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Quantifying Antibody Binding Kinetics on Fixed Cells and Tissues via Fluorescence Lifetime Imaging
Prerit Mathur1,2, Anna Fomitcheva Khartchenko1,2, Stavros Stavrakis1
1Institute for Chemical and Bioengineering, Department of Chemistry & Applied Biosciences, Eidgenössische Technische Hochschule (ETH Zürich), Vladimir-Prelog-Weg 1-5/10, 8093 Zürich, Switzerland.
Insights
We developed a fluorescence imaging method to monitor antigen-antibody binding on cells and tissues. This technique measures binding kinetics, revealing differences in p53 expression in ovarian cancer tissues.
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.
Abstract:
We present a method for monitoring spatially localized antigen-antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates. This enables measurement of binding constants (kon) on surface-bound antigens and on cell blocks using model biomarkers. Finally, we directly measure p53 kinetics with differential biomarker expression in ovarian cancer tissue sections, observing that the degree of expression corresponds to the changes in kon, with values of 3.27-3.50 × 103 M-1 s-1 for high biomarker expression and 2.27-2.79 × 103 M-1 s-1 for low biomarker expression.
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