Establishment And Detection Of Clenbuterol Residues In Annimal Foods By Rapid Immunoassay

Clenbuterol,a substituted Phenylamino ethanol that has beta-2 adrenomimetie ProPertiesat very low doses. CL can enha animal's nervous exeitation, reduce the fat to deposit and may also lead to the development of muscle Proten, because of these, CL was used as an adjunet to animalfeeds. In recent years,a lot of news about food poisoning resulting from CL residues in food has been reported. Currently,there is appreciable concern about the potential consumer health hazards associated with the illegal use of clenbuterol as a growth promoting agent. Though the policies and regulations of the country have already been forbidden strictly using it as the additive of feed, there is application in violation of rules and regulations.So,in order to guarantee meat produet quality and human health,it is necessary and signifieant to set up a method which is fast,simple and convenient to monitor. In this study,the polyclonal antibodies were developed for the establishment of competitive enzyme-linked immunosorbent assay(ELISA) and Membrane based colloidal gold immunoassay for detecting CL.In order to develop a fast ELISA detection kit, CL was diazotized firstly and then coupled to bovine serum albumin(BSA)and ovalbumin(OVA)to synthesis immunogen and coating antigen respectively. The BSA-CL emulsified in freund's complete adjuvant were chosen to immunize 6 to 8 weeks old female New Zealand white rabbits. The assay results showed that the rabbit's antiserum titre is as highly as 12800.After purified by saturated ammonium sulphate,ion exchange and reverse immunoaffinity chromatography in turn.Based on this purified antibody,an competitive indirect ELISA was developed. This reseacher indicated that ELISA was very sensitive and specific,IC50 was 2.18 ng/mL, detection limit against CL was as low as 0.05 ng/ml,recoveries between 84.1 and 89.0 percent and a working range in the 0.32-200 ng/mL.And showed lower than 10% cross activity towards Salbutamol,Tulobuterol and Ractopamine.Moreover,it was compared with the GC-MS,and showed that this ELISA kit can be applied to practical detection for clenbuterol.One-step membrane-based colloidal gold immunoassay in flow-through format for the rapid detection of CL was developed. Nitro-cellulose membrane strip was separately coated with goat-rabbit IgG (control line) and CL-OVA conjugate (test line). Membrane based colloidal gold immunoassays had a visual detection limit of 10 ng/mL for CL.Using 4% NaCl-0.1 M HCl-methanol as extraction solvent ,the matrix effect could fairly removed in analyzing animal tissue samples .the method was simple and time-saving with the detection time. A positive reaction could be easily seen by visual detection, so that it is suitable for rapid testing to CL in various food anmples on-site.