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 for flow cytometry have inherent limitations and are often experiment-specific.

Purpose of the Study:

• To develop and validate a new method for quantifying antigen molecules per cell using flow cytometry.
• To overcome the limitations of equilibrium-based and calibration-dependent quantification methods.

Main Methods:

• Utilized flow cytometry to analyze 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 the antibody-antigen binding rate constant for quantification, independent of calibration beads.
• Determined the binding site radius of CD8 antibody molecules, enabling recalculation of binding rates.
• Demonstrated the method's applicability to low and high affinity antibodies under various conditions, including human blood samples.

Conclusions:

• The dynamic, model-based approach using binding kinetics offers a robust alternative to traditional antigen quantification.
• This method provides accurate cell surface molecule counts without reliance on specific calibration standards or beads.
• The findings are applicable to diverse antibody-antigen systems and experimental conditions in flow cytometry.