QUANTIFICATION OF ANTIGEN MOLECULES USING DYNAMIC FLOW CYTOMETRY

摘要

The described invention provides a method for quantifying cellular antigens that is independent of specially prepared calibration beads and antibody reagents. The described method can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions.

主权项

1. A method for quantifying a cellular antigen using flow cytometry without calibration beads, comprising:
(a) isolating cells from a subject; (b) measuring concentration of the isolated cells; (c) mixing the isolated cells with a fluorescently-labeled antibody that specifically binds to an antigen expressed by the isolated cells to initiate an antibody-antigen binding reaction; (d) collecting samples of the antibody-antigen binding reaction at multiple time intervals; (e) stopping the antibody-antigen binding reaction in the collected samples; (f) analyzing the samples by flow cytometry of (e) and obtaining a measured time-series of mean fluorescence intensity (MFI); calculating reaction rate constant k, for the antibody-antigen binding reaction based on the measured time-series of mean fluorescence intensity (MFI); and calculating amount of antigen n from k+.