Methods for measurement of lymphocyte function
View Patent ↗A method for measuring the responses of sets or subsets of lymphocytes to mitogens or antigens in a sample is disclosed comprising incubating a population of cells with a mitogen or antigen, separating the desired subset of cells by means of the interaction of a specific binding reagent that is attached to the solid phase with a cell surface determinant that is present on the cell subset of interest, lysing the separated cells, and measuring an intracellular component that is increased if the cells have responded to the stimulus. The method provides a convenient, simple, and reliable method for measuring immune function in a variety of conditions.
1. A method for detecting activation of lymphocytes comprising the steps of:
a) incubating a sample containing a mixed population of cell types including a plurality of subsets of lymphocytes where each subset includes lymphocytes with characteristic determinants that distinguish one subset from another, with an inducing agent selected from the group consisting of mitogens and antigens; then
b) separating a selected subset of lymphocytes from said sample by:
i) contacting said sample with a solid support having a specific binding substance, said specific binding substance being specific for at least one characteristic determinant of said selected subset of lymphocytes, and said contacting step resulting in the formation of a complex of cells, binding substance and solid support; and
ii) separating said complex from a remainder of said sample; then
c) lysing lymphocytes in said selected subset to release an activation-correlated intracellular component selected from the group consisting of ATP, NADP, and PCNA; then
d) measuring a level of said activation-correlated intracellular component released from said lymphocytes and a level of said activation-correlated intracellular component in a control sample; and
e) determining activation of lymphocytes for said selected subset of lymphocytes from said level of said activation-correlated intracellular component measured in said measuring step, wherein said lymphocytes are activated if said level of said activation-correlated intracellular component is compared to said level of said activation-correlated intracellular component in said control sample.
2. A method as in claim 1 where said antigen is a virus or a bacteria or a subcomponent thereof selected from the group consisting of O fever cells, PPD, tetanus toxoid, OSPA, OSPB, OSPC, gp 120 protein, and peptides derived from gp 120.
3. A method according to claim 1 wherein said inducing agent is selected from the group consisting of drugs, organic chemicals, inorganic chemicals, metals, tumor cell proteins, and proteins from transplanted organs.
4. A method according to claim 1 wherein said subset of lymphocytes is selected from the group consisting of T lymphocytes, helper T lymphocytes, natural killer T lymphocytes, and cytotoxic T lymphocytes.
5. A method according to claim 1 wherein said characteristic determinant is a characteristic determinant of T cells and is selected from the group consisting of a functional marker, a marker of a particular differentiation stage, and an activation marker.
6. A method according to claim 1 , wherein said solid support comprises magnetic or paramagnetic material.
7. A method as recited in claim 6 wherein the step of separating said complex is performed by magnetic separation.
8. A method according to claim 1 , wherein said solid support comprises polystyrene.
9. A method according to claim 1 wherein said detecting step includes the step of adding luciferin to said intracellular component released from said lymphocytes in said subset of lymphocytes.
10. A method according to claim 1 , wherein the said specific binding substance is an antibody.
11. A method according to claim 1 , wherein said specific binding substance is a cytokine.
12. A method according to claim 1 wherein said intracellular component is ATP.
13. A method according to claim 12 wherein said control sample comprises liposomes containing ATP.