Detection of shed CD31, diagnosis of atherothrombosis and autoimmune disorders, and methods for analyzing signaling pathways

摘要

The present invention stems from the finding that the extracellular domain of CD31 proteins present on blood leukocytes is shed and released in the circulation as a soluble form of CD31. A method for detecting shed CD31 is further disclosed. The invention therefore relates to a method for detecting a shed ectodomain of a transmembrane protein such as CD31 and to the use of such a method as a diagnostic tool. The invention further provides methods for determining whether a candidate protein is part of a molecular complex.

主权项

1. A method for detecting a shed ectodomain of a CD31 transmembrane protein among soluble forms of said transmembrane protein in a biological sample, wherein said soluble forms include a soluble splice variant of said transmembrane protein and optionally said shed ectodomain, which comprises the steps of:
a) providing a bead linked to a capture ligand, which capture ligand specifically binds to a region that is present both on said shed ectodomain and on said splice variant; b) providing a first discriminating antibody, which first discriminating antibody is a first type of fluorescently-labelled antibody which specifically binds to an epitope located in a region that is present both on said shed ectodomain and on said splice variant; c) providing at least one second discriminating antibody, which second discriminating antibody is a second type of fluorescently-labelled antibody which specifically binds to any one of an epitope selected from the group consisting of (i) an epitope located in a region that is present on said shed ectodomain and absent from said splice variant, and (ii) an epitope which is present on said splice variant and absent from said shed ectodomain; d) contacting said bead and antibodies with a biological sample likely to contain said soluble forms of said transmembrane protein; e) measuring by flow cytometry the fluorescent signal for each fluorescent label; and f) comparing the signal obtained for each fluorescent label,wherein a difference in the signals measured at step (e) indicates that the biological sample comprises said shed ectodomain.