Determination of biological activity of gonadotropins hCG and FSH by Förster resonance energy transfer based biosensors

Abstract

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 protein ^TEpac^VV in 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.

Figure 1. Generation of the BacMam delivered cAMP detection-based functional assay system for quantification of biologically active gonadotropins using living cells.

Figure 2. Time dependent change of FRET signal in response to different concentrations of hCG and FSH.

COS7 cells expressing recombinant LH/CG receptor (A) and KGN cells expressing the endogenous FSH receptor (B) were transduced with BacMam-^TEpac^VV virus and incubated with serial concentrations of recombinant hCG (A) and FSH (B) for indicated time. The graphs show data points with connecting lines from a representative experiment performed in triplicate (n = 4).

Figure 3. Change of FRET signal at different concentrations of hCG and FSH.

COS7 cells with LH/CG receptor (A) and KGN cells with FSH receptor (B), transduced with BacMam-^TEpac^VV virus were incubated with different concentrations of recombinant hCG (A) and FSH (B) for 60 min. The graphs show data points from a representative experiment performed in triplicate (n = 4). The insets show the linear area of the response that was used for estimation of the limit of detection and quantification.

Figure 4

Influence of heat treatment on activities of recombinant hCG (A) and FSH (B) as measured by cAMP biosensor assay, electrochemiluminescence immunoassay (ECLIA) and enzyme-linked immunosorbent assay (ELISA). The hormone preparations were incubated at room temperature (untreated), at 65 °C or at 95 °C for 30 min and then their activities were measured. The concentration of hCG was 30 pM (cAMP assay) or 150 pM (ECLIA and ELISA) and the concentration of FSH was 300 pM. Data were normalized against activities of hormones kept at room temperature. Data are presented as mean ± SEM of 2 independent experiments carried out in triplicates.