Cooperativity in the antibody binding to surface-adsorbed antigen

Area of Science:

• Immunology
• Biochemistry
• Surface Chemistry

Background:

• Antibody-antigen interactions are crucial in immunology and diagnostics.
• Understanding antibody binding to surface-adsorbed antigens is key for various applications.
• Intrinsic antibody affinity alone does not fully explain binding to surface-bound antigens.

Purpose of the Study:

• To investigate the binding behavior of monoclonal antibodies (mAbs) with varying affinities to surface-adsorbed dinitrophenyl (DNP) antigen.
• To analyze the influence of epitope density and antibody concentration on binding cooperativity.
• To elucidate factors beyond intrinsic affinity affecting heterogeneous antibody-antigen binding.

Main Methods:

• Calibrated Enzyme-Linked Immunosorbent Assay (ELISA) to measure antibody binding.
• Scatchard plot analysis to determine binding concentration-dependence and cooperativity.
• Dissociation assays using tritium-labelled antibodies and unlabeled antibodies in solution.

Main Results:

• Positive cooperativity observed at low surface antibody concentrations, particularly for low-affinity mAbs at high epitope densities.
• Enhanced positive cooperativity for low-affinity clones at lower epitope densities.
• Negative cooperativity indicated at higher surface antibody concentrations, supported by increased dissociation rates.
• Binding behavior is influenced by surface concentration, epitope density, and antibody affinity.

Conclusions:

• Antibody binding to surface-adsorbed antigens is a heterogeneous process.
• Binding is not solely determined by intrinsic antibody affinity but also by antibody self-interaction and surface conditions.
• Cooperativity effects (positive and negative) play a significant role in the overall binding reaction.