A sensitive competitive solid‐phase enzyme immunoassay for the detection of tert‐butylic beta‐agonist drugs was developed. An antiserum was raised in rabbits against the diazo‐conjugate of clenbuterol and the human serum albumin. In the assay, the anticlenbuterol antibody was incubated with the salbutamol‐enzyme conjugate and unlabelled standard or samples in microtitre wells precoated with affinity‐purified antirabbit IgG. The absolute detection limit for clenbuterol and mabuterol was 0.3 ng/ml (15 pg/well) and 50% relative binding at 1.0 ng/ml (50 pg/well). The absolute detection limits for salbutamol and terbutaline were 0.9 and 1.3 ng/ml, respectively. The cross‐reactivity of iso‐propylic parent compounds was quite low (<1%). Urine and sera were tested without any extraction step. Sample blanks (96 urine and 76 sera from five different sources) showed low matrix effect, suggesting a limit of decision for bovine urine of 0.6 ng/ml clenbuterol equivalent. Mean recoveries of clenbuterol in spiked urine and serum samples ranged from 90 to 120% (mean recovery 96%). Comparative analysis by gas chromatography/mass spectrometry showed that false negative results do not occur. The assay is simple, rapid, sensitive, multiresidual, reliable and cost‐effective. The test is adaptable to high sample throughput laboratories as well as field test for rapid screening of a few samples in slaughterhouses.
