# US 8,771,971 B2 · Methods and kits for measurement of lymphocyte function > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,771,971 B2 - Document ID: US8771971B2 - Status: Granted - Kind: B2 - Application number: 12/375,135 - Filed: 2007-07-30 - Published: 2010-07-29 - Granted: 2014-07-08 - Earliest claimed: 2006-07-28 - Assignees: The Research Foundation of State University of New York - Applicants: Allen J. Norin - Inventors: Allen J. Norin - Web page: https://patentgrep.com/patent/US8771971B2 ## CPC - G01N 33/56972: {White blood cells} - Y10T 436/25: including sample preparation - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract The present invention provides simple and rapid methods for measuring the function of a desired subset of lymphocytes, for example, T cells, B cells or NK cells. In addition, the present invention provides an all-in-one kit that contains reagents which permit a rapid and reliable analysis of the functions of T cells, B cells and NK cells obtained directly from whole blood or cord blood. ## Claims ### Claim 1 A method for determining the function of a selected subset of lymphocytes, comprising: contacting a sample containing subsets of lymphocytes with a stimulating agent configured to activate at least one selected subset of lymphocytes; contacting the sample with an isolation agent that specifically binds said stimulated selected subset of lymphocytes, wherein the isolation agent is coated onto magnetic beads of one to five microns and wherein the stimulating agent used to activate at least one selected subset of lymphocytes is the same agent as the isolation agent used to specifically bind the selected subset of lymphocytes; separating from the remainder of the sample, said selected subset of activated lymphocytes, which are bound to said isolation agent; and measuring cells from the separated subset of activated lymphocytes for functional activity, gene expression, or lymphocyte subset-specific activation marker, wherein the measurement is compared with that from unstimulated lymphocytes. ### Claim 2 A method for determining the function of a selected subset of lymphocytes, comprising: contacting a sample containing subsets of lymphocytes with an isolation agent that specifically binds said selected subset of lymphocytes to separate said selected subset of lymphocytes from the remainder of the sample, wherein the isolation agent is coated onto magnetic beads of one to five microns; contacting the separated, selected subset of lymphocytes with a stimulating agent for a time sufficient to activate said lymphocytes, wherein the stimulating agent is configured to activate the at least one selected subset of lymphocytes and is the same agent as the isolation agent used to specifically bind the selected subset of lymphocytes; and measuring the activated and isolated subset of lymphocytes for a functional activity, gene expression, or lymphocyte subset-specific activation marker to determine the function of said selected subset of activated lymphocytes, wherein the measurement is compared with that from unstimulated lymphocytes. ### Claim 3 (depends on 1, 2) The method of claim 1 or 2, wherein said selected subset of lymphocytes is composed of T lymphocytes. ### Claim 4 (depends on 3) The method of claim 3, wherein said isolation agent comprises the magnetic beads immobilized with an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-T cell receptor antibody, an antigen-binding fragment of any of the foregoing antibodies, or a combination of any of the foregoing antibodies or fragments. ### Claim 5 (depends on 1, 2) The method of claim 1 or 2, wherein the stimulating agent is coated onto magnetic beads. ### Claim 6 (depends on 5) The method of claim 5, wherein said stimulating agent is selected from the group consisting of phorbol esters, phytohemagluttinin (PHA), Concanavalin A (ConA), an anti-T cell receptor antibody bound to magnetic beads, an anti-CD3 antibody bound to magnetic beads, an antigen-binding fragment of an anti-CD3 antibody bound to magnetic beads, anti-CD28 antibody bound to magnetic beads, an antigen-binding fragment of anti-CD28 antibody bound to magnetic beads, an anti-CD3 antibody or antigen-binding fragment thereof bound to magnetic beads in combination with interleukin 2 (IL-2), and a combination thereof. ### Claim 7 (depends on 5) The method of claim 5, wherein said stimulating agent is selected from the group consisting of PansorbinR, Protein A, an anti-IgM antibody or an antigen-binding fragment thereof bound to beads, and a combination thereof. ### Claim 8 (depends on 5) The method of claim 5, wherein said stimulating agent is selected from the group consisting of interleukin 2 (IL-2), interleukin 15 (IL-15), Haymaker, an anti-CD16 antibody or an antigen-binding fragment thereof, and a combination thereof. ### Claim 9 (depends on 1, 2) The method of claim 1 or 2, wherein said selected subset of lymphocytes is composed of B lymphocytes. ### Claim 10 (depends on 9) The method of claim 9, wherein said isolation agent is Protein A bound to magnetic beads, an anti-CD19 antibody or an antigen-binding fragment thereof bound to magnetic beads, an anti-CD20 antibody or an antigen-binding fragment thereof bound to magnetic beads, an anti-IgM antibody or an antigen-binding fragment thereof bound to magnetic beads. ### Claim 11 (depends on 1) The method of claim 1 wherein the magnetic beads are one to two micron. ### Claim 12 (depends on 2) The method of claim 2 wherein the magnetic beads are magnetic beads of one to two microns. ### Claim 13 (depends on 1, 2) The method of claim 1 or 2, wherein said selected subset of lymphocytes is composed of natural killer (NK) cells. ### Claim 14 (depends on 13) The method of claim 13, wherein said isolation agent is an anti-CD16 antibody or an antigen-binding fragment thereof, bound to magnetic beads. ### Claim 15 (depends on 1, 2) The method of claim 1 or 2, wherein said sample is a whole blood sample obtained from a human subject. ### Claim 16 (depends on 1, 2) The method of claim 1 or 2, wherein the selected subset of cells are lysed after stimulation and separation, and at least one of the following is measured: the level of adenosine triphosphate (ATP) from cell lysate, or the expression of a gene encoding interleukin 2 (IL-2), a cytokine receptor, perforin, Granzyme B, gamma interferon, nuclear factor (NF) kappa B, or nuclear factor of activated T cells (NFAT). ### Claim 17 (depends on 1, 2) The method of claim 1 or 2, wherein the selected subset of cells is analyzed by fluorescent activated flow cytometry for an activation marker selected from the group consisting of CD25, CD69, IL-2, perforin and Gramzyme B. ### Claim 18 A kit for determining the function of a selected subset of lymphocytes, comprising a stimulating agent configured to activate a selected subset of lymphocytes, an isolation agent that specifically binds and isolates said selected subset of lymphocytes, wherein the isolation agent is coated onto magnetic beads of one to five microns, wherein the stimulating agent used to activate the at least one selected subset of lymphocytes is the same agent as the coated isolation agent used to specifically bind the selected subset of lymphocytes, a lysis solution, and reagents for measuring the adenosine triphosphate (ATP) level from lysed lymphocytes, reagents for detecting changes in gene expression, or reagents for detecting activation markers based on flow cytometry. ### Claim 19 A kit for propagating T cells, B cells or natural killer (NK) cells comprising a stimulating agent configured to activate a selected subset of lymphocytes, an isolation agent that specifically binds and isolates said selected subset of lymphocytes, wherein the isolation agent is coated onto magnetic beads of one to five microns, at least one of one micron anti-CD3/CD28 coated magnetic beads, one micron Protein A coated magnetic beads, and one micron anti-CD16 coated magnetic beads, respectively, wherein the stimulating agent used to activate the at least one selected subset of lymphocytes is the same agent as the coated isolation agent used to specifically bind the selected subset of lymphocytes.