Problems and pitfalls with measurement of antibody affinity using solid phase binding in the ELISA

Area of Science:

• Immunology
• Biochemistry
• Analytical Chemistry

Background:

• Enzyme-linked immunosorbent assays (ELISA) are widely used for antibody affinity constant estimation.
• Current ELISA methodologies often assume homogeneous antibody binding to solid-phase antigens, which may not reflect biological reality.

Purpose of the Study:

• To theoretically derive antibody binding curves assuming homogeneous binding.
• To experimentally validate these theoretical curves using monoclonal antibodies against fibronectin.
• To investigate and explain deviations from homogeneous binding observed in experimental data.

Main Methods:

• Theoretical derivation of antibody binding curves under homogeneous binding conditions.
• Experimental ELISA to measure binding of monoclonal antibodies to solid-phase fibronectin.
• Comparative analysis of theoretical and experimental binding curves to identify discrepancies.

Main Results:

• Some monoclonal antibodies exhibited binding patterns consistent with theoretical homogeneous binding.
• Other antibodies showed significant departures from homogeneous binding, attributable to various surface effects.
• Identified specific surface effects that can influence antibody-antigen interactions on solid phases.

Conclusions:

• The assumption of homogeneous antibody binding in ELISA can lead to inaccurate affinity constant estimations.
• Surface effects significantly impact antibody binding kinetics and thermodynamics in ELISA.
• Current ELISA methods for antibody affinity ranking have inherent limitations due to non-homogeneous binding phenomena.