Luminescence 2003;18:72-78 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: lO.1002/bio.708 ORIGINAL RESEARCH A rapid and sensitive 384-well microtitre format chemiluminescent enzyme immunoassay for 19- nortestosterone Aldo Roda,1* Anna Chiara Manetta,2 Ottavio Portanti,2 Mara Mirasoli,1 Massimo Guardigli,1 Patrizia Pasini' and Rossella Lelli2 'Oepartment of Pharmaceutical Sciences, University of Bologna, Bologna, Italy 4stituto Zooprofilattico Sperimentale dell' Abruzzo e del Molise 'G. Caporale', Teramo, ltaly Received 16 July 2002; revised 19 August 2002; accepted 23 August 2002 ABSTRACT: We developed a competitive chemiluminescent (CL) enzyme immunoassay for rapid, sensitive analysis of 19- nortestosterone(19-NT) in bovine urine. Anti-19-NT polyclonal antibodies were raised in rabbits using a 19-NT-hemisuccinate derivative conjugated with ovalbumin; the derivative was also conjugatedwith horseradish peroxidase (HRP) as a label. Antibodies were immobilized on 384-well black polystyrenemicrotitre plates and HRP-labelled 19-NT activity was measured using an efficient chemiluminescent substrate (SuperSignal@ ELISA Femto) after 3 min incubation. Emitted light was recordedusing a conventional, photomultiplier-tube-based microtitre plate readeror a sensitiveback-illuminated,cooled CCD camera. The developed methodfulfils alI the requirements of precision(intra- and inter-assay CV < 10%)and accuracy (mean recovery 94-112%), with a detectionlimit of 0.03 ppb (1.1 x 10-9 mol/L) in a urine matrix. Chemiluminescence enhances detectability of the HRP-labeUed tracer (thus lowering the limit of detection with respect to colorimetry) and reducesanalysislime. The 384-weUmicrotitre plate cuts the sample/reagent volume (20 ~L), a five-fold reduction with respect to the conventional96-weUmicrotitre plate. The developed method is suitable for high-throughput screening of 19-NT in urine samples,with reduced costs as compared with conventional colorimetric enzyme immunoassays. Copyright <ç) 2003 John Wiley & Sons, Ltd. KEYWORDS: growth promoters; 19-nortestosterone; enzyme immunoassay; chemiluminescence; urine INTRODUCTION of sample collection, urine is commonly chosenas the targetmatrix. The National ResidueMonitoring Plans of 19-Nortestosterone is used in veterinary as well as in the European Union suggested an action level of 2 ppb human medicine for the treatment of protein deficiency for 19-nortestosterone in urine. diseases, osteoporosis and advancedbreastcancer. This Radioimmunoassays and enzyme immunoassays bave anabolic androgenicsteroid has also been employed as a been widely used for routine screening(4-8) and many doping agent to boostmuscular strength andperformance kits afe commercially available for 19-NT. Conventional in sport and borse racing, and as a growth-promoting chromatographicmethòds, such as gas chromatography agent to accelerate weight gain and improve feeding (GC) or liquid chromatography (HPLC) coupled to mass efficiency in cattle (1). Use of growth-promoting drugs spectrometry (MS) and tandem mass spectrometry (MS- for fattening livestock has beenbanned in the European MS), afe then used as confirmatory methodsbecauseof Union since 1986 (2) to protect consumers from possible the high specificity ofthe information from the molecular harrnful effects due to the intake of hormoneresidues and structure of the analyte (9-11). their metabolites. However, illegal use of 19-NT as a In order to provide a more rapid and straightforward growth promoter has been widely reported throughout method for screening of 19-NT in urine samples suitable Europe (3), thus prompting continuous ministerial for large-scale surveillance programs, we developed a surveillance to control its abuse and the import of quantitative enzyme immunoassayusing a chemilumi- affected animals.This hasresulted in the development of nescent (CL) substrate for the measurementof HRP- a broad range of methods for deiecting residues of this labelled 19-NT activity. We used a 384-well microtitre drug and its metabolites in biological matrices. Due to plate format, which allows the analysis of many samples pharmacokinetic considerationsand to the relative ease per analytical run (high-throughput screening) , and a five-fold reduction in reagent quantity, compared with *Correspondence to: A. Roda, Dipartimento di Scienze Farmaceu- conventional 96-well microtitre plates. The CL signals tiche, Università degli Studi di Bologna, Via Belmeloro 6, 40126 from the 384 wells can be measured in a few minutes by Bologna, Ita1y. f . al .. l l . Email: roda@a1ma.unibo.it means O a convention IDlcrotitre p ate uID1nometer, or http://www.unibo.it/anchem within 1 min if a sensitiveCCD 10w-light imaging device Published online 6 January 2003 Copyright @ 2003 John Wiley & Sons, Ltd.