Submitted to: Food and Agricultural Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/22/2005
Publication Date: 9/1/2005
Citation: Shelver, W.L., Keum, Y., Li, Q.X., Fodey, T.L., Elliott, C.T. 2005. Development of an immunobiosensor assay for the beta-adrenergic compound zilpaterol. Food and Agricultural Immunology 16(3):199-211. Interpretive Summary: Zilpaterol is a compound recently approved in South Africa and Mexico for use to promote cattle growth and produce leaner meat. This compound is not approved for use in the United States or the European Union. In this paper, a biosensor method is developed to measure zilpaterol in sheep urine (one case of an illegal use scenario). The method is sensitive, user friendly, capable of high throughput and measures zilpaterol accurately and with minimal interference from other substances. Because of its favorable analytical properties, the method could be used to detect the illegal use of this compound. Prevention of the illegal use of zilpaterol can assure markets which do not allow the use of these compounds in food.
Technical Abstract: A surface plasmon resonance biosensor method was developed to measure zilpaterol residues in sheep urine. A CM-5 sensor chip previously reacted with ethylenediamine to produce an aminoethyl group was coupled with 4-carboxybutyl zilpaterol activated using EDC/NHS. Five polyclonal and four monoclonal antibodies were screened for their suitability to detect low levels of zilpaterol using the biosensor technology. Total binding was greater for polyclonal than monoclonal antibodies, but a more concentrated antibody solution was required for polyclonal antibodies. A fixed antibody concentration and various concentrations of zilpaterol were injected to obtain a standard curve for each antibody to allow for B0 and IC50 determination. The stability of the assay was assessed by the consistency of B0 in repeated experiments extending at least 6 hours. A measure of non-specific binding allowed the assessment of the specificity of the antibody-immobilized ligand interaction. The effect of varying concentrations of urine on B0 and IC50 was evaluated to assess the degree of “matrix” effect that would be present in an assay. Based on these criteria the most promising antibody (2E10) was selected for further evaluation. This antibody had good sensitivity with IC50 = 4.47 +/- 0.41 ng/mL (n=11). Both intra- and inter-assay variation studies showed excellent recovery and reproducibility for concentrations between 2 ng/mL and 8 ng/mL. A comparison of the biosensor method with a previously developed ELISA demonstrated that both methods give equivalent results (slope of the correlation plot = 1.02) with a high correlation (r2 = 0.91) between them.