Whole blood assay for measuring AMPK activation
View Patent ↗A method of sample analysis is provided. In certain embodiments, the method comprises: a) labeling cells of a blood sample using an antibody that specifically binds to phospho-AMPK or a phosphorylated target thereof, to produce a labeled sample; and b) measuring antibody binding by a population of blood cells of the labeled sample using flow cytometry. In particular embodiments, the method may further comprise, prior to the labeling step: contacting blood with a test agent ex vivo or in vivo; and comparing the evaluation to results obtained from a reference sample of blood cells.
1. A method for determining the metabolic health of a subject, comprising:
a) obtaining a blood sample from a subject and permeablizing the cells of the blood sample;
b) contacting the permeabilized cells of the blood sample with a fluorescently detectable antibody that specifically binds to phospho-adenosine monophosphate kinase (AMPK);
c) measuring the amount of the fluorescently detectable antibody bound to phospho-AMPK in a plurality of the permeabilized cells in the blood sample, wherein the measuring comprises detecting the level of fluorescence signal on a single-cell basis, using flow cytometry; and
d) calculating a geometric mean fluorescence value of the blood sample based on the measurements obtained in (c), wherein the geometric mean fluorescence value provides an indication of the level of AMPK activation in said plurality of permeabilized cells that correlates with the metabolic health of the subject.
2. The method of claim 1 , wherein said plurality of permeabilized cells is a plurality of permeabilized white blood cells.
3. The method of claim 2 , wherein said plurality of permeabilized cells is composed of a plurality of permeabilized lymphocytes or granulocytes.
4. The method of claim 2 , wherein the calculating comprises calculating a geometric mean fluorescence value in a subset of the plurality of permeabilized cells.
5. The method of claim 4 , wherein the subset of the plurality of permeabilized cells comprises lymphocytes or granulocytes.
6. The method of claim 1 , wherein said method further comprises:
prior to said contacting step: b), contacting blood with a first amount of a test agent ex vivo or in vivo; and
comparing said geometric mean fluorescence value to a geometric mean fluorescence value obtained by the method of claim 1 from a reference sample of blood cells that is either not contacted with the test agent, or contacted with a second amount of the test agent wherein the first amount of the test agent and the second amount of the test agent are different, thereby determining a change in the level of AMPK activation in said plurality of permeabilized cells induced by the test agent.
7. The method of claim 6 , wherein said contacting blood with a first amount of the test agent comprises administering said test agent to a subject and then drawing blood from said subject after a specified period of time.
8. The method of claim 6 , wherein said contacting blood with a first amount of the test agent comprises drawing blood from a subject and then contacting said test agent with the drawn blood for a specified period of time.
9. The method of claim 6 , wherein said test agent is known to change AMPK activation level.
10. The method of claim 6 , wherein it is not known if said test agent induces a change in AMPK activation level.
11. The method of claim 6 , wherein said reference sample comprises blood cells obtained from the same subject as said blood sample that is contacted with the test agent.
12. The method of claim 6 , wherein said reference sample has not been contacted with said test agent.
13. The method of claim 6 , wherein said blood sample is contacted with a first amount of said test agent and the reference sample is contacted with a second amount of said test agent, wherein the first amount and second amount are different.
14. A method of determining the effect of a change in lifestyle on AMPK activation in a subject, comprising:
a) subjecting a subject to a change in lifestyle;
b) obtaining a blood sample from said subject and permeablizing the cells of the blood sample;
c) contacting the permeabilized cells of the blood sample with a fluorescently detectable antibody that specifically binds to phospho-adenosine monophosphate kinase (AMPK);
d) measuring the amount of the fluorescently detectable antibody bound to phospho-AMPK in a plurality of permeabilized cells in the blood sample, wherein the measuring comprises detecting the level of fluorescence signal on a single-cell basis, using flow cytometry;
e) calculating a geometric mean fluorescence value of the blood sample based on the measurements obtained in (d), wherein the geometric mean fluorescence value provides an indication of the level of AMPK activation in said plurality of permeabilized cells that correlates with the metabolic health of the subject; and
f) comparing said level of AMPK activation to the level of AMPK activation obtained from a reference sample of blood cells from the subject before the change in lifestyle, thereby determining the effect of said change in lifestyle on AMPK activation of said subject.
15. The method of claim 14 , wherein said change in lifestyle is a diet.
16. The method of claim 14 , wherein said change in lifestyle is increased exercise.