Development of a polyclonal indirect ELISA with sub-ng g(-1) sensitivity for the analysis of clenbuterol in milk, animal feed, and liver samples and a small survey of residues in retail animal products

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with sub-ng g(-1) sensitivity for clenbuterol was developed. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. All four antisera displayed high sensitivity with IC50 and limit of detection (LOD) values lower than 0.9 and 0.03 ng ml(-1), respectively. The most sensitive ELISA was established with IC50 and LOD values of 0.1-0.3 and 0.01-0.02 ng ml(-1), respectively, which are more than ten times lower than those reported in the literature. The cross-reactivity (CR) values of the four antisera with salbutamol, another frequently used beta-agonist of similar molecular structure to clenbuterol, were estimated to be within 25-46%. No binding (CR<0.01%) of nine other drugs which are frequently used in animal feeds was observed. The superior ELISA at optimal assay conditions was used for the analysis of five clenbuterol-fortified samples and the results were confirmed by high-performance liquid chromatography (HPLC). Acceptable recovery rates of 92.2-97.0% and intra-assay coefficients of variation of 1.3-5.3% were obtained. The proposed ELISA was highly correlated with HPLC (R-2 = 0.9893, n = 5). Another sixteen samples were randomly collected from local markets and analysed by ELISA. The highest clenbuterol residues found in milk, feed, swine and chicken livers were 5.33, 64.04, 2.28 and 0.74 ng g(-1), respectively.