Development of a competitive enzyme immunoassay for 17 alpha-19-nortestosterone.
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.
Van Look
L J
LJ
Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, State University of Ghent, Belgium.
Jansen
E H
EH
van den Berg
R H
RH
Zomer
G
G
Vanoosthuyze
K E
KE
Van Peteghem
C H
CH
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002417:Cattle; D007124:Immunoenzyme Techniques; D009277:Nandrolone / Q000276:immunology / Q000652:urine*
D000906:Antibodies; D009277:Nandrolone