Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2005
Publication Date: 4/2/2005
Citation: Shelver, W.L., Kim, H., Li, Q.X. 2005. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the beta-adrenergic agonist zilpaterol. Journal of Agricultural and Food Chemistry 53:3273-3280. Interpretive Summary: Zilpaterol, a beta-adrenergic agonist, is a compound recently approved to use as a growth promoter in the animal husbandry industry in South Africa and Mexico. Because of its ability to improve feed efficiency and produce leaner meat, this agent has a potential to be used illegally. Current analysis method for this compound is complicated, needs to be performed by highly trained personnel, and requires sophisticated instruments. This paper develops a methodology that is user friendly, has high throughput, fast turn around time, and has potential to be used as an on site detection method.
Technical Abstract: Zilpaterol is a beta-adrenergic agonist approved for use as a growth promoter in cattle in South Africa and Mexico but not in the EU, USA, or in Asia. Here, we report the development of a monoclonal antibody based ELISA for zilpaterol. Mice immunized with zilpaterol-butyrate-KLH were utilized for monoclonal antibody generation whereas zilpaterol-butyrate-BSA was used as a coating antigen for ELISA. Thirteen clones were isolated and after the initial sensitivity and isotyping experiments, three clones were selected for further ELISA optimization. Studies of pH indicated the optimum was near 7.4. Clone 3H5 had the highest sensitivity to zilpaterol, and some interaction with clenbuterol and terbutaline at high concentrations, but not other N-alkyl [bamethane, (-) isoproterenol, (+) isoproterenol, metaproterenol, or salbutamol,] or N-arylalkyl (dobutamine, fenoterol, isoxsuprine, ractopamine, or salmeterol) beta-agonists tested. However, clone 3H5 was not functional at high salt concentrations which precluded further development for urine matrix. Clone 2E10 showed increased sensitivity as salt concentrations were increased, and did not cross-react with any of the structural analogues tested. However, its salt and urine sensitivity could cause high variability. Clone 7A8 showed good sensitivity and only a modest change when the salt concentrations vary. Clone 7A8 also demonstrated smaller changes in IC50 and B0 with increasing sheep urine concentrations in comparison to clones 2E10 or 3H5, and thus was selected for further development. The IC50 of the antibodies showed exponential increases when increasing solvent concentrations, making it desirable to keep solvent levels to a minimum. In conclusion, a sensitive, specific zilpaterol monoclonal antibody based ELISA has been developed that can serve as a rapid screening assay.