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Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 1/17/2002 Publication Date: 7/1/2002 Citation: Shelver, W.L. and Smith, D.J. 2002. Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals. Journal of Agricultural and Food Chemistry 50(10):2742-2747. Interpretive Summary: Ractopamine is a beta-adrenergic agonist that recently has been approved for use as a swine growth promoter in the U.S., Central America, and Asia. Because the European Union has prohibited the usage of this class of compounds and because ractopamine is only approved for usage in pigs, there is a need for an analytical method capable of screening large numbers of samples in a short period of time. This assay should be usable for on-sit monitoring. Immunoassay can provide the advantage of user friendliness, high throughput, and field portability in comparison to standard instrumental methods. Before the immunoassay can be used for these purposes, the assay must be validated for its accuracy relative to the instrumental method. This paper described the successful validation of an immunoassay to determine ractopamine levels in cattle or sheep urine and in cattle liver. Technical Abstract: A monoclonal antibody based ractopamine immunoassay has been applied to incurred samples from sheep and cattle. Results obtained by immunoassay results were compared with HPLC results. Three sets of samples containing primarily unmetabolized ractopamine were analyzed. Correlation of HPLC with ELISA for beef liver samples gave an r2=0.98 despite rather low ractopamine concentrations (range: 1.1 - 13.4 ng/mL, n=6). Ractopamine concentrations in cow urine samples treated by SPE, to remove ractopamine metabolites, also showed a high correlation between HPLC and the ELISA results (r2 = 0.95, range 1.0 - 74.8 ng/mL, n=61). In contrast, HPLC and ELISA analysis of ractopamine in sheep urine were not well correlated (r2 = 0.58, range 4 - 52.2 ng/mL, n = 34). When ractopamine conjugates in urine samples were hydrolyzed with hydrolytic enzymes ELISA and HPLC methods were highly correlated [r2 = 0.94 for sheep (range 123 - 10,554 ppb, n = 60) and dan r2 = 0.98 for cattle (range 14 - 8,159 ppb, n = 62)]. Tissues containe only minute amounts of ractopamine, and after seven-day withdrawal periods, less than 1 ppb free ractopamine was detected. Ractopamine was rapidly metabolized in both cattle and sheep. The difference in ractopamine concentration of urine samples after hydrolysis indicated that only 1-5% of ractopamine was excreted unmetabolized. Results from this study indicate that the monoclonal antibody could be useful for a sensitive, quantitative or qualitative ractopamine screening assay. |
