| Patulin (PAT) is a toxic secondary metabolite produced by some fungal species. It hasbeen isolated from a variety of foods especially from apples and apple products. Patulin maycause various health risks, including acute and chronic toxicity, immune toxicity, mutation,teratogenic and carcinogenic effects. In order to ensure safety and quality of foods,establishing rapid and sensitive methods for detection of PAT has always been concerned. Thecurrent methods for the detection of PAT are mainly chromatographic analytical methods suchas TLC, HPLC and GC. These methods can examine PAT qualitatively and quantitatively,however, expensive analytical instrumentation is necessary to accomplish it and extensiveprotocols of sample cleanup are required prior to the analysis. Therefore, more rapid andeconomical methods for the determination of PAT in foods are needed. An immunochemicalanalytical method, based on highly specific antigen-antibody interactions, would be desirable,offering several advantages compared to conventional techniques, i.e., low cost per sample,high selectivity, high throughput and it is particularly suitable for large quantities of sampletest. Thus it has been applied widely in detecting mycotoxin. At present, there are few reportsabout detecting patulin with immunochemical analytical method. This is mainly due to theelectrophilic properties of patulin and its reactivity toward thiol groups and amino groupsexisted in the blood proteins. Consequently it is not stable in vivo and can not produce orproduce inefficient antibodies.In the present study L-Arabinose was used as the precursor, after five steps chemicalreaction, a new patulin derivative, named P2, was synthetized. It still maintains the originalskeleton of the natural toxin, but exhibits a higher chemical stability. The structure of thederivatives was determined by HNMR. Then P2was activated through active ester method,and conjugated to ovalbumin (OVA) and bovine serum albumin (BSA) respectively, and usedas detecting antigen P2-OVA and immunogen P2-BSA. PAT was modified and conjugated to OVA to make another detecting antigen P1-OVA. Inorder to establish direct ELISA, themodified patulin was also connected to horseradish peroxidase, and this enzyme labled haptenwas called P1-HRP.New Zealand rabbit and BABL/C mice were immunized with prepared immunogenfollowing a standard protocol. After the third time immunization, titer of serum wasdetermined by indirect ELISA. To obtain polyclonal antibodies, the rabbit was sacrificed andthe anti-serum was collected from the blood by centrifugation, which was then purified byProtein G resin. For preparation of monoclonal antibodies, mice spleen cells were taken andfused with myeloma cells. And then, hybridoma cell lines that producing PAT monoclonalantibodies were obtained by limitedly dilution cloning method. The titer of purified rabbitpolyclonal antibody was1:6400for P1-OVA and1:12800for P2-OVA. Specific monoclonalantibodies after purified through protein G resin, can reach the titer of1:3200for P1-OVA and1:6400for P2-OVA.Based on the prepared specific antibodies, three typies of ELISA were attempted toestablish, among them indirect competitive ELISA was the most appropriate one. And then,the analysis conditions of indirect ELISA including reaction pH, different substrate,antigen-antibody optimum dilution concentration and substrate response time, were optimized.With the optimized conditions, the standard curve of indirect ELISA for detection PAT wasdeveloped. Colloidal gold immunochromatographic assay for PAT was also developed in thisstudy, and it could be used for potentially detection of patulin in food samples. |
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