Synthesis Of Complete Hapten And The Preparation Of Polyclonal Against Patulin

Patulin is a mycotoxin and produced by a number of molds such as Penicillium, Aspergillus and Byssochlamys. Patulin has the potential carcinogenicity, pregnant dams and teratogenicity. So, how to assay patulin rapidly and efficiently in fruit juice has being one of the most interesting researching area at home and abroad. There are many methods of detecting the toxin such as classical colorimetric method, the TLC method, the GC-MS method, the HPLC method and so on. As immunoassay mothed is cheap, rapid and easy, it has been the focus of patulin detection. In this experiment, we modificated the structure of patulin to produce complete hapten and developed the polyclonal antibody. Meanwhile, we also appraised the antibody. The research establishes the basis of imnumoassay of patulin.To increase the connective efficiency of patulin and carrier protein, PAT was modified by succinic anhydride, and identified by GC-MS. Meanwhile carrier protein bovine serum album(BSA) were modified by ethylene diamine(EDA) and ovalbumin(OVA) to prepare the immune antigen PAT-HS-EDA-BSA and the detection antigen PAT-HS-OVA by l-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC) method respectively.Mice were immunized with PAT-HS-EDA-BSA every other 14d. The day after immunized 3 times, the titer was tested by indirect ELISA. When the titer was high enough, rats were killed and the serum was collected to separate antiserum. The anti-serum was purified by caprylic acid-ammonium sufate precipitation. The titer of antiserum and the optimal Ag coating concentration were confirmed by indirect ELISA. The titer of antiserum and the optimal Ag coating concentration and were 1 : 40000 and 0.5 ug/mL respectively. The specificity of antiserum was confirmed by indirect ELISA.The results showed that the conjugated antigens were successfully obtained and could be used to prepare the polyclonal antibody against PAT with immune antigen PAT-HS-EDA-BSA and detection antigen PAT-HS-OVA.Analysised by indirect ELISA, Anti-PAT polyclonal antibodies showed good sensitivity. This research formed a foundation of production monoclonal antibody against patulin and immunoassay of patulin.