Determination of biological activity of gonadotropins hCG and FSH by Förster resonance energy transfer based biosensors.
Determination of biological activity of gonadotropin hormones is essential in reproductive medicine and pharmaceutical manufacturing of the hormonal preparations. The aim of the study was to adopt a G-protein coupled receptor (GPCR)-mediated signal transduction pathway based assay for quantification of biological activity of gonadotropins. We focussed on studying human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH), as these hormones are widely used in clinical practice. Receptor-specific changes in cellular cyclic adenosine monophosphate (cAMP, second messenger in GPCR signalling) were monitored by a Förster resonance energy transfer (FRET) biosensor proteinTEpacVVin living cells upon activation of the relevant gonadotropin receptor. The BacMam gene delivery system was used for biosensor protein expression in target cells. In the developed assay only biologically active hormones initiated GPCR-mediated cellular signalling. High assay sensitivities were achieved for detection of hCG (limit of detection, LOD: 5 pM) and FSH (LOD: 100 pM). Even the small-scale conformational changes caused by thermal inactivation and reducing the biological activity of the hormones were registered. In conclusion, the proposed assay is suitable for quantification of biological activity of gonadotropins and is a good alternative to antibody- and animal-testing-based assays used in pharmaceutical industry and clinical research.
University of Tartu, Institute of Chemistry, Tartu, Estonia.|Competence Centre on Health Technologies, Tartu, Estonia.
Olga
O
Mazina
University of Tartu, Institute of Chemistry, Tartu, Estonia.
Anni
A
Allikalt
Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
Juha S
JS
Tapanainen
Competence Centre on Health Technologies, Tartu, Estonia.|Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.|Department of Obstetrics and Gynaecology, University of Tartu, Tartu, Estonia.|Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia.
Andres
A
Salumets
University of Tartu, Institute of Chemistry, Tartu, Estonia.
Ago
A
Rinken
D015374:Biosensing Techniques; D002470:Cell Survival / Q000187:drug effects; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006801:Humans; D011994:Recombinant Proteins / Q000494:pharmacology; D013997:Time Factors
D006063:Chorionic Gonadotropin; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP