Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics

Tolulope A. Aweda, Claude F. Meares

Research output: Contribution to journalArticle

3 Scopus citations

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 8M -1; K D<10 -8M), 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.

Original languageEnglish (US)
Pages (from-to)145-153
Number of pages9
JournalMethods
Volume56
Issue number2
DOIs
StatePublished - Feb 2012

Keywords

  • Antibody
  • Binding
  • ITC
  • Kinetics
  • Ligand
  • Thermodynamics

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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