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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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