Enzyme-Linked Immunosorbent Assay-Based Detection of Free Trenbolone in Bovine Bile JANE FITZPATRICK, ²,‡,§ BERNADETTE MANNING, ², | AND RICHARD O’KENNEDY* ,²,‡ School of Biotechnology, and National Centre for Sensor Research (NCSR), Dublin City University, Glasnevin, Dublin 9, Ireland A rapid antibody-based detection system has been developed for the presence of free trenbolone in bovine samples. Polyclonal antibodies were produced that showed specificity toward epitopes located around the steroidal A-ring of the trenbolone molecule. These antibodies were shown to have little or no recognition for many closely related compounds. The antibodies were utilized as the specific biorecognition molecules in competitive and inhibitive enzyme-linked immunosorbent assay systems. While both assays were able to detect low nanogram concentrations of trenbolone in bovine bile, the competitive format was more sensitive (2.41 vs 17.15 ng/mL for TRAb2 and 3.31 vs 30.73 ng/mL for TRAb1). This format was also more accurate and the data produced by this assay fitted more closely to the four parameter equation used to calculate the standard curve. This was a common finding with both of the polyclonal antibodies, suggesting that this was a characteristic of the format used. KEYWORDS: Trenbolone; ELISA; bovine bile INTRODUCTION Trenbolone acetate is a powerful synthetic steroidal androgen, which is used as a growth promoter in cattle. It is rapidly hydrolyzed to its metabolite 17-trenbolone after administration to cattle (1). This is the active form of trenbolone acetate, and it is the main metabolite found in muscle and fat tissues of implanted cows (2). Trenbolone (TR) and trenbolone acetate cannot be used as growth promoters within the European Union while the U.S. FDA allows the administration of trenbolone acetate to cattle. Testing regimes are required within the EU to enforce this ban. Many assays have been described for the detection of TR in different animal matrices, including urine, bile, muscle, liver, and faeces (3). These assays include radioimmunoassays (4), enzyme immunoassays (3), and immunoaffinity chromatography followed by high-performance liquid chromatography (HPLC)- thin-layer chromatography (TLC) detection (5). Each of these methods had its own advantages and disadvantages, but they all required lengthy sample extraction and/or cleanup before the detection of the compound can be carried out. The assay described here uses a diluted bile sample directly applied to an enzyme-linked immunosorbent assay (ELISA) system to give specific and quick detection of TR in bile with high sensitivity. MATERIALS AND METHODS The materials were purchased from Sigma-Aldrich Chemicals (Dublin) unless otherwise stated. All reagents were of analytical grade. Distilled water was used throughout the work. The phosphate-buffered saline (PBS) used was sourced from Oxoid and was Dulbecco’s A PBS, pH 7.4. The steroids used to determine the cross-reactivity of the antibodies were generous gifts from Dr. Christopher Elliott and Dr. Steven Crooks, Veterinary Sciences Division, Northern Ireland. Preparation and Use of Bovine Bile. Bovine bile was collected from Kepak, Co. (Dublin), centrifuged, and stored at -20 °C until required. The samples were defrosted and filtered through a 0.45 µm syringe filter before use. The standards were prepared by spiking the bile with the steroid from an ethanolic stock and performing dilutions to achieve a wide concentration range. Derivatization of TR to TR-17-Hemisuccinate (HS). The method used was a modification of the procedure used by Jondorf in 1980 (6). A 100 mg amount of TR was dissolved in 3 mL of dichloromethane and 2 mL of dry pyridine. A 187 mg amount of succinic anhydride was added, and the reaction was left at room temperature for 48 h. The reaction was evaporated to dryness using a rotary vacuum evaporator with a heat gun, and the residue was dissolved in 10 mL of chloroform. The residue was washed twice with water (2 × 5 mL) to remove excess succinic anhydride, and the chloroform was then removed under vacuum. The residue was redissolved in ethanol and stored in the dark at room temperature. Production of TR-Thyroglobulin (THY) Conjugate Using EDC/ NHS Chemistry. The production of this conjugate was carried out with a modification of the carbodiimide procedure described by van Look et al. (5). A 5 mg of TR-HS was dissolved in 500 µL of dioxane in a glass vial. Solid NHS was added to give a final molarity of 0.1 M. A 5 mg amount of EDC was dissolved in 250 µL of distilled water and added to the steroid solution. This was incubated for 10 min with stirring. A solution containing 10 mg of THY in 700 µL of 0.05 M phosphate buffer, pH 7.8, was added to the reaction mixture. The reaction was allowed to proceed for 75 min. The resultant mixture was dialyzed for 48 h against four changes of PBS at 4 °C. Production of TR-Bovine Serum Albumin (BSA) Conjugate Using Mixed Anhydride Chemistry. This was prepared using a modification of the mixed anhydride procedure described by Nambara * To whom correspondence should be addressed. Tel: +353 1 7005313. Fax: +353 1 7005412. E-mail: richard.okennedy@dcu.ie. ² School of Biotechnology. ‡ NCSR. § Current address: National Food Centre, Teagasc, Ashtown, Dublin 15, Ireland. | Current address: Schering-Plough, Ringaskiddy, Cork, Ireland. J. Agric. Food Chem. 2004, 52, 4351-4354 4351 10.1021/jf0352531 CCC: $27.50 © 2004 American Chemical Society Published on Web 06/18/2004