title: Development of a competitive enzyme immunoassay for 17 alpha-19-nortestosterone.
abstract: Because 17 beta-19-nortestosterone and its esters are frequently used anabolic steroids in cattle in Europe, there is a need for an assay that can also detect certain metabolites. The enzyme immunoassay described here involves the detection and quantitation of the major metabolite 17 alpha-19-nortestosterone in urine. The assay is based on the coating of an antibody raised in a rabbit against 17 alpha-19-nortestosterone-3-carboxy-methyloxime-bovine serum albumin (17 alpha-19-NT-3-CMO-BSA), the competitive incubation of 17 alpha-19-NT and the 17 alpha-19-nortestosterone-3-CMO-horseradish peroxidase label, followed by the detection of the blue colour developed by the action of the enzyme on tetramethylbenzidine. The 3-CMO conjugate of 17 alpha-19-nortestosterone was used to produce an antibody with selective affinity for the 17 alpha-epimer. For the optimization of the assay, different coatings and incubation conditions were tested. The standard curve ranged between 0.98 and 4000 pg per well, with a B/B0 50% of +/- 65 pg per well. Colour was measured with a microtitre plate reader. The method was validated by means of certified blank and spiked cattle urine samples.
authors.affiliation: Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, State University of Ghent, Belgium.
authors.forename: L J
authors.initials: LJ
authors.lastname: Van Look
authors.forename: E H
authors.initials: EH
authors.lastname: Jansen
authors.forename: R H
authors.initials: RH
authors.lastname: van den Berg
authors.forename: G
authors.initials: G
authors.lastname: Zomer
authors.forename: K E
authors.initials: KE
authors.lastname: Vanoosthuyze
authors.forename: C H
authors.initials: CH
authors.lastname: Van Peteghem
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002417:Cattle; D007124:Immunoenzyme Techniques; D009277:Nandrolone / Q000276:immunology / Q000652:urine*
chemical_list: D000906:Antibodies; D009277:Nandrolone