific against T-2, and the cross reactivity with HT-2 was 4%. It had no cross reactivity with mycotoxins FB1, DON and ZEN. Comparing with related antibodies reported, 2C5 is the best monoclonal antibody on sensitivity and specificity.An indirect competitive ELISA was developed for T-2 toxin detection, and the limit of detection was 0.015 ng.mL-1.The detection range(IC20-IC80) of this method was 0.05-57.6 ng.mL-1.The recoveries of spiked T-2 toxin, in 0.05-57.6 ng.mL-1,were 93.5%-107.5%.The developed method can be used for T-2 toxin detection in many grain and oil products, such as soybean, corn and peanut.Nanobody for aflatoxin B1 was achieved by train, extraction, purification and lyophilization. Express condition of nanobody basis on the strains which have been obtained in our laboratory. The expression conditions of nanobody, such as induction temperature, induction time and inducer concentration, were optimized. The optimal induction temperature was 33℃, the optimal induction time wsa 10 h,and the best inducer concentration was 1mmol/L. Nanobody was achieved mass production with fermenter. The antibody has a high sensitivity(expressed by IC50), which was 0.75 ng.mL-1,and showed good specificity. The main technical parameters of nanobody remained the same to miniprepped nanobody, while the production of antibodies has been greatly improved.A new type of immunoaffinity column was developed. The column capacity of this immunoaffinity column was 200 ng. Reuse experiments show that nanobody immunoaffinity column with good resistance to organic solvents, and this feature does not affect the elution of the target substance.