Studies On The Enzyme Immunoassay To Detect Clebuterol Residuals In Food

Food safety has already become the global focus of attention. In recent ten years, a lot ofnews about food poisoning resulting from clenbuterol residues in food has been reported.Currently, there is appreciable concern about the potential consumer health hazards associatedwith the illegal use of clenbuterol as a growth promoting agent. Enzyme-linked immunosorbent assay (ELISA) and chemiluminescent enzymeimmunoassay (CLEIA) are two kinds of convenient and fast enzyme immunoassay methodsto screen large samples. But now, mainly depending on expensive foreign kits hinder theapplication of these two assay methods, so it is urgent to develop ELISA fast kit forclenbuterol with own independent property right. In order to develop a fast ELISA detection kit, clenbuterol was diazotized firstly and thencoupled to bovine serum albumin (BSA) and ovalbumin (OVA) to synthesis immunogen andcoating antigen respectively. According to an immune protocol, three New Zealand whiterabbits were immuned 5 times by CL-BSA in three months. The assay results showed that oneof the three rabbits' antiserum titre is as highly as 3.2×105. After purified by saturatedammonium sulphate, ion exchange and reverse immunoaffinity chromatography in turn,monospecific antibody was obtained and its average affinity constant is about 4.5×10-9 mol/L.Based on this purified antibody, an competitive indirect ELISA was developed. By optimizingthe immunoreagent conditions such as the pH and ionic strength of buffers, concentration ofTween-20 and so on, ELISAs provided a limit of detection of 0.096 ng/mL, recoveries (bodyfluid and edible tissues) between 80 and 120 percent and a working range in the 0.1-8.1ng/mL. Preliminary evaluation of assay performance through following items: reproducibility,sensitivity, precision, and accuracy showed that this ELISA kit can be applied to practicaldetection for clenbuterol. Moreover, it was compared with the foreign ELISA kit andhigh-performance liquid chromatography.Meanwhile, some exploring researches have been employed to study the CLEIA method.By changing certain immunoreagent conditions of ELISA for clenbuterol, CLEIA gave a limitof detection of 0.01ng/mL, lower ten times than that of ELISA, which demonstrated theadvantage of CLEIA method.