Drug Testing and Analysis

Research article Received: 23 May 2014

Revised: 9 July 2014

Accepted: 21 July 2014

Published online in Wiley Online Library

(www.drugtestinganalysis.com) DOI 10.1002/dta.1705

Detection of tetracosactide in plasma by enzyme-linked immunosorbent assay (ELISA) Laurent Martin, Ayman Chaabo and Françoise Lasne* As a synthetic analogue of adrenocorticotropic hormone (ACTH), tetracosactide is prohibited in sport by the World Anti-Doping Agency (WADA). An enzyme-linked immunosorbent assay (ELISA) method is proposed for detection of this drug in plasma. Since its structure corresponds to the 24 N-terminal of the 39 amino acids of the natural endogenous peptide ACTH, tetracosactide can be detected with a commercial ELISA kit for ACTH that uses antibodies, the epitopes of which are located in the 1 24 part of ACTH. However, an essential condition for detection specificity is the preliminary total clearance of endogenous ACTH in the plasma samples. This is achieved by a preparative step based on cation-exchange chromatography before ELISA. The method is specific and sensitive (LOD: 30 pg/mL) and may be used as a screening analysis in anti-doping control. The pre-analytical conditions are shown to be of the upmost importance and recommendations for blood collection (EDTA tubes), sample transport (4 °C) and plasma sample storage (-20 °C) are presented. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: tetracosactide; synacthen; ELISA; doping; sport

Introduction Tetracosactide is a synthetic peptide that exerts the same biological effects as the endogenous adrenocorticotropic hormone (ACTH) produced by the anterior pituitary gland. Its structure reproduces the first 24 of the 39 amino acids that compose ACTH. Like ACTH, the natural corticotropin, it induces the synthesis of glucocorticoids, mineralocorticoids and, to a lesser extent, androgens by the adrenal cortex. Both glucocorticoids and androgens are on the list of prohibited substances in sport from the World Anti-Doping Agency (WADA) and thus corticotropins are prohibited as well.[1] Tetracosactide is produced by the pharmaceutical industry as Synacthen and Synacthen Depot, a slow-release form in which the pharmacologically active substance is adsorbed on an inorganic zinc complex. While the therapeutic indications for these drugs are limited, both are used for the investigation of adrenocortical function in diagnostic tests.[2–4] The potential for misuse of these drugs in sport has led to the development of anti-doping control analysis for the detection of tetracosactide in plasma[5,6] and urine[7] samples. These methods are based on mass spectrometry. A different approach based on ELISA is presented here and is suggested as a method for screening.

The ACTH(1-24) was tetracosactide (Synacthen®) from Novartis Pharma (Rueil-Malmaison, France). The ACTH(1-39) was from Tocris Bioscience (Bristol, UK). Poly-Prep chromatography columns (0.8 x 4 cm) were from BioRad (Hercules, CA, USA). CM Sephadex C-25 was from Sigma-Aldrich (St Louis, MO, USA). Protein determinations were performed using the Coomassie Blue Protein Assay Reagent Kit from Pierce (Rockford, IL, USA). Two ELISA kits were used. The first one, ACTH (1-24) Enzyme Immunossay Kit from Peninsula Laboratories (San Carlos, CA, USA) was necessary for the analysis of prepared plasma samples and was a competitive immunoassay using an antiserum to human ACTH (124), thus recognizing both ACTH(1-39) and tetracosactide. The buffer (named EIA buffer), pH 7.5, supplied in this kit for dilution of the antiserum was in the form of a 20x concentrate. This kit could not be used directly with plasma and required a prior extraction process. The second kit, ACTH ELISA from Biomerica (Irvine, CA, USA), was only used here to demonstrate the effective removal of ACTH from prepared plasma samples and was an immunometric assay (‘sandwich’ assay) using two different antibodies. The epitope of the capture antibody was situated in the 34 39 fragment of human ACTH so that only ACTH(1-39) but not tetracosactide was detected by this kit. This kit could be used directly with plasma. The Protein LoBind tubes (2 mL) were from Eppendorf (Hamburg, Germany).


Sample preparation


The principle of the method was to use the ELISA kit from Peninsula, which reacts with amino acids 1 24 of ACTH (and thus

Blood samples were obtained from healthy volunteers. Depending on the experiment, different Vacutainer tubes from Becton Dickinson (Franklin Lakes, NJ, USA) were used for blood collection. BD vacutainers K2E (additive: K2EDTA), K3E (additives: K3EDTA and Aprotinin), LH (additive: lithium Heparin) were used for the experiments with plasma and Vacutainer gel tubes SST II Advance (with gel separator, additive: clot activator) were used for the experiments with serum.

Drug Test. Analysis (2014)

* Correspondence to: Françoise Lasne, Agence française de lutte contre le dopage, Département des analyses, 143, avenue Roger Salengro, Châtenay-Malabry 92290 France. E-mail: [email protected] Agence Française de Lutte contre le Dopage, Département des Analyses, 143 avenue Roger Salengro, Châtenay-Malabry 92290 France

Copyright © 2014 John Wiley & Sons, Ltd.

Drug Testing and Analysis

L. Martin, A. Chaabo and F. Lasne

tetracosactide) after total elimination of endogenous ACTH from the tested plasma samples. In such conditions, ELISA became specific for tetracosactide. The sample preparation consisted in removing most of the proteins and all traces of ACTH from plasma. This was achieved by cation-exchange chromatography. For this, 0.4 mL of CM Sephadex C-25 conditioned in 0.1 M glycine/NaOH buffer, pH 10, were introduced into as many columns as samples. Plasma samples (0.5 to 2 mL) were diluted 1:10 in the same buffer and applied to the columns. The effluents were collected and reintroduced into the columns so that the samples were applied three times. Sephadex was then washed twice with 10 mL of the same buffer and finally once with 10 mL of 0.1 M NaCl. Elution of the bound material from Sephadex was performed with 0.9 mL of 15 mM NaOH, 0.5 M NaCl. The eluates were collected in pre-prepared LoBind tubes containing 50 μL of EIA buffer concentrate (20x) from the Peninsula ELISA kit and 50 μL of 187 mM HCl. In these conditions, considering the dilution of the elution solution by the bed volume of the gel, the final composition of the eluates was EIA buffer (at the right dilution for ELISA) containing 380 mM NaCl. These eluates were stored at 4 °C or -20 °C until they were assayed by ELISA, depending on whether the delay was less or more than 48 h, respectively. Tetracosactide assay by ELISA for ACTH(1-24) To ensure that the standards and samples were in the same diluent, the standards were prepared by dilution of ACTH(1-24) in EIA buffer enriched with NaCl (380 mM final molarity). A range of 7 standards was established corresponding to 0, 0.01, 0.039, 0.156, 0.625, 2.5, and 10 ng/mL. The assay was then performed according to the manufacturer’s instructions. Briefly, after 1 h of incubation of standards and samples (50 μL) with antiserum (25 μL) in the wells of a plate coated with antibodies capturing the antiserum, biotinylated tracer ACTH (1-24) (25 μL) was added. After 2 h of incubation, the plates were thoroughly washed with EIA buffer (not enriched with NaCl) and then incubated with streptavidin-horseradish peroxidase (100 μL) for 1 h. After the plate was thoroughly washed with EIA buffer, the substrate solution (Tetramethybenzidine) (100 μL) was added. The developing blue colour was read at 650 nm. When the absorbance reached approximately 0.6 (usually in 30 60 min), the reaction was stopped with 100 μL of 2 N HCl and the absorbance was then read at 450 nm. The calibration curve was established using a sigmoid mode.

Results and discussion Analytical sensitivity of the ELISA kit for ACTH (1-24) The sensitivity of the ELISA kit for ACTH(1-24) was defined as the concentration corresponding to 3 standard deviations below (negative slope of the calibration curve) the mean optical density of 10 replicates of the zero standard and was 15 pg/mL. This meant that any signal corresponding to less than 15 pg/mL had to be considered as noise. Limit of detection The limit of detection (LOD) was defined as the lowest concentration in a sample giving rise to a significant signal (i.e., corresponding


to more than 15 pg/mL). To determine the LOD, three batches of six samples were spiked with ACTH(1-24) at decreasing concentrations of 50, 30, and 20 pg/mL. All the samples spiked at 50 and 30 pg/mL but only three of the six samples spiked at 20 pg/mL gave rise to significant signals. The LOD was thus evaluated at 30 pg/mL. Specificity Thirty-eight control plasma samples devoid of tetracosactide were prepared and assayed by ELISA for ACTH(1-24). None of the samples gave rise to a significant signal. In fact, the essential condition for the specificity of the method is the total elimination of ACTH(1-39) and this point was especially investigated. Due to the lack of the COOH terminal part (25-39), tetracosactide presents a higher pI (11) than ACTH (9.5), which comprises 5 additional acidic amino acids. Preparation by cation-exchange chromatography at pH 10 takes advantage of this difference. Whereas ACTH(1-24) is bound, ACTH(1-39) and a high proportion of the plasma proteins pass straight to the ion exchanger. The efficiency of the preparative step was checked by enriching 1 mL of a plasma sample with ACTH(1-39) at a final concentration of 68 ng/mL; that is, approximately 1000 times more than the physiological levels.[8] This plasma sample was then submitted to preparation by CM-Sephadex chromatography as described above. The enriched plasma was diluted 1:10 in 0.1 M glycine/NaOH buffer, pH 10, before chromatography. The effluent containing the unbound material, the two washing fractions and the final eluate were assayed for total protein and ACTH(1-39) content using the ELISA from Biomerica. In addition, the final eluate was assayed for ACTH (1-39) by the ELISA from Peninsula. As shown in Table 1, about 93% of the ACTH(1-39) introduced into the plasma sample and the same proportion of total proteins initially present in this sample were recovered in the effluent containing the unbound material and the first washing fraction. Less than 0.4% of the initial ACTH amount initially introduced into the plasma sample was recovered in the final eluate of the preparative step. Though a high variability for plasma ACTH has been reported, depending on the immunoassays used,[9] it is generally assumed that physiological levels of ACTH do not exceed 50 pg/mL from 6 to 9 am (peak of the circadian rhythm).[8] Such physiological levels would provide eluates containing less than 0.1 to 0.4 pg in 1 mL, depending on the amount of plasma, ranging from 0.5 to 2 mL, that had been prepared. Such low concentrations

Table 1. ACTH and total protein amounts in 1 mL of enriched plasma during the preparative step by cation exchange chromatography. nd: not determined due to the impossibility of using the * Peninsula kit without previous extraction of ACTH. : the total protein in the eluate was corrected for the amount of proteins present in the 50 μL of EIA buffer concentrate present in the LoBind tube. This corresponds to the amount of proteins from the plasma sample not removed by the preparative step ACTH(1-39) (Biomerica)

Plasma Effluent st Washing (1 ) nd Washing (2 ) Eluate

ACTH(1-24) (Peninsula)

Total protein






68.4 63.9 — —

Detection of tetracosactide in plasma by enzyme-linked immunosorbent assay (ELISA).

As a synthetic analogue of adrenocorticotropic hormone (ACTH), tetracosactide is prohibited in sport by the World Anti-Doping Agency (WADA). An enzyme...
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