Tracking growth hormone abuse in sport: performance of marker proteins in a controlled setting.

Human growth hormone (hGH) abuse in sport is a challenge at present. The current strategy used, known as direct method, is based on the quantification of hGH variants in serum. An alternative strategy, known as indirect method, focuses on serum markers such as insulin-like growth factor I (IGF-I) and procollagen type III N-terminal propeptide (P-III-NP). The indirect method allows a longer window of detection (WOO) of hGH abuse. To evaluate the performance of the indirect method, in parallel to the direct method, a clinical trial with recombinant hGH (rhGH) was conducted on healthy male subjects during 7 days (0.026 mg(-1) kg(-1) person(-1) day(-1)). The data were fit to the discriminant formula proposed in the previously published GH-2000 project. The low sensitivity of the scores, judged from the high number of false negative outcomes, imposed a new discriminant analysis, standarised using local population subjects demographically similar to the ones of the study. The sensitivity of the method significantly increased, highlighting the importance of the standardisation. The indirect method allowed extended window of opportunity (WOO), although two false positive evaluations were observed derived from elevated basal IGF-I and P-III-NP concentrations stressing the need for an independent confirmation method. When direct and indirect methods were combined the best selectivity and sensitivity were achieved.

Tracking growth hormone abuse in sport: a comparison of distinct isoform-based assays.

Detecting recombinant human growth hormone (rhGH) abuse in sport remains one of the major challenges in doping control. We have compared two different approaches to detect the hGH (human growth hormone) abuse. The first measures the concentrations of the 22 kDa hGH isoform (rec assay) and pituitary derived isoforms (pit assay) and a ratio rec/pit is obtained. The second measures the concentrations of 22 and 20 kDa hGH isoforms and also a ratio 22/20 kDa is derived. Using a single set (nine healthy male subjects, 7 days, 0.026 mg/kg/day of rhGH, 2 week wash out period) both approaches were compared. To quantify the agreement between the immunoassays, B.A. (Bland-Altman) analysis and P.r. (Pearson correlation) were used. To fully understand the assay readings, all relevant antibodies were characterised by surface plasmon resonance (SPR). In either approach the ratio numerator produces similar results and the denominator determines both signal-amplitude and time-frame of possible application. The rec vs pit approach displays a higher distinctive capacity to detect hGH abuse but the complex binding properties of the capture antibodies make it very difficult to evaluate the precise contributions of the individual hGH variants to the assay result. In the 22 vs 20 approach, the 20 kDa hGH concentration measures determine its applicability. Both approaches are based on a different principle, should be preferably applied within 24 h after rhGH administration, and are perfectly comparable given the results obtained. The reduced time frame of application indicates that their principle application should be preferably in an out-of-competition setting.

Surface plasmon resonance immuno assays - A perspective.

Human growth hormone (GH) represents an extremely challenging task from an anti-doping viewpoint. GH is an endogenously produced substance, present at very low levels in circulation (for the most abundant 22kDa isoform approximately 50pM in plasma and 100fM in urine) either as monomer or homo- and heterodimers, comprises a family of distinct isoforms, and obeys a pulsatile secretion routine that is affected by many different internal and external factors. Upon administration of the recombinant, single-isoform pharmaceutical, the feedback mechanism reduces the endogenous heterogeneity resulting in altered ratios between the different GH isoforms. Thus, measuring the isoform ratios through immuno assays appears the approach of choice. Conventional assays do not provide information on isoform-specific association and dissociation events of the individual primary antibody-isoform or isoform-secondary antibody interactions. This particular information can be obtained using the technology of surface plasmon resonance (SPR) which enables monitoring of biomolecular interactions in a dynamic and label-free setting. In this paper the different aspects of SPR are described, how the technology may be beneficial for understanding today's anti-GH immunoassays, and whether the approach could be employed for measuring GH in the near future.