Biological complex specific for Alzheimer's disease detection in vitro and use thereof

Patent metadata

Field	Value
Publication number	US-9377472-B2
Application number	US-201313874052-A
Country	US
Kind code	B2
Filing date	Apr 30, 2013
Priority date	Apr 30, 2012
Publication date	Jun 28, 2016
Grant date	Jun 28, 2016

Abstract

A biomarker for Alzheimer's disease (AD) comprising a complex of an Aβ amyloid peptide and a cell or a cellular membrane. Non-invasive methods for diagnosing Alzheimer's disease or monitoring its development or progression using this biomarker.

First claim

The invention claimed is:

1. A complex comprising an isolated Aβ amyloid peptide and a cell or a cell membrane, wherein said complex is obtainable by incubating a cell or cell membrane with 0.1 to 2 micromolars of an isolated Aβ amyloid peptide in a biological fluid or an iso-osmotic medium and at a temperature ranging between 4 and 42° C.

2. The complex of claim 1 , wherein said isolated Aβ amyloid peptide comprises at least six contiguous amino acids of Aβ1-42 of human, mouse or rat Aβ amyloid peptide of SEQ. ID. NOS: 1, 2 or 3, respectively.

3. The complex of claim 1 , wherein said isolated Aβ amyloid peptide is selected in the group consisting of human Aβ1-42 of SEQ. ID. NO: 4, human Aβ1-40 of SEQ. ID. NO: 5, human Aβ17-25 of SEQ. ID. NO: 6 or Aβ25-35 of SEQ. ID. NO: 7.

4. The complex of claim 1 , wherein said isolated Aβ amyloid peptide is tagged or labeled with a fluorophore, radioisotope, stable isotope, spin marker, enzyme, conjugated residue, or other detectable marker.

5. The complex of claim 1 , wherein said cell or cell membrane is a mammalian cell or cell membrane selected from the group consisting of
a red blood cell or red blood cell membrane;
a platelet or platelet cell membrane;
a non-RBC, non-platelet anuclear cell or the membrane of such an anuclear cell;
white blood cell or white blood cell membrane; and
a cell derived from the nervous system or a membrane of such a cell;
an epithelial cell or membrane of epithelial cell; and
a mucosal cell or a membrane of such a cell.

6. A method of diagnosing Alzheimer's disease or a disease or disorder associated with amyloid deposition comprising detecting the complex of claim 1 in a sample.

7. A method for identifying a therapeutic agent or compound for treating Alzheimer's disease or a disease or disorder characterized by amyloid deposits comprising:
identifying an agent or compound that inhibits membrane priming by beta amyloid peptide or a molecule derived therefrom.

8. A composition or kit comprising at least one of:
one or more reagents for incubating the cells or cell membranes with a tagged or labelled beta amyloid peptide or a molecule derived from a beta amyloid peptide,
one or more reagents for measuring a complex of the labelled beta amyloid peptide or a molecule derived therefrom, with said cells or cell membranes,
one or more reagents for isolating or purifying a cell or cell membrane to which a beta amyloid peptide has bound,
a device to detect or quantify the amount of complex formation,
software for detecting, quantifying or otherwise analyzing complex formation, and/or
written instructions or a user manual for using the composition or kit to detect or assess the risk of Alzheimer's disease.

9. A method for detecting the presence of a complex of claim 1 comprising:
a) purifying or isolating cells or cell membranes from a biological sample of a subject suspected of having, or at risk of developing, Alzheimer's disease or a disease characterized by deposits of amyloid protein in brain or nervous system,
b) contacting the purified or isolated cells or cell membranes with a tagged or labeled Aβ amyloid peptide that comprises at least six contiguous amino acids of Aβ1-42 of a mammalian Aβ amyloid peptide at a concentration between 0.1 and 2 micromolars for a time and under conditions suitable for complex formation, said conditions include an iso-osmotic medium and a temperature comprised between 4 and 42° C. and an incubation time comprised between 10 minutes and 3 hours, and
c) detecting complex formation between the cells or cell membranes and the labeled Aβ amyloid peptide by measuring tagged or labeled Aβ amyloid peptide bound to said cells or cell membranes.

10. The method of claim 9 , further comprising:
d) comparing the amount of complex formation to the amount of complex formation in a normal subject, in a subject not having Alzheimer's disease, or to a normal control value, and diagnosing the subject as having Alzheimer's disease or as being at risk of developing Alzheimer's disease when complex formation is higher than that in the normal subject, subject not having Alzheimer's disease, or normal control value.

11. The method of claim 9 , wherein the subject is being treated for Alzheimer's disease or for a disease or disorder characterized by the deposit of amyloid.

12. A noninvasive method according to claim 9 for diagnosing a subject as having Alzheimer's disease or for being at risk of developing or progressing for Alzheimer's disease or a disease or disorder characterized by the deposit of amyloid in the brain or nervous system comprising:
a) purifying or isolating cells from a biological sample of a subject suspected of having, or at risk of developing or progress to, Alzheimer's disease,
b) contacting the purified or isolated cells with a tagged or labeled molecule sensitive to calcium concentration under conditions including an iso-osmotic medium temperature comprised between 4 and 42° C. and an exposure time, which allows said molecule to be loaded inside the cells,
c) contacting the purified or isolated cells with an Aβ amyloid peptide that comprises at least six contiguous amino acids of Aβ1-42 of a mammalian Aβ amyloid peptide at a concentration between 0.1 and 2 micromolars for a time and under conditions sufficient for the Aβ amyloid peptide to modulate intracellular calcium concentration, said conditions include an iso-osmotic medium and a temperature comprised between 4 and 42° C.,
d) detecting the intracellular calcium concentration in the cells contacted with the Aβ amyloid peptide by measuring said tagged or labeled molecule sensitive to calcium concentration,
e) comparing the intracellular calcium concentration in the cells to that obtained by an otherwise identical method from sample obtained from a normal subject, a subject not having Alzheimer's disease, or to a normal control value, and
f) diagnosing the subject as having Alzheimer's disease or as being at risk of developing or progressing for Alzheimer's disease when intracellular calcium concentration is higher than the intracellular calcium concentration determined for the normal subject, subject not having Alzheimer's disease, or normal control value.

13. A non-invasive method according to claim 12 wherein said Aβ amyloid peptide is tagged or labeled and wherein step d) also comprises the detection of complex formation between the cells and the tagged or labeled Aβ amyloid peptide by measuring tagged or labeled Aβ amyloid peptide bound to said cells.

14. A method according to claim 9 for screening an agent or compound that inhibits priming for use as a therapeutic agent for Alzheimer's disease or a disease or disorder characterized by amyloid deposits, comprising:
a) isolating cells or cell membranes,
b) contacting the isolated cells or cell membranes with an agent or compound to be tested and with 0, 1 to 100 nanomolars of an amyloid peptide under conditions sufficient to prime the cell or cell membrane of a control that is not contacted with the agent or test compound, wherein said amyloid peptide comprises at least six contiguous amino acids of Aβ1-42 of a mammalian Aβ amyloid peptide, said conditions include an iso-osmotic medium and a temperature comprised between 4 and 42° C. and an incubation time between 10 minutes and 24 hours,
c) contacting the purified or isolated cells or cell membranes from step b) with a tagged or labeled Aβ amyloid peptide that comprises at least six contiguous amino acids of Aβ1-42 of a mammalian Aβ amyloid peptide at a concentration between 0.1 and 2 micromolars for a time and under conditions suitable for complex fo

Assignees

• Amoneta Diagnostics
• Centre Nat Rech Scient
• Ct Nat De La Rech Schientifique

Inventors

• Mbebi-Liegeois Corinne
• De Barry Jean
• Sellal Francois

Classifications

• A01K67/0275

  Genetically modified vertebrates, e.g. transgenic · CPC title

• A01K2217/072

  maintaining or altering function, i.e. knock in · CPC title

• C07K14/4711

  Alzheimer's disease; Amyloid plaque core protein · CPC title

• G01N2333/4709

  Amyloid plaque core protein · CPC title

• G01N2800/50

  Determining the risk of developing a disease · CPC title