Measurement of the functional affinity constant of a monoclonal antibody for cell surface receptors using kinetic exclusion fluorescence immunoassay

J Immunol Methods. 2005 Sep;304(1-2):1-14. doi: 10.1016/j.jim.2005.04.009.


Measuring a protein-ligand interaction in solution, away from the ligand's cellular environment, may not provide an affinity value applicable in vivo. Here, we present a simple, accurate and highly sensitive method for determining the antibody affinity to cell surface receptor, hIGFR, and compare this data to affinity determined for the soluble receptor. Measurements were performed on both full-length bivalent IgG and the monovalent Fab fragments to assess possible differences in apparent affinity introduced by avidity of the bivalent IgG. Affinities determined for soluble hIGFR were 4 x 10(-12) M for the bivalent IgG and monovalent Fab. Comparable affinities of 6 x 10(-12) M and 1 x 10(-11) M for the bivalent IgG and Fab, respectively, were also determined for full-length hIGFR on cell surface. The method described allows estimation of reactant concentrations (anti-IGFR antibody) relative to one known reference concentration (the concentration of soluble hIGFR in our case) allowing us to estimate the average receptor density on the cell surface. Taken together, we believe these data can provide valuable insight into antibody behavior in vivo, especially in the case of insoluble or difficult to purify transmembrane receptors.

Publication types

  • Comparative Study

MeSH terms

  • Antibodies, Monoclonal / metabolism*
  • Antibody Affinity*
  • Binding Sites, Antibody
  • Carbocyanines
  • Fluorescent Antibody Technique, Direct / methods*
  • Fluorescent Dyes
  • Humans
  • Immunoglobulin Fab Fragments / metabolism
  • Immunoglobulin G / metabolism
  • Kinetics
  • Receptor, IGF Type 1 / immunology*


  • Antibodies, Monoclonal
  • CY5.5 cyanine dye
  • Carbocyanines
  • Fluorescent Dyes
  • Immunoglobulin Fab Fragments
  • Immunoglobulin G
  • Receptor, IGF Type 1