| The mycotoxin zearalenone (ZEN) is a fungal metabolite, mainly produced by F.graminearum and F.culmorum, which are known to colonize corn, barley, wheat, oats and sorghum. ZEN show marked estrogenic properties in rats, mice, poultry and in swine. ZEN and its related compounds can cause hyperoestrogenism and reproductive and infertility problems in animals, especially in swine. It also has genotoxicity, immunoxicity and carcinogenicity to experimental animals. ZEN has estrogenic effect and possible carcinogenicity on human. In recent years, corn is frequently made into animal feeds in our country, mould development of corn often leads to active chronic toxicosis. So it is very important for ensuring food safety to inhance the detection of ZEN.At present, there are several methods to detect the content of ZEN, including Thin Layer Chromatography, High Performance Liquid Chromatography and immunological method. Thin Layer Chromatography is insensitive, inspecific, time-consuming, and easily interfered by fluorescent material. High Performance Liquid Chromatography is sensitive and accurate, howerever, the equipment is very expensive and the pre-processing is complicated. Compared with the two methods introduced before, Enzyme-linked immunosorbent assay (ELISA) can be used to screen a large number of samples, meanwhile, it is a sensitive, special and rapid method.To establish the ELISA method for detecting ZEN, 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 ZEN in corn was stable and reliable.1. The synthesis and identification of completely antigen. O-(Carboxymethyl) hydroxylamine hemihydrochloride and ZEN in pyridine reacted and generated ZEN oximation, then the latter conjugated with protein forming complete antigen. In the course of reaction, TLC method was used to monitor the convertion of ZEN in real time. In addition, mass spectrometry method was used to test the synthesis ZENO structure , UV scanning methd to identify the effect of synthesis, and the ratio of ZEN to protein was calculated on the basis of the result of UV scanning. The results show: After the process of composition improved, the oximation ratio of ZEN increased a lot and reached 79.3% respectively. UV-scanning identified the conjugates. The ratio of haptens to carrier protein is 12:1 and 21:1 for ZEN-BSA and ZEN-OVA.2. The preparation of the Monoclonal Antibodies against Zearalenone. ZEN-BSA was used to immunize BALB/c mouse, the titer of blood serum collected after the fifth immunization reached 1:10,000. Through the monoclonal antibody technology and modern cell fusion technique, one monoclonal hybridoma cell line called 3F11 was obtained. Stability experiments showed that this cell line could secrete monoclonal antibody against ZEN 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 1:32,000. ZEN standard curve was prepared by 3F11 ascites, with the linear equation: y =0.2989x-0.0292(R2=0.9898), LOD 2.16 ng/ml, LOQ 22.37 ng/ml. The cross reaction experiment of congeneric mycotoxin showed that the ZEN monoclonal antibody has good specificity, with less than 0.5 percent inhibition rate anti other mycotoxins.4. The addition and reclamation test of ZEN in the corn. To cut down the interference of extract and raise extraction rate, the method of extracting ZEN from corn was optimized, intervention disappeared when the extrace were diluted eight times. In the range from 1 to 500 ng/ml, the linear equation was y =0.3076x-0.05(R2=0.9931)and LOD was 1.9 ng/ml. The recovery ratio were between 72.5%～92.9% when adding 50,100 and 500μg/kg ZEN to corn. The intraassay coefficient of variation were between 2.46%～5.73%, and the interassay coefficient of variation were between 4.37%～9.54%. The reproducibility was fine.
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