Detection of spectrin and spectrin proteolytic cleavage products in assessing nerve cell damage
View Patent ↗Methods for detecting a cell damage relate to the discovery that proteases are selectively activated in subjects suffering from nervous system damage compared to samples from healthy subjects. Breakdown products reflecting activation of proteases that degrade spectrin are produced. A cell injury is detected by providing a biological sample derived from the subject; detecting in the sample the presence of these breakdown products generated by multiple proteases, and correlating the presence of these breakdown products with the presence or type of cell damage.
1. A mixture comprising:
a biological sample isolated from a subject suspected of having a damaged nerve cell, the biological sample being a fluid in communication with the nervous system of the subject prior to being isolated from the subject; and
at least two added antibodies that specifically and independently bind to at least two markers selected from αII-spectrin and an αII-spectrin breakdown product (SBDP) selected from at least one of SBDP150i, SBDP150, SBDP145 and SBDP120 generated from proteolytic cleavage of αII-spectrin by at least one protease selected from the group consisting of caspase-3 and calpain.
2. The mixture of claim 1 , wherein the markers consist of αII-spectrin, SBDP150i, SBDP150, SBDP145, and SBDP120.
3. The mixture of claim 1 , wherein the at least two antibodies specifically and independently bind to αII-spectrin and at least one of SBDP150i, SBDP150, SBDP145, and SBDP120.
4. The mixture of claim 1 , wherein the at least two antibodies specifically and independently bind to SBDP145 and to only one of the group consisting of αII-spectrin, SBDP150i, SBDP150, and SBDP120.
5. The mixture of claim 1 , wherein the subject is human.
6. The mixture of claim 1 , wherein the SBDP's are immobilized on a substrate.
7. The mixture of claim 1 , further comprising a detectable label.
8. The mixture of claim 7 , wherein the detectable label is conjugated to the at least two antibodies.
9. The mixture of claim 7 , wherein the detectable label is conjugated to a substance that specifically binds to the at least two antibodies.
10. The mixture of claim 1 , wherein the antibodies specifically and independently bind to SBDP120, SBDP150i, SBDP145 and optionally to αII-spectrin.
11. The mixture of claim 1 , wherein the antibodies specifically and independently bind to SBDP145, SBDP150, SBDP150i and optionally to αII-spectrin.
12. The mixture of claim 1 , wherein the antibodies specifically and independently bind to SBDP145, SBDP150i and optionally to αII-spectrin.
13. The mixture of claim 1 , wherein the antibodies specifically and independently bind to SBDP145, SBDP150 and optionally to αII-spectrin.
14. The mixture of claim 1 , wherein the antibodies specifically and independently bind to SBDP145, SBDP120 and optionally to αII-spectrin.
15. The mixture of claim 1 , wherein the antibodies specifically and independently bind to αII-spectrin and SBDP145.
16. The mixture of claim 1 , wherein the at least two added antibodies specifically and independently bind only to SBDP145.
17. A kit for analyzing cell damage in a subject, the kit comprising:
(a) a substrate for holding a biological sample isolated from a human subject suspected of having a damaged nerve cell, the biological sample being a fluid in communication with the nervous system of the subject prior to being isolated from the subject;
(b) antibodies that specifically and independently bind to at least two markers selected from αII-spectrin and an αII-spectrin breakdown product (SBDP) selected from the group consisting of SBDP120, SBDP145, SBDP150 and SBDP150i wherein the SBDPs are generated from proteolytic cleavage of αII-spectrin by at least one protease selected from the group consisting of caspase-3 and calpain; and
(c) printed instructions for reacting the antibodies with the biological sample or a portion of the biological sample to detect the presence or amount of the markers in the biological sample.
18. The kit of claim 17 , wherein the markers detected are αII-spectrin and an SBDP selected from the group consisting of SBDP150i, SBDP150, SBDP145, and SBDP120.
19. The kit of claim 18 , wherein the antibodies detect the presence and amount of αII-spectrin, SBDP150i, SBDP150, SBDP145, and SBDP120.
20. The kit of claim 19 , wherein the antibodies specifically detect αII-spectrin, SBDP150, SBDP145, and SBDP120.
21. The kit of claim 17 , wherein the subject is a human.
22. The kit of claim 17 , further comprising a detectable label.
23. The kit of claim 22 , wherein the detectable label is conjugated to at least one antibody.
24. The kit of claim 22 , wherein the detectable label is conjugated to a secondary antibody that specifically binds to at least one antibody.
25. A kit for analyzing cell damage in a subject, the kit comprising:
(a) a substrate for holding a biological sample isolated from a subject suspected of having a damaged nerve cell, the biological sample being a fluid in communication with the nervous system of the subject prior to being isolated from the subject;
(b) an antibody that specifically and independently binds to a marker identified as αII-spectrin breakdown product (SBDP) 145 kDa generated from proteolytic cleavage of αII-spectrin by at least one protease selected from the group consisting of caspase-3 and calpain; and
(c) printed instructions for reacting the antibody with the biological sample or a portion of the biological sample to detect the presence or amount of the 145 kDa marker in the biological sample.
26. The kit of claim 25 , further comprising one or more antibodies that specifically and independently bind to an SBDP selected from the group consisting of SBDP150i, SBDP150 and SBDP120.
27. The kit of claim 26 , further comprising an antibody that specifically and independently binds to αII-spectrin.
28. The kit of claim 25 , further comprising an antibody that specifically and independently binds to αII-spectrin.
29. The kit of claim 25 , wherein the subject is human.