Development of an immunobiosensor assay for the beta-adrenergic compound zilpaterol*
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 less diluted 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 B 0 and IC 50 determination. The stability of the assay was assessed by the consistency of B 0 in repeated experiments extending at least six 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 B 0 and IC 50 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, a monoclonal antibody) was selected for further evaluation. This antibody had good sensitivity with IC 50 =4.47±0.41ng/ml ( n =11) in buffer). Both intra- and inter-assay variation studies showed excellent recovery and reproducibility for concentrations between 2ng/ml and 8ng/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 (r 2 =0.91) between them.
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Document Type: Research Article
USDA-ARS Biosciences Research Laboratory, Fargo, USA
Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, USA
Department of Agriculture and Rural Development, Veterinary Sciences Division, Belfast, Northern Ireland, UK
September 1, 2005
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