Detection of platelet-derived shed CD31
The present invention relates to various soluble forms of CD31, including a novel form which is shed by activated platelets and released into the circulation. Methods for detecting said soluble forms of CD31 are disclosed, as are methods of specifically 1 detecting said platelet-derived shed CD31 and the use of such methods as a diagnostic tool.
1. A method for detecting a platelet-derived shed ectodomain of a cluster of differentiation 31 (CD31) protein among soluble forms of CD31 in a biological sample, wherein said soluble forms include a soluble splice variant of said CD31 protein, wherein said platelet-derived shed ectodomain of a CD31 protein is a CD31 fragment (i) comprising the 1st to 5th extracellular immunoglobulin-like (Ig-like) domains and the whole or a part of the 6th extracellular Ig-like domain comprising an amino acid sequence corresponding to residues 494 to 501 of human CD31 protein and (ii) not comprising the intracellular domain of said full-length protein, the method comprising the steps of:
a) providing a first discriminating antibody, wherein the antibody specifically binds to an epitope located in a region that is present on said splice variant and on said platelet-derived shed ectodomain, wherein the antibody is labeled with a detectable label;
b) providing a second discriminating antibody, wherein the antibody specifically binds to an epitope located in a region that is present on said splice variant and absent from said platelet-derived shed ectodomain, wherein the antibody is labeled with a detectable label;
c) contacting said antibodies with the biological sample;
d) measuring the signal obtained from each detectable label;
wherein said first discriminating antibody binds to the 6th Ig-like extracellular domain of said CD31;
and wherein said second discriminating antibody binds to the intracellular domain of said CD31.
2. The method according to claim 1 , further comprising the step of calculating the percentage and/or the amount of said soluble forms that corresponds to said platelet-derived shed ectodomain, and/or the step of calculating either the ratio of platelet-derived shed ectodomain to soluble forms, or the ratio of soluble splice variant to soluble forms.
3. The method of claim 1 , wherein said method comprises a step of providing a capture antibody before step a), wherein said capture antibody specifically binds to an epitope located in a region that is present both on said a platelet-derived shed ectodomain and on said splice variant.
4. The method of claim 3 , wherein said capture antibody is immobilized on a solid support.
5. The method of claim 3 , wherein said capture antibody binds to a region within the 1 st , 2 nd , 3 rd , 4 th , 5 th or 6 th Ig-like extracellular domain of said CD31.
6. The method according to claim 1 , wherein the signal in step c) is measured by flow cytometry.
7. The method according to claim 1 , wherein said biological sample is plasma, blood or urine.
8. The method according to claim 1 , wherein said biological sample is obtained from an individual suffering from or at risk of suffering from a thrombotic or an autoimmune disorder.
9. The method of claim 1 , wherein said first discriminating antibody is selected from the group consisting of the antibodies MBC78.2, PECAM1.1, PECAM 1.2 and HC1/6.
10. The method of claim 1 , wherein said second discriminating antibody is selected from the group consisting of the antibodies MBC235.1 and SP38.
11. The method of claim 3 , wherein said capture antibody is selected from the group consisting of PECAM 1.1, PECAM 1.2, HC1/6, MBC78.2, MEM-05, MBC78.1, JC70A, 9G11, WM59 and MBC78.3.
12. A method for detecting a platelet-derived shed ectodomain of a cluster of differentiation 31 (CD31) protein among soluble forms of CD31 in a biological sample, wherein said soluble forms include a soluble splice variant of said CD31 protein, wherein said platelet-derived shed ectodomain of a CD31 protein is a CD31 fragment (i) comprising the 1 st to 5 th extracellular immunoglobulin-like (Ig-like) domains and the whole or a part of the 6 th extracellular Ig-like domain comprising an amino acid sequence corresponding to residues 494 to 501 of human CD31 protein and (ii) not comprising the intracellular domain of said full-length protein, the method comprising the steps of:
a) providing a first discriminating antibody, wherein the antibody specifically binds to an epitope located in a region that is present on said splice variant and on said platelet-derived shed ectodomain, wherein the antibody is labeled with a detectable label;
b) providing a second discriminating antibody, wherein the antibody specifically binds to an epitope located in a region that is present on said splice variant and absent from said platelet-derived shed ectodomain, wherein the antibody is labeled with a detectable label;
c) contacting said antibodies with the biological sample;
d) measuring the signal obtained from each detectable label;
wherein said first discriminating antibody binds to the 6 th Ig-like extracellular domain of said CD31 and wherein said first discriminating antibody is selected from the group consisting of the antibodies MBC78.2, PECAM1.1, PECAM 1.2, HC1/6 and antibodies binding to the same epitope;
and wherein said second discriminating antibody binds to the intracellular domain of said CD31, and wherein said second discriminating antibody is selected from the group consisting of the antibodies MBC235.1, SP38 and antibodies binding to the same epitope.
13. The method of claim 12 , wherein said method comprises a step of providing a capture antibody before step a), wherein said capture antibody specifically binds to an epitope located in a region that is present both on said platelet-derived shed ectodomain and on said splice variant and wherein said capture antibody is selected from the group consisting of PECAM 1.1, PECAM 1.2, HC1/6, MBC78.2, MEM-05, MBC78.1, JC70A, 9G11, WM59, MBC78.3 and antibodies binding to the same epitope.
14. A method of detection of platelet aggregation in a subject, said method comprising the detection of a CD31 protein fragment wherein said fragment is a platelet-derived shed ectodomain of a CD31 protein comprising (i) the whole or a part of the 6th extracellular Ig-like domain of said full-length CD31 protein comprising an amino acid sequence corresponding to residues 494 to 501 of human CD31 protein and (ii) not comprising the intracellular domain of said full-length CD31 protein in a biological sample from a subject, wherein the step of detecting said CD31 protein fragment is performed according to the method as defined in claim 1 .
15. A method of diagnosis of thrombosis
said method comprising the detection of a CD31 protein fragment wherein said fragment is a platelet-derived shed ectodomain of a CD31 protein comprising (i) the whole or a part of the 6th extracellular Ig-like domain comprising an amino acid sequence corresponding to residues 494 to 501 of human CD31 protein and (ii) not comprising the intracellular domain of said full-length CD31 protein in a biological sample from a subject, wherein the step of detecting said CD31 protein fragment is performed according to the method as defined in claim 1 .