Assays for beta-amyloid processing
OwnerRebecca Pruss, John Huggins +4inventors
InventorsRebecca Pruss, John Huggins, Guy Rautmann, Barbara Cordell, Jan Marian Scardina, Ronald P. Mischak
Life of this patent
Estimating the term…
Abstract
The invention relates to the design, construction and use of eukaryotic cell lines useful in the identification of inhibitors of β-amyloid processing. More specifically, the invention relates to in vitro assays capable of identifying or quantifying a 4.2 kDa β-amyloid protein. The present invention also provides for DNA and protein molecules for the design, construction and use of eukaryotic cell lines useful in the identification of inhibitors of β-amyloid processing.
Claims86
An eukaryotic cell line having a exogenous gene construction, said exogenous gene construction comprising a cytomegalovirus promoter encoding sequence, a strong ribosome binding site encoding sequence, a amyloid precursor protein encoding sequence, a selectable marker encoding sequence, and a poly-adenylation signal encoding sequence.
A eukaryotic cell line selected from the group consisting of 21-N-1, 21 -N-2, 21-N-3, 21-N-4, 21-N-5, 21-N-6, 21-N-7 21-N-8, and 21-N-9 or a derivative thereof.
An eukaryotic cell line capable of producing greater than 30 ng 4.2 kDa β-amyloid protein molecule per mg protein.
An eukaryotic cell line having a exogenous gene construction, said exogenous gene construction comprising a cytomegalovirus promoter encoding sequence, a ribosome binding site encoding sequence, a β-amyloid precursor protein encoding sequence, a selectable marker encoding sequence, and a poly-adenylation signal encoding sequence.
A substantially purified nucleic acid molecule that encodes, in order:
- a cytomegalovirus promoter;
- a ribosome binding site;
- a β-amyloid precursor protein molecule;
- a selectable marker;
- and
- a poly-adenylation signal.
A substantially purified nucleic acid molecule that encodes, in order:
- a cytomegalovirus promoter;
- a strong ribosome binding site;
- a β-amyloid precursor protein molecule;
- a selectable marker;
- and
- a poly-adenylation signal.
A nucleic acid molecule comprising pCMV-IRES-βAPP695.
A method for identifying an inhibitor of β-amyloid processing pathway comprising:
- administering said inhibitor to a eukaryotic cell line having a exogenous gene construction, said exogenous gene construction comprising a cytomegalovirus promoter encoding sequence, a strong ribosome binding site encoding sequence, a β-amyloid precursor protein encoding sequence, a selectable marker encoding sequence, and a poly-adenylation signal encoding sequence;
- and
- quantifying a protein molecule that is indicative of inhibition of β-amyloid processing pathway.
A composition comprising a eukaryotic cell line and an inhibitor of a β-amyloid processing pathway, said eukaryotic cell line having a exogenous gene construction, said exogenous gene construction comprising a cytomegalovirus promoter encoding sequence, a strong ribosome binding site encoding sequence, a β-amyloid precursor protein encoding sequence, a selectable marker encoding sequence, and a poly-adenylation signal encoding sequence.
A composition comprising a media having a protein molecule that is indicative of inhibition of a β-amyloid processing pathway, said media incubated with a eukaryotic cell line having a exogenous gene construction, said exogenous gene construction comprising a cytomegalovirus promoter encoding sequence, a strong ribosome binding site encoding sequence, a β-amyloid precursor protein encoding sequence, a selectable marker encoding sequence, and a poly-adenylation signal encoding sequence.
A composition comprising a media having a protein molecule that is indicative of inhibition of β-amyloid processing pathway, said media supplemented with biotinylated 4.2 kDa β-amyloid protein molecule, said media incubated with a eukaryotic cell line and an inhibitor of β-amyloid processing pathway, said eukaryotic cell line having a exogenous gene construction, said exogenous gene construction comprising a cytomegalovirus promoter encoding sequence, a strong ribosome binding site encoding sequence, a β-amyloid precursor protein encoding sequence, a selectable marker encoding sequence, and a poly-adenylation signal encoding sequence.
A method for identifying an inhibitor of β-amyloid processing pathway comprising,
- (a) incubating an inhibitor of β-amyloid processing and a eukaryotic cell line to produce a protein molecule that is indicative of inhibition of β-amyloid processing pathway in a medium containing a labeled amino acid, said eukaryotic cell line having a exogenous gene construction, said exogenous gene construction comprising a cytomegalovirus promoter encoding sequence, a strong ribosome binding site encoding sequence, a β-amyloid precursor protein encoding sequence, a selectable marker encoding sequence, and a poly-adenylation signal encoding sequence, wherein said exogenous gene construction is capable of stably expressing a β-amyloid precursor protein molecule;
- (b) separating said eukaryotic cell line and said medium;
- (c) separating said protein molecule from said eukaryotic cell line and said medium;
- and
- (d) quantifying said protein molecule.
A high throughput assay for identification of an inhibitor of β-amyloid processing comprising:
- (a) incubating an inhibitor of β-amyloid processing and a eukaryotic cell line to produce a protein molecule that is indicative of inhibition of β-amyloid processing pathway, said eukaryotic cell line having a exogenous gene construction, said exogenous gene construction comprising a cytomegalovirus promoter encoding sequence, a strong ribosome binding site encoding sequence, a β-amyloid precursor protein encoding sequence, a selectable marker encoding sequence, and a poly-adenylation signal encoding sequence;
- and
- (b) quantifying said protein molecule produced by the incubation step.
A composition comprising, a β-amyloid 108.1 42 specific monoclonal antibody, a β-amyloid 1101.1 4.2 kDa-specific monoclonal antibody and a media having a protein molecule that is indicative of inhibition of β-amyloid processing pathway, said media incubated with a eukaryotic cell line and an inhibitor of β-amyloid processing pathway, said eukaryotic cell line having a exogenous gene construction, said exogenous gene construction comprising a cytomegalovirus promoter encoding sequence, a strong ribosome binding site encoding sequence, a β-amyloid precursor protein encoding sequence, a selectable marker encoding sequence, and a poly-adenylation signal encoding sequence, wherein said β-amyloid 108.1 42 specific monoclonal antibody and said β-amyloid 1101.1 4.2 kDa-specific monoclonal antibody specifically bind said protein molecule that is indicative of inhibition of β-amyloid processing pathway.
A eukaryotic cell line ATCC No CRL 12329 or a derivative thereof.
A monoclonal antibody selected from the group consisting of monoclonal antibody 108.1 or a derivative thereof, monoclonal antibody 1101.1 or a derivative thereof, and monoclonal antibody 1702.1 or a derivative thereof.
Dependencies are inferred from the claim text.