Quantification of antigen molecules using dynamic flow cytometry
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsDarya Y. Orlova, David R. Parks, Leonore A. Herzenberg, Andrei V. Chernyshev, Alexander E. Moskalensky
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Abstract
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.
Claims17
A method for quantifying a cellular antigen using flow cytometry without a calibration system, 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 at the time of sample collection;
- (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+, wherein the amount of antigen is quantified without a calibration system.
A method for quantifying a cellular antigen using flow cytometry without a calibration system, comprising:
- (a) isolating cells from a subject, wherein the cells are selected from the consisting of cytotoxic T-cells, stem cells, granulocytes, monocytes, T-lymphocytes, B-lymphocytes, thrombocytes and natural killer cells;
- (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 at the time of sample collection;
- (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+, wherein the amount of antigen is quantified without a calibration system.
Dependencies are inferred from the claim text.