Enzyme-linked Immunoassay For The Determination Of Deoxynivalenol In Agricultural Products

A direct competitive enzyme-linked immunosorbent assay (cdELISA) for the determination of deoxynivalenol (DON) in agricultural products was developed in this paper.Firstly, the coupling agent, succinic anhydride was used for the synthesis of the hapten3-HS-DON. The immunogen was prepared following a scheme that employs active ester method for the conjugation of the haptens to BSA(Bovine Serum Albumin), and the EDC assay was used for the conjugation of the hapten to OVA(Albumin from Chicken Egg White) and HRP(Horseridish Peroxidase). Polyclonal antibodies against deoxynivalenol were raised in rabbits after immunization with immunogen.Secondly, On the basis of studying the effects of different chemical conditions, the competitive direct ELISA standard curve of DON was established. The sensitivity and limit of detections were0.03mg/kg and0.003mg/kg. The DON antibody specificity was checked on the basis of the50%inhibition levels, relative cross-reactivities with3-AC-DON was found to be500%, with15-AC-DON, T-2, OTA and ZEN were found to be very low.Thirdly, Barley, wheat, oat, maize, rice, feed, flour, beef and milk were selected as samples which were proved as DON negative. To reduce matrix interferences, the extractions with doubly deionized water from the samples were centrifuged and then diluted by PBS in some fold. The recoveries range of DON was70%～100%at three different levels. For the validation of the ELISA tests, samples were analysed by HPLC. And good correlations(R2=0.9613) was obtained in the correlation studies between ELISA and HPLC.In the practical application of the method, the sample involved in cereal, feed, milk and livestock products. A simple and efficient pre-treatment method was established. The maximal limit of detection in samples was0.48mg/kg, which is lower than the national standards MRL:1mg/kg. This method can meet the need for rapid detection of DON in a variety of farm products.