Enhancing the Sensitivity of the 2D-PAGE detection assay for hGH doping - follow up study

Code: 10B10MT

The misuse of recombinant growth hormone in elite sports is well known from confiscations and confessions and additionally, the first two positive samples were reported in 2009 using the luminescence immunoassay developed by Bidlingmaier and Strasburger. The luminescence immunoassay (LIA) provides a powerful screening tool but a complementary method for confirmation providing more detailed information would be desirable. Therefore, a method based on immunoaffinity purification, 2D-PAGE and immunoblotting was developed which detects discrete endogenous variants of growth hormone. After successful development and validation, the methods´ sensitivity and robustness need to be optimized.

The project is planned to improve the sensitivity to be similar to that reached by the LIA to yield a powerful confirmation method. This can be done by optimizing a) the immunoaffinity purification e.g. by coupling specific antibodies directly to magnetic beads, b) the blotting conditions or the immunodetection, e.g. by using different secondary antibodies for the visualization and detection. Furthermore, the robustness of the method should be improved by providing another primary antibody which could replace the currently used one to ensure continuous availability.

After optimization, another follow-up project could include the measurement of a reference population to allow the calculation of reference values.

Main Findings

Human endogenous growth hormone (hGH) is one of the most important growth promoting hormones in the human body. It regulates bone growth in childhood and has an important impact on many metabolic processes such as muscle growth and increased fat consumption. Recombinant growth hormone (rGH) is supposedly misused by various athletes as performance enhancing agent because of its lipolytic and anabolic effects. It is a protein composed by 191 amino acids with a molecular weight of 22 kDa and is produced in the pituitary gland. Alternative splicing results in a smaller isoform of 20 kDa missing the amino acids 32-46, and different posttranslational modifications such as phosphorylation, acylation, glycosylation as well as proteolytic cleavage and dimerization lead to a large heterogeneity. The detection of discrete variants of hGH by immunoaffinity purification, 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and immunoblotting could serve as a complementary detection assay to uncover the misuse of hGH in sport. The aim of this project was to enhance the sensitivity of an existing 2D-PAGE detection assay for hGH doping by optimizing different steps of the sample preparation protocol plus tests for alternative primary antibodies. Out of four tested antibodies, one proved to be an adequate alternative as it was not only able to detect rGH amounts down to 0.25 ng but also to bind all endogenous variants of hGH. The antibody was subjected to protein A purification and the sample preparation protocol was optimized by modifying antibody concentrations, incubation times, secondary antibodies, amplification systems and fluorescence detection. Finally, an optimized protocol was composed comprising immunoaffinity purification, 2D-PAGE and immunoblotting (with secondary antibody amplification); however, due to these new aspects and requirements of the methodology, further evaluation of the performance and applicability to authentic administration study samples might be required. In the absence of technical alternatives (e.g. MS-based methodologies) to immunologically driven assays, the use of monoclonal antibodies (as employed in the currently routinely used LIA test system) might be preferable to ensure constant quality and comparability of analytical results.