Development Of Indirect Competitive Enzyme-Linked Immunosorbent Assays(ic-ELISAs) For The Detection Of Two New β-Agonists Brombuterol And Cimbuterol

β-agonists, commonly known as lean meat powder, have the function of the nutrition redistribution so as to increase animals’ lean meat percentage. The illegal use of β-agonist in livestock production has led to many poisoning incidents after the consumption of animal tissue. Therefore, β-agonists are banned as feed additives for growth promotion in animals by the Ministry of Agriculture of the People’s Republic of China. Clenbuterol, ractopamine and salbutamol are the most frequently used β-agonists. A new β-agonist phenylethanolamine A appeared in recent years. Fortunately, people have established a series of methods to detect these four β-agonists. However, as new clenbuterol-like β-agonists, brombuterol and cimbuterol are forbidden in animal husbandry with limited detection methods. In case of potential food safety hazard from β-agonists, it is essential to establish rapid detection/screening methods for brombuterol and cimbuterol.In our research, indirect competitive enzyme-linked immunosorbent assays(ELISAs) with high sensitivity and specificity for the detection of brombuterol and cimbuterol in tissues and feed were developed.Considering structural similarity between brombuterol and cimbuterol, the two chemicals were coupled to carrier proteins with diazobenzidine method. After immunizing rabbits with brombuterol-BSA and cimbuterol-BSA respectively, ic-ELISAs with high sensitivity and specificity were established on the obtained antisera. Both ELISAs were confirmed by high performance liquid chromatography(HPLC). Under optimum reaction conditions, it was found that IC50 of ELISA for brombuterol was in the range of 0.165-0.243 ng·mL-1 and that for cimbuterol was 0.218 ng·mL-1, indicating high sensitivity of the ELISAs. The cross-reactivity rates of the ELISAs also demonstrated the assays exhibited high specificity. To investigate the accuracy and precision of the assays, swine meat, liver and feed samples were spiked with brombuterol and cimbuterol respectively and detected by ELISA. For brombuterol analysis, reasonable recovery rates of 91.9–115.4% and intra-assay coefficients of variation of 1.5–9.5%(n=3) were achieved. While for cimbuterol analysis, reasonable recovery rates of 95.41–114.5% and intra-assay coefficients of variation of 2.2–9.3%(n=3) were obtained. Both ELISAs were confirmed by HPLC with high correlation coefficients(R2). Besides, an Au nanopaticles based immunochromatography assay(GICA) for cimbuterol detection was established and comparied with the proposed ELISA. The naked-eye LOD of ICA for cimbuterol was about 3 ng·m L-1, which was much higer than that of ELISA. The proposed two ELISAs were proven to be feasible quantitative/screening methods for brombuterol and cimbuterol analysis with the properties of high sensitivity and specificity, high sample throughput and low price.