Analysis of urinary human growth hormone (hGH) using hydrogel nanoparticles and isoform differential immunoassays after short recombinant hGH treatment: Preliminary results

Abstract

Successful application clinical-grade human growth hormone (hGH) immunoassays to the discovery of illegal doping cases has been rare. Indeed, the preferred biological matrix in doping control is urine, where the estimated baseline concentration of hGH falls well below the linear range and sensitivity threshold of all commercially available immunoassays, including hGH isoform differential immunoassays which can discriminate pituitary endogenous hGH from recombinant hGH. We employed hydrogel nanoparticles as a pre-processing step that concentrate urinary hGH into the linear range of isoform differential immunoassays. We explored the characteristics of immunoassays in urine spiked with both phGH or rhGH, after pre-treatment with the nanoparticles. Subsequently, pre-treatment was applied to urine obtained from 3 healthy volunteers administered during three days with daily subcutaneous injections of 0.026 mg/kg/day rhGH, Genotonorm®. Linearity between both rhGH and phGH concentrations in urine measured by a chemoluminescent assay (Immulite) and in the particle eluate was evident for differential immunoassays (R square higher than 0.999). In case of treated individuals the recombinant/pituitary concentration ratios remained above the established World Anti-Doping Agency (WADA) criterion for hGH misuse up to 24 h after the last administration dose, using both assays for volunteer 1 and 2 while in case of volunteer 3 results were inconclusive. The use of nanoparticles appears to open the possibility of assessing rhGH misuse in urine.

Direct and label‐free detection of the human growth hormone in urine by an ultrasensitive bimodal waveguide biosensor

“…To detect hGH in urine at clinically relevant levels, different methodologies have already been developed but normally they require fluorescent or magnetic amplification . The most sensitive method to detect hGH in urine involves the use of hydrogel nanoparticles to concentrate urinary hGH obtaining detection limits (LOD) around pg mL –1 . However, this method cannot be employed as a cost‐effective and immediate hGH urine test due to the complexity of the steps undertaken.…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…Bosch et al employed hydrogel-based nanoparticles to enrich GH isoforms from urine and to probe for the possibility to use the differential immunoassay on this matrix. [104] In a pilot study with spiked urine samples as well as administration study specimens, volumes of 20 mL of urine allowed for applying the isoform test, which yielded results of comparable ratios found in timematched serum samples in case of elimination study specimens.…”

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

“…In one step, in solution, the nanoparticles perform a rapid (within minutes) sequestration and concentration of low molecular weight analytes 10,11,13,[15][16][17][18] . Proteins are subsequently eluted from the nanoparticles and detected using western blotting [19][20][21] , mass spectrometry 10,11,13,15,18,22,23 , immunoassays/ELISA 10,11,15,18 , or reverse phase protein microarray 16,24 assays. Nanoparticles functionalized with chemical bait, and presenting a core or core shell architecture, capture and concentrate proteins based on the bait/shell physicochemical properties.…”