Detection of recombinant growth hormone in human plasma by a 2‐D PAGE method

Abstract

Human growth hormone (GH) has several central metabolic functions including bone growth in childhood, and its anabolic and lipolytic effects in particular are assumed reasons for the abuse of GH by athletes. Human endogenous GH consists of a main 22 kDa variant and several isoforms. In contrast, recombinant GH consists of only one variant being identical to the main endogenous isoform. The method presented here separates different isoforms by 2-D PAGE after isolation of GH from plasma using an immunoaffinity purification system. While samples containing endogenous GH yield up to four isoforms, samples with recombinant GH contain the main 22 kDa spot only. Normalized spot volumes (NSV) are calculated after addition of an internal standard and a discrimination limit was determined at 0.52 for the NSV of the main 22 kDa spot. Above this value, samples containing endogenous GH show at least the main 22 kDa isoform and the 20 kDa splice variant. In contrast, samples with a NSV >0.52 and only one spot are suspicious to contain recombinant GH. This method detects discrete isoforms of GH from plasma and discriminates endogenous GH from its recombinant analog, which makes it useful for doping control purposes.

Tag7 (PGLYRP1) in Complex with Hsp70 Induces Alternative Cytotoxic Processes in Tumor Cells via TNFR1 Receptor

“…The sheep polyclonal antibodies against TNFR1 were prepared as described (25). The rabbit polyclonal antibodies to the murine TNFR1, TNF-␣, and soluble sTNFR1 were from Sigma-Aldrich.…”

Passing the anaerobic threshold is associated with substantial changes in the gene expression profile in white blood cells

“…These finding are in agreement with reported data on the effect of intensive physical exercise on secretion of these mediators into blood. GH (Felsing et al 1992;Kohler et al 2008;Sakharov et al 2008) plays an important role in adaptation to stress, activating the system of growth factors and affecting proliferation of muscle cells (Giannoulis et al 2008;Sakharov et al 2008). eHSP70 Sakharov et al 2009) is a signal molecule within the immune system, which activates macrophages, neutrophils, mast cells and dendritic cells, and upregulates the production of cytokines (Gastpar 2005;Mayer et al 2005).…”

Detecting Growth Hormone Abuse in Athletes

“…In addition, it has been proposed to use immunoassays based on surface plasmon resonance to investigate isoform composition in an athlete's samples. 40 Other new developments include the use of 2-dimensional gel electrophoresis to visualize the isoforms present in a sample, 10 and also to identify the isoforms from the spots in the gel via mass spectrometry. 11 Overall, the field of methods based on the isoform approach is developing, and it might be expected that in the future more than one method will be used to increase the possibility to detect doping with GH.…”

“…Today, application of MS to the analysis of peptide hormones is a rapidly developing field, and also for GH promising data have recently been reported. 10,11 However, the extremely low concentrations together with the complex biological matrix represent problems for these methods, and immunoassays are still the most frequently used methods for measuring peptide hormones in clinical medicine. The approaches to detect GH doping described later in this article are based on immunoassay measurement of GH or GH-dependent peptide markers.…”