Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/12/2004
Publication Date: 3/17/2004
Citation: Shelver, Weilin L. and Smith, David J. 2004. Enzyme-linked immunosorbent assay development for the beta-adrenergic agonist zilpaterol. Journal of Agricultural and Food Chemistry. 52:2159-2166.
Interpretive Summary: Zilpaterol, a beta-adrenergic agonist, is a compound that has been approved as a growth promoter in the animal husbandry industry in South Africa and Mexico. This compound is banned from the US, European Union, and other countries. Because of its leanness-enhancing action, this agent has a potential to be used illegally. Current analysis methods for this compound are complicated, need to be performed by highly trained personnel, and require sophisticated instruments. This paper develops a methodology that is user friendly, has a high turnaround time, and has potential to be used as an on-site detection method.
Technical Abstract: Zilpaterol is an -adrenergic agonist approved for use in cattle in South Africa and Mexico as a growth promoter. It is not currently approved for use in the EU, USA, or in Asia. Here we report the development of an ELISA for zilpaterol. Zilpaterol was reacted with ethyl 4-bromobutyrate followed by refluxing in 0.1 M potassium hydroxide. The resulting hapten was reacted with two carrier proteins, bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as an activating agent. Immunization of goats with the zilpaterol-butyrate-KLH resulted in an antibody useful for an ELISA. We utilized zilpaterol-butyrate-BSA as a coating antigen for ELISA development. The average IC50 derived from the developed zilpaterol immunoassay was 3.94 0.48 ng/mL (n = 25). The antibody did not cross react with N-alkyl [bamethane, clenbuterol, (-) isoproterenol, (+) isoproterenol, metaproterenol, or salbutamol,] or N-arylalkyl (dobutamine, fenoterol, isoxsuprine, ractopamine, or salmeterol) -agonists. The assay was tolerant of up to 10% (v/v) of acetone, ethanol, or methanol, and 15% (v/v) of acetonitrile or DMSO. Salt concentrations ranging from 0.05-1.0M minimally affected B0 or IC50 values. When buffer pH was <7 or >8.8, the IC50s increased in comparison to those measured at pH 7.4. In conclusion, a sensitive, specific zilpaterol ELISA has been developed that can serve as a rapid screening assay.