Summary of traditional biologic assays for GLP-1 analogs
| Molecular/Cellular Measurement | Description/Outcome | References |
|---|---|---|
| Receptor-binding assays: | ||
| Radioligand competition | Determines binding affinity (IC50 or Kd) by measuring radioactivity remaining on the receptor after competitive inhibition of radioligand with a GLP-1 analog. | Mathi et al., 1997; Tibaduiza et al., 2001; Xiao et al., 2001 |
| Time-resolved fluorescence resonance energy transfer (FRET) | Determines binding affinity (IC50) by measuring a decrease in FRET signal between Tb-labeled receptor and fluorescent ligand after competitive inhibition with a GLP-1 analog. | Maurel et al., 2008; Zwier et al., 2010; Roed et al., 2014 |
| Circular dichroism and fluorescence spectroscopies | Determines binding affinity by measuring conformational changes of a receptor protein upon ligand binding. | Runge et al., 2007 |
| Isothermal titration calorimetry | Determines thermodynamic parameters of binding, such as the dissociation constant, enthalpy change, entropy change, and reaction stoichiometry by measuring heat changes during receptor–ligand interaction. | Wiseman et al., 1989; Bazarsuren et al., 2002; Donnelly, 2012 |
| Total-internal reflection fluorescence imaging | Determines equilibrium constants and dissociation rates by monitoring fluorescence-labeled single molecule on lipid bilayer. | Fox et al., 2009; Myers et al., 2012 |
| Surface plasmon resonance | Determines dissociation constants by real-time measurement of receptor–ligand interaction. | Bazarsuren et al., 2002; Schroder-Tittmann et al., 2010; Locatelli-Hoops et al., 2013 |
| Photoaffinity labeling | Identifies residues of peptide and receptor at binding interface that are spatially proximal to each other. | Chowdhry and Westheimer, 1979; Ji et al., 1997; Vodovozova, 2007; Chen et al., 2009; Miller et al., 2011 |
| Receptor functional assays: | ||
| Homogeneous time-resolved fluorescence (HTRF) or alpha screen cAMP assays | HTRF technology is an immunoassay based on a FRET between a Tris-bipyridine europium cryptate used as a long-lived fluorescent donor and a chemically modified allophycocyanin used as acceptor. Alpha technology is a bead-based proximity assay. | Gesellchen et al., 2006; Einhorn and Krapfenbauer, 2015 |
| Radioimmunoassay | Determines receptor-stimulating potency (EC50) by quantitative analysis of cAMP production with immobilized anti-cAMP or anti-cGMP antibodies and radiolabeled cAMP/cGMP. | Farmer et al., 1975; Wheeler et al., 1995 |
| FRET-based cAMP assay | Determines receptor-stimulating potency (EC50) by measuring a decreased FRET signal between fluorescent proteins (CFP and YFP) and Epac protein. | Holz, 2004; Nikolaev et al., 2004; Landa et al., 2005; Harbeck et al., 2006; Sloop et al., 2010 |
| Luciferase reporter assay | Determines receptor-stimulating potency (EC50) by measuring luminescence that is increased by transcription of transfected luciferase reporter plasmid linked to cAMP response element. | Grynkiewicz et al., 1985; Cullinan et al., 1994; Bode et al., 1999; Miranda et al., 2008; Murage et al., 2008; Smale, 2010 |
| Intracellular calcium ion | Determines receptor activation by measuring intracellular Ca2+ level with calcium-sensitive dye, Fura-2. | Grynkiewicz et al., 1985; Cullinan et al., 1994; Bode et al., 1999 |
| Determination of incretin effects: | ||
| Glucose tolerance test (GTT; oral GTT, OGTT; intraperitoneal GTT, IPGTT; intravenous GTT, IVGTT) | Determines insulinotropic action of GLP-1 analogs by measuring glucose level after their administration. | Kreymann et al., 1987; Toft-Nielson et al., 1996 |
| Insulin secretion | Determines potency of GLP-1 analogs by measuring insulin secretagogue action. | Albano et al., 1972; Andersen et al., 1993; Goke et al., 1993b; Toft-Nielson et al., 1996; Kjems et al., 2003; Peyot et al., 2009 |