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US 5,206,143 AGranted
Method and reagents for performing subset analysis using quantitative differences in fluorescence intensity
AssigneeSmithKline Beecham Corporation
InventorsPaul K. Horan, Sue E. Slezak
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Abstract
Methods for distinguishing multiple subpopulations of biological particles in a single sample based upon quantitative differences in the fluorescence intensity attributable to one or two fluorochromes with which the biological particles are labelled. The method is used with flow cytometric particle counting techniques to count and sort and biological particles such as the formed elements of blood and other tissue cells. Also disclosed are reagents containing fluorochrome-conjugated antibodies used in the methods.
Claims42
A method for distinguishing at least two subpopulations of biologic particles in a sample comprising the steps of
- a. selecting a first reagent that comprises:
- a first amount of a first labelled antibody that is labelled solely with a first fluorochrome, or
- a mixture of the first amount of the first labelled antibody and a first unlabelled antibody that is free of any fluorochrome,
- wherein the first labelled antibody and the first unlabelled antibody have the same antigen specificity and are specifically reactive to a first subpopulation biologic particle in the same, and
- wherein the first labelled antibody, or the first labelled antibody and the first unlabelled antibody, are present in a quantity sufficient to obtain maximum binding to the first subpopulation biologic particle and sufficient to confer upon the first subpopulation biologic particle a first fluorescence intensity,
- b. selecting a second reagent that comprises:
- a second amount of a second labelled antibody that is labelled solely with the first fluorochrome, or
- a mixture of the second amount of the second labelled antibody and a second unlabelled antibody that is free of any fluorochrome,
- wherein the second labelled antibody and the second unlabelled antibody have the same antigen specificity and are specifically reactive to a second subpopulation biologic particle in the sample, and
- wherein the second labelled antibody, or the second labelled antibody and the second unlabelled antibody, are present in an quantity sufficient to obtain maximum binding to the second subpopulation biologic particle and sufficient to confer upon the second subpopulation biologic particle a second fluorescence intensity,
- wherein the first and second amounts of the labelled antibodies are adjusted, relative to each other, so that one subpopulation biologic particle is distinguished from another subpopulation biologic particle based upon the difference in their fluorescence intensities,
- c. mixing the reagents with the sample,
- d. inducing fluorescence of the fluorochrome, and
- e. quantitatively measuring the intensity of fluorescence of each biologic particle in the sample.
11 more elements
A diagnostic kit for distinguishing at least two subpopulations of biologic particles in a sample comprising:
- a first reagent that comprises a first labelled antibody that is labelled solely with a first fluorochrome,
- a first diluent that comprises a first unlabelled antibody that is free of any fluorochrome,
- a second reagent that comprises a second labelled antibody that is labelled solely with the first fluorochrome, and
- a second diluent that comprises a second unlabelled antibody that is free of any fluorochrome,
- wherein the first labelled antibody and the first unlabelled antibody have the same antigen specificity and are specifically reactive to a first subpopulation biologic particle in the sample,
- wherein the second labelled antibody and the second unlabelled antibody have the same antigen specificity and are specifically reactive to a second subpopulation biologic particle in the sample, further comprising:
- a first additional reagent that comprises a first additional labelled antibody that is labelled solely with a second fluorochrome, and
- a first additional diluent that comprises a first additional unlabelled antibody that is free of any fluorochrome,
- wherein the first additional labelled antibody and the first additional unlabelled antibody have the same antigen specificity and are specifically reactive to a first additional subpopulation biologic particle in the sample, and
- wherein the first and second fluorochromes are inducible to fluoresce at different wavelengths.
7 more elements
Dependencies are inferred from the claim text.