Biointeraction analysis by high-performance affinity chromatography: Kinetic studies of immobilized antibodies

Mary Anne Nelson, Annette Moser, David S. Hage

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


A system based on high-performance affinity chromatography was developed for characterizing the binding, elution and regeneration kinetics of immobilized antibodies and immunoaffinity supports. This information was provided by using a combination of frontal analysis, split-peak analysis and peak decay analysis to determine the rate constants for antibody-antigen interactions under typical sample application and elution conditions. This technique was tested using immunoaffinity supports that contained monoclonal antibodies for 2,4-dichlorophenoxyacetic acid (2,4-D). Association equilibrium constants measured by frontal analysis for 2,4-D and related compounds with the immobilized antibodies were 1.7-12 × 106 M-1 at pH 7.0 and 25 °C. Split-peak analysis gave association rate constants of 1.4-12 × 105 M-1 s-1 and calculated dissociation rate constants of 0.01-0.4 s-1 under the application conditions. Elution at pH 2.5 for the analytes from the antibodies was examined by peak decay analysis and gave dissociation rate constants of 0.056-0.17 s-1. A comparison of frontal analysis results after various periods of column regeneration allowed the rate of antibody regeneration to be examined, with the results giving a first-order regeneration rate constant of 2.4 × 10-4 s-1. This combined approach and the information it provides should be useful in the design and optimization of immunoaffinity chromatography and other analytical methods that employ immobilized antibodies. The methods described are not limited to the particular analytes and antibodies employed in this study but should be useful in characterizing other targets, ligands and supports.

Original languageEnglish (US)
Pages (from-to)165-171
Number of pages7
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Issue number2
StatePublished - Jan 15 2010


  • Antibody-antigen interactions
  • Biointeraction analysis
  • Frontal affinity chromatography
  • High-performance affinity chromatography
  • Immunoaffinity chromatography
  • Kinetic studies
  • Peak decay analysis
  • Split-peak analysis

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology


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