Detection of SHED CD31, Diagnosis of Atherothrombosis and Autoimmune Disorders, and Methods for Analyzing Signaling Pathways
AssigneeInstitut National De La Sante Et De La Recherche Medicale (Inserm)
InventorsGiuseppina Caligiuri, Antonino Nicoletti
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Abstract
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.
Claims24
A method for detecting a shed ectodomain of a 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 first type of bead linked to an antibody which specifically binds to an epitope located in a region that is present both on said shed ectodomain and on said splice variant (first discriminating antibody);
- b) providing at least one second type of bead linked to an antibody which specifically binds to an epitope located in a region that is either present on said shed ectodomain and absent from said splice variant, or present on said splice variant and absent from said shed ectodomain (second discriminating antibody);
- c) providing a fluorescently-labelled ligand which specifically binds to a region that is present both on said shed ectodomain and on said splice variant (signaling ligand);
- d) contacting said antibodies with a biological sample likely to contain said soluble fauns of said transmembrane protein;
- e) for each type of bead, measuring the signal obtained with said florescent label by flow cytometry;
- and
- f) comparing the signal obtained for each type of bead;
- wherein a difference in the signals measured at step (e) indicates that the biological sample comprises said shed ectodomain.
Canceled
A method for diagnosing whether an individual suffers, or is at risk of suffering, from a thrombotic or an autoimmune disorder, which comprises the step of detecting a shed ectodomain of CD31 in a biological sample of said individual, wherein the presence of said shed ectodomain of CD31 indicates that said individual suffers from or is at risk of suffering from said a thrombotic or autoimmune disorder.
A method for monitoring the response of a patient to a drug, said method comprising the steps of:
- a) detecting shed ectodomains of CD31 in a biological sample of said patient before and after onset of a treatment of said patient with said drug;
- b) comparing the levels of shed ectodomains of CD31 detected at step (a);
- and, optionally,
- c) correlating a difference in said levels of shed ectodomains of CD31 with the effectiveness of the drug for treating said patient.
A diagnostic kit comprising:
- i) a first type of bead linked to an antibody that specifically binds to an epitope located in a region that is present both on a shed ectodomain of a transmembrane protein and on a soluble splice variant of said transmembrane protein;
- ii) a second type of bead linked to an antibody that specifically binds to an epitope located in a region that is either present on said shed ectodomain and absent from said soluble splice variant, or present on said soluble splice variant and absent from said shed ectodomain;
- and
- iii) a fluorescently-labelled antibody that specifically binds to an epitope located in a region that is present both on said shed ectodomain and on soluble said splice variant.
(canceled)
A method for determining whether a candidate molecule is member of a molecular complex which comprises the steps of:
- a) providing a bead linked to an antibody that specifically binds to a member of said molecular complex;
- b) contacting said bead with a biological sample containing said molecular complex;
- c) contacting said beads contacted with the biological sample with at least one type of fluorescently-labelled antibody that specifically binds to said candidate molecule;
- and
- d) detecting the fluorescence by flow cytometry;
- wherein:
- the detection of a signal at step (d) indicates that said candidate molecule is a member of said molecular complex;
- and
- if more than one type of fluorescently-labelled antibody is used at step (c), said antibodies are labelled with different fluorescent labels.
A kit for analyzing a molecular complex which comprises:
- i) a bead linked to an antibody that specifically binds to a member of said molecular complex;
- and
- ii) at least one type of fluorescently-labelled antibody that specifically binds to a molecule likely to be member of said molecular complex;
- wherein, if the kit comprises more than one fluorescently-labelled antibody, said antibodies are labelled with different fluorescent labels.
Dependencies are inferred from the claim text.