| Fumonisins(FB) are a group of structurally related mycotoxins produced by a number of Fusarium species. The most prevalent of these mycotoxins in contaminated corn is FB1, which is believed to be the most toxic. FB1 has been detected in maize and maize-based products all over the world. FB1 is known to be the cause of equine leukoencephalomalacia(ELEM) and porcine pulmonary edema(PPE) syndrome. Available epidemiological evidence has suggested a link between dietary fumonisinB1 exposure and human oesophageal cancer in some locations with high disease rates.Several analytical methods have been used for the analysis of FB1 in food are sensitive and specific, but need too much time and money. Enzyme-linked immunosorbant assay (ELISA) for detection of FB1 which is based on monoclonal work. There are ELISA kits for FB1 abroad which are very expensive. To develop hybridoma cell lines excreting monoclonal antibodyies against FB1 and develop rapid detection kit that possessing patent of our country are important.To establish the ELISA method for detecting FB1, this research synthesized complete antigen, developed high-grade antibodies using the technology of monoclonal antibody. The addition and reclamation test of corn samples proved that the established CiELISA method for detecting FB1 in corn was stable and reliable.1. The synthesis and identification of completely antigen. Method for the synthesis of FB1 immunoconjugates and for the preparation of conjugated with protein forming complete antigen. Keyhole limpet hemocyanin-fumonisinB1 (KLH-FB1) was synthesized using both the classical glutaraldehyde(GA) protocol and 2-step GA protocol. UV scanning method to identify the effect of synthesis and the results show:after the process of composition improved, UV-scanning identified the conjugates.2. The preparation of the Monoclonal Antibodies against FB1. FB1-KLH was used to immunize BALB/c mouse, the titer of blood serum collected after the fifth immunization reached 1:8000. Through the monoclonal antibody technology and modern cell fusion technique, one monoclonal hybridoma cell line called 4E10 was obtained. Stability experiments showed that this cell line could secrete monoclonal antibody against FB1 stably.3. The foundation of ELISA method. Through the foundation of indirect competition ELISA method, the best work concentration of ascites was got, which is 1:20000. The titer of ascites reached 1:106. FB1 standard curve was prepared by 4E10 ascites, with the linear equation:y=0.8065x+0.2533(R2=0.9659), IC50 0.5μg/mL, LOD 0.051μg/mL, LOQ 5.39μg/mL. The cross reaction experiment of congeneric mycotoxin showed that the FB1 monoclonal antibody has good specificity, with less than 0.5 percent inhibition rate anti other mycotoxins.4. The addition and reclamation test of FB1 in the corn. To cut down the interference of extract and raise extraction rate, the method of extracting FB1 from corn was optimized, intervention disappeared when the extrace were diluted eight times. In the range from 0.05 to 4μg/mL, the linear equation was y=1.089x+0.2268 (R2=0.9351). The recovery ratio were between 80.2%-93.4% when adding 50,100 and 500μg/kg FB1 to corn. The intra assay coefficient of variation were between 2.43%-5.71%, and the interassay coefficient of variation were between 4.31%-9.52%, the reproducibility was fine. |
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