Dynamic quantification of antigen molecules with flow cytometry.

Area of Science:

• Immunology
• Biophysics
• Analytical Chemistry

Background:

• Traditional antigen quantification relies on equilibrium-based fluorescence detection, limiting accuracy with low-affinity antibodies.
• Existing calibration methods have inherent limitations, restricting the broad applicability of flow cytometry for molecule counting.

Purpose of the Study:

• To develop and validate a new flow cytometry approach for quantifying antigen molecules on cells.
• To overcome the limitations of equilibrium-based calibration methods in antigen quantification.

Main Methods:

• Utilized flow cytometry to monitor mean fluorescence intensity over time during antibody-antigen binding.
• Applied a diffusion-reaction mathematical model with nonlinear least squares fitting to experimental data.
• Compared results with the Quanti-BRITE calibration system for validation.

Main Results:

• Developed a method using binding rate constants to quantify antigen molecules, independent of calibration beads.
• Determined the binding site radius for CD8 antibody molecules, enabling recalculation of binding rates under varying conditions.
• Successfully demonstrated the method on human blood samples for CD8α antigen quantification on T cells.

Conclusions:

• A dynamic, time-resolved flow cytometry method offers accurate antigen quantification without experiment-specific calibration.
• The binding rate constant approach is versatile, applicable to various antibody affinities and binding conditions.
• This method provides a robust alternative for cell surface antigen measurement in diverse research settings.