| Background: The mycotoxin zearalenone (ZEN) is a fungal metabolite, mainly produced by F.graminearum and F. culmorum, which are known to colonize maize, 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. Occurrence of the Fusarium mycotoxin zearalenone is widespread in the world, and it is an important mycotoxin in both temperate and warm regions of the world. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) and EU member states have studied ZEN thoroughly in 2000 and 2002, respectively. They have surveyed ZEN contamination levels in different foods and countries, and concluded the nature of the toxicological of ZEN, and provided the maximum tolerate level (tolerable daily intake, TDI), and assessed dietary intake of ZEN by the population in various country. Now nine countries have set maximum tolerable levels for ZEN, but the levels and the methods of determination are different in different countries and the lack of certified reference materials (CRMs) makes the results between different laboratories can not be compared. Thus the trade obstacles between the countries can easily affect cereals trade. ZEN mainly contaminates cereals that are the major food in our country, and China is the large country of importing and exporting cereals, so ZEN brings potential harm to the health of consumers. But now our country has not established the standard testing method for ZEN in cereals and not surveyed the ZEN contamination levels in cereals and not studied the dietary intake of ZEN by the population of China, thus we cannot assess the harm of ZEN to population of China. Cereals contaminated with ZEN may go into our country because we have not set the maximum tolerated levels of ZEN in food and the cereals can be harmful to the health of consumers , and it may has an adverse effect on our county's cereals trade. So in order to protect both the health of the population and the economic benefit, it is urgent to survey the contamination levels of ZEN in cereals. Purpose: 1 To establish quick, sensitive, specific, and relatively inexpensive enzyme-linked immunosorbent method (ELISA) and developing ELISA-kit, as well as applied for as standard testing method. 2 To compare and analyze the nine counties'guidelines ( maximum tolerable levels) for ZEN in food.3 To develop reversed-phase HPLC method. Methods: 1 Two hybridoma cell lines excreting monoclonal antibodies against Zearalenone, were obtained by hybridoma technology. The ascites containing monoclonal antibodies against ZEN was gained via inoculation of the hybridoma cell into abdominal cavity of BALB/c mice. Character of the antibodies were identified by ELISA. 2 ELISA-kit for ZEN were developed with the monoclonal antibodies and the parameters of the kit were studied. 3 Standards about ZEN were analysed and compared. 4 Corn and wheat samples were extracted with chloroform and the extract was filtrated with filter and applied to HPLC with fluorometric detection(λex=280nm, λem=460nm) using methanol-water(80:20) as mobile phase. So a quantitative reversed-phase HPLC method determing ZEN in corn and wheat was developed. Results: 1 Two hybridoma cell lines excreting against Zearalenone, coded Z2A4 and Z8D6, respectively, were obtained. 2 The titer of antibodies in ascites was 1: 1.6×106 and 1:3.2×106, the MW was 150kD and the working concentration was 1:3.2×105 and 1:6.4×105. IgG concentration in purified ascites yielded by hybridoma cell with the code number of Z2A4 and Z8D6 reached 34mg/mL and 39 mg/mL, respectively, and there is no cross reactions between the monoclonal antibodies against ZEN and the other mycotoxins. Affinityconstant of Z2A4 and Z8D6 was 5.52×108L/mol and 4.68×109L/mol. 3 ELISA-kit for ZEN... |
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