Variable domain-identical antibodies exhibit IgG subclass-related differences in affinity and kinetic constants as determined by surface plasmon resonance

Mol Immunol. 1994 Jun;31(8):577-84. doi: 10.1016/0161-5890(94)90165-1.


We have analysed the binding of variable domain-identical mouse monoclonal antibodies (mAb) of the IgG3, IgG1 and IgG2b subclasses, as well as F(ab')2 fragments derived from the IgG3 and IgG1 mAb, to a multivalent glycoprotein target. Using a biosensor device (BIAcore, Pharmacia Biosensor) that measures the mass of the antibody (or other receptor molecule) deposited on a sensor chip displaying the relevant epitopes, we found that the IgG3 mAb binds more effectively than the other antibody species at a high but not a low epitope density. The greater functional affinity associated with the IgG3 mAb, at high epitope density, was correlated with both slower dissociation rate constants and faster association rate constants in comparison with the IgG1 and IgG2b mAb and the F(ab')2 fragments derived from the IgG3 and IgG1 mAb. Evidence for slower dissociation kinetics for the IgG3 mAb versus the IgG1 and IgG2b mAb was also obtained by ELISA and flow cytometry. These results demonstrate that: (1) differences in heavy chain constant (CH) domains can significantly influence apparent functional affinity for multivalent antigen, as determined without the use of covalently modified primary or secondary antibodies; (2) differences in CH domains can alter both association and dissociation rate constants for interactions between IgG antibodies and multivalent antigen; and (3) these effects of CH domains depend on epitope density. The effect of constant region differences on the apparent association rate constants suggests new approaches for achieving better binding or functional effectiveness through antibody engineering.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylglucosamine / analogs & derivatives
  • Acetylglucosamine / immunology*
  • Allosteric Regulation
  • Animals
  • Antibodies, Monoclonal / classification
  • Antibodies, Monoclonal / immunology*
  • Antibody Affinity
  • Antigen-Antibody Reactions / physiology*
  • Biosensing Techniques
  • Cell Wall / immunology
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes / immunology
  • Flow Cytometry
  • Immunoglobulin G / classification
  • Immunoglobulin G / immunology*
  • Immunoglobulin Variable Region / immunology*
  • Kinetics
  • Mice
  • Models, Chemical
  • Serum Albumin, Bovine / immunology


  • Antibodies, Monoclonal
  • Epitopes
  • Immunoglobulin G
  • Immunoglobulin Variable Region
  • N-acetylglucosamine-bovine serum albumin conjugate
  • Serum Albumin, Bovine
  • Acetylglucosamine