title: Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics.
abstract: For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (K(A)>10(8)M(-1); K(D)<10(-8)M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate.
authors.affiliation: Chemistry Department, University of California, Davis, CA 95616, USA.
authors.forename: Tolulope A
authors.initials: TA
authors.lastname: Aweda
authors.forename: Claude F
authors.initials: CF
authors.lastname: Meares
mesh_terms: D000906:Antibodies / Q000737:chemistry; D000915:Antibody Affinity; D000936:Antigen-Antibody Complex / Q000032:analysis / Q000737:chemistry*; D002151:Calorimetry / Q000295:instrumentation / Q000379:methods*; D002614:Chelating Agents / Q000737:chemistry; D005453:Fluorescence; D007700:Kinetics; D008024:Ligands; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D046911:Macromolecular Substances / Q000032:analysis / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D012680:Sensitivity and Specificity; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D002614:Chelating Agents; D008024:Ligands; D046911:Macromolecular Substances; D009942:Organometallic Compounds; C551023:yttrium 2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetate