Methods of detecting anti-leptin neutralizing antibodies
Provided herein are methods for detecting neutralizing antibodies to leptins, including metreleptin, as well as identifying subjects having such neutralizing antibodies.
1. A method of detecting neutralizing antibodies to leptin in a test sample obtained from a human subject, wherein the test sample is a blood, serum, or plasma sample, the method comprising the steps of:
(a) inactivating leptin present in the test sample;
(b) adding a known quantity of labeled leptin to the test sample;
(c) contacting the sample containing labeled leptin of (b) with a substrate capable of binding the labeled leptin;
(d) washing the substrate to remove unbound labeled leptin;
(e) measuring the label signal produced by the substrate-bound labeled leptin;
(f) measuring a negative control signal, said negative control signal produced by completing steps (a)-(e) in a control blood, serum, or plasma sample that does not contain antileptin neutralizing antibodies; and
(g) comparing the signal from (e) with the signal from (f), wherein if the level of signal measured in (e) is greater than or equal to the level of signal measured in (f), then the sample does not contain neutralizing antibodies to leptin, and wherein if the level of signal measured in (e) is less than the level of signal measured in (f), then the sample contains neutralizing antibodies to leptin.
2. A method of identifying human subjects having neutralizing antibodies to leptin in blood, serum, or plasma from the subject comprising the steps of:
(a) inactivating leptin present in the sample of blood, serum, or plasma;
(b) adding a known quantity of labeled leptin to the sample;
(c) contacting the sample containing labeled leptin of (b) with a substrate capable of binding the labeled leptin;
(d) washing the substrate to remove unbound labeled leptin;
(e) measuring the label signal produced by the bound labeled leptin;
(f) measuring a negative control signal, said negative control signal produced by completing steps (a)-(e), wherein the sample of blood, serum, or plasma does not contain antileptin neutralizing antibodies; and
(g) comparing percent difference in the signal from (e) and the signal from (f) with a cutpoint, wherein if the percent difference in the signal from (e) and the signal from (f) is greater than the cutpoint, then the subject tests positive for anti-leptin neutralizing antibodies.
3. The method of claim 2 , wherein the cutpoint is between about 5% and about 40%.
4. The method of claim 3 , wherein the subject is a healthy subject, and the cutpoint is between about 7% and about 8%.
5. The method of claim 1 , wherein the leptin is labeled with horseradish peroxidase, or ruthenium tris-bipyridine.
6. The method of claim 1 , wherein the inactivation step (a) is accomplished by heating the sample.
7. The method of claim 1 , wherein said substrate capable of binding the labeled leptin comprises:
i. a coated matrix;
ii. a tagged leptin receptor bound to the matrix of (i).
8. The method of claim 7 , wherein the leptin receptor is tagged with biotin.
9. The method of claim 1 , wherein said substrate capable of binding the labeled leptin comprises:
i. a coated matrix;
ii. an antibody bound to the matrix of (i); and
iii. a tagged leptin receptor bound to the antibody of (ii).
10. The method of claim 9 , wherein the leptin receptor is tagged with a tag suitable for affinity purification.
11. The method of claim 9 , wherein the antibody has suitable affinity for the leptin receptor tag.
12. The method of claim 9 , where the antibody is labeled with a tag suitable to bind to the coated matrix.
13. The method of claim 2 , wherein the inactivation step (a) is accomplished by heating the sample.
14. The method of claim 2 , wherein said substrate capable of binding the labeled leptin comprises:
i. a coated matrix;
ii. a tagged leptin receptor bound to the matrix of (i).
15. The method of claim 14 , wherein the leptin receptor is tagged with biotin.
16. The method of claim 2 , wherein said substrate capable of binding the labeled leptin comprises:
i. a coated matrix;
ii. an antibody bound to the matrix of (i); and
iii. a tagged leptin receptor bound to the antibody of (ii).
17. The method of claim 16 , wherein the leptin receptor is tagged with a tag suitable for affinity purification.
18. The method of claim 16 , wherein the antibody has suitable affinity for the leptin receptor tag.
19. The method of claim 16 , where the antibody is labeled with a tag suitable to bind to the coated matrix.