Development Of A Rapid Immunoassay For Detection Of Dapsone In Animal Foods

Dapsone, an antibacterial drug, always be used as an antibiotic in animals to prevent and treat diseases and shows strong sterilization effect in large dose. But, the most prominent side effects of this drug are dose-related haemolysis, liver damage, mental disorders and can cause the majority of deaths because of its toxicity. For these reasons, it has been prohibited from use in food-producing animals according to the notice of the Ministry of Agriculture and the Council Directive 90/2377/EC and 37/2010/EC. In this study, DDS artificial antigen was prepared and the polyclonal antibodies were obtained for the establishment of dc ELISA and GICA for detecting DDS. Two methods have good specificity and sensitivity for rapid detection of DDS.DDS was coupled to bovine serum albumin(BSA) to synthesis immunogen antigen by diazotization method, DDS was coupled to ovalbumin(OVA) to synthesis coating antigen by glutaraldehyde method and DDS was labeled with horseradish peroxidase by sodium periodate oxidation method. The DDS-BSA was used to immunize two healthy New Zealand white rabbits. Three months later, results showed that the rabbit’s antiserum titre is as highly as 12800. The antiserum was purified by Chromatography on Protein A-Sepharose 4B gel column and the resulte showed that lots of interfering substances were removed and the sensitivity was improved.Several conditions were optimized, such as coating concentration and enzyme-labelled antigen dilution, blocking solution, ions concentration, p H, acetonitrile concentration. Under the optimum conditions, a method of dc ELISA was developed. And it was very sensitive and specific, IC50 was 2.96 μg/L, IC15 was as low as 0.02 μg/L and showed lower than 15% cross activity towards sulfobenzide, 4, 4- dihydroxy diphenyl sulfone, sulfanilamide and sulfadiazine, the coefficient of variation was between 3.47-12.16%. Milk need to be diluted 10 times with PBS, honey need to be diluted 5 times, pork and egg need to be dried under nitrogen and redissolved, the matrix effect could be removed and the recoveries between 75.45 and 90.48 percent. Moreover, it was verified by HPLC and the result showed that this ELISA can be applied to practical detection for dapsone.Nitro-cellulose membrane strip was separately coated with DDS-OVA conjugate(test line) and goat-rabbit Ig G(control line). Under the optimum conditions, Membrane based colloidal gold immunoassays had a visual detection limit of 20 μg/L for DDS. Milk need to be diluted 5 times with PBS, pork and honey need to be diluted 10 times and egg need to be diluted 20 times, the matrix effect could be removed. The strip was so simple and time-saving that it is widely used to detect DDS in various food samples on-site.