Sensitive and specific detection of a new β-agonist brombuterol in tissue and feed samples by a competitive polyclonal antibody based ELISA

Brombuterol, a new beta-adrenergic agonist which can enhance the lean meat-to-fat ratio, is forbidden as an additive in animal feeds for livestock production due to its adverse effects on consumers. In this study, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for the detection of brombuterol in tissue and feed samples was developed. The immunogen and coating antigen were prepared by directly linking brombuterol to carrier proteins using the diazobenzidine method. The antisera against brombuterol were obtained from immunized rabbits. The IC50 and limit of detection (LOD) values of the superior ELISA were 0.165 ng mL(-1) and 0.007 ng mL(-1), respectively. The cross-reactivity (CR) values of ELISA with clenbuterol, terbutaline and cimbuterol were less than 6.2%; there was no CR of ELISA with nine other compounds. To investigate the accuracy and precision of the assay, liver, meat and feed samples were spiked with brombuterol at different concentrations and analyzed by ELISA. Acceptable recovery rates of 91.9-115.4% and intra-assay coefficients of variation of 1.5-9.5% (n = 3) were achieved. The ELISA was also validated by HPLC. As revealed, both methods were highly correlated (R-2 = 0.9927-0.9994, n = 6). The proposed ELISA was proven to be a feasible method for quantitative analysis of brombuterol in tissue and feed samples.