Quantifying Antibody Binding Kinetics on Fixed Cells and Tissues via Fluorescence Lifetime Imaging

Area of Science:

• Biophysics
• Biochemistry
• Immunology

Background:

• Monitoring antigen-antibody interactions is crucial for understanding biological processes and disease.
• Existing methods often lack spatial resolution or are limited to specific sample types.
• Accurate measurement of binding kinetics on physiological substrates remains a challenge.

Purpose of the Study:

• To present a novel method for monitoring spatially localized antigen-antibody binding events.
• To measure binding kinetics on physiologically relevant substrates like cell and tissue sections.
• To investigate p53 kinetics in ovarian cancer tissue sections with differential biomarker expression.

Main Methods:

• Utilizing fluorescence lifetime imaging to differentiate between free and bound fluorescently tagged antibodies.
• Employing a microfluidic probe format to minimize mass transport effects and localize analysis.
• Measuring binding constants (kon) on surface-bound antigens, cell blocks, and ovarian cancer tissue sections.

Main Results:

• The method successfully monitors spatially localized antigen-antibody binding events.
• Binding constants (kon) were measured on model biomarkers and in ovarian cancer tissue.
• p53 kinetics correlated with biomarker expression levels, with distinct kon values for high and low expression.

Conclusions:

• Fluorescence lifetime imaging with a microfluidic probe offers a robust method for studying antigen-antibody binding kinetics on biological substrates.
• The technique provides insights into biomarker expression and its impact on molecular interactions in cancer tissues.
• This approach has potential applications in diagnostics and drug development.