Preparation Of Monoclonal Antibody Against Zearalenone And Development Of ELISA Method For Zearalenone

Zearalenone is a kind of mycotoxin which is produced by Fusarium graminearum, Fusarium culmorum and other kinds of Fusarium. It mainly causes the pollution of corn, wheat, barley and other cereals, and functions as female hormone. It may cause estrogen excess syndrome of pigs, rats, mice, poultry and many other animals, and also can cause estrogen excess syndrome of human. Besides, it has the immunity toxicity, heredity toxicity and suspicious carcinogenicity and so on. In order to prevent the harm to the human and animals, there are at least 19 countries in the world which have drawn up the tolerance limit of ZEN in food by 2003, and then EU also set up the tolerance limit of ZEN in food in 2005. National standard detection method of ZEN in food has not been established in our country at present. In order to protect the economic interest of our country in the international grain trade and the health of consumers, it is urgently necessary to establish the national standard detection method of ZEN, and draw up the tolerance limit of ZEN in food. The present research aims to laid the groundwork of development of chemiluminescence enzyme immunity analysis kit for ZEN detection.ZEN was conjugated to HAS and BSA by the active lipid method respectively, and ZEN-HSA and ZEN-BSA was obtained and identified by the thin layer chromatography. The BALB/c mice were immunited with ZEN-HSA. Spleen cells from the immuned mouse were fused with cells Sp2/0 by routine method. The positive cell lines that secret anti-ZEN McAb were selected by ELISA. After 4 times clones, 5 hybridoma cell lines named 3F1, 2D1, 1D4, 5B4 and 5H10 were obtained. The McAb secreted by 3F1 and 2D1 was identified as IgG1, 5B4 as IgG2a, 1D4 as IgG2b and 5H10 as IgM respectively. The titer of 3F1 and 2D1 was 1:1.28×106 and 1:6.4×105 respectively. There were no cross reactions between the anti-ZEN McAbs and the other mycotoxins and the carrier proteins. Affinity constants of 3F1 and 2D1 were 5.6×108 L/mol and 3.7×108 L/mol. 3F1 was selected to establish the indirect compete ELISA.The limit of detection concentration of ZEN was 2 ng/mL, and the linear range of standard curve was 2~200 ng/mL, and the linear equation was y=-0.3397x+0.8827 (R2=0.9929).