Methods for the detection of soluble .beta.-amyloid peptide

• US 5,593,846 A
• Filed: 05/09/1995
• Issued: 01/14/1997
• Est. Priority Date: 07/10/1992
• Status: Expired due to Term

• Current Assignee: Eli Lilly and Company, Athena Neurosciences Incorporated (Perrigo Company PLC)
• Original Assignee: Eli Lilly and Company, Athena Neurosciences Incorporated (Perrigo Company PLC)
• Inventors: Schenk, Dale B., Seubert, Peter A., Vigo-Pelfrey, Carmen
• Primary Examiner(s): Knode, Marian C.
• Assistant Examiner(s): DUFFY, PATRICIA ANN
• Application Number:
  US08/437,067
• Time in Patent Office: 616 Days
• Field of Search: 435/7.9, 435/7.92, 435/7.94, 435/967, 435/975, 436/518, 436/548, 436/811
• US Class Current: 435/7.9
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/4711 Alzheimer's disease; Amyloid plaque core protein
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: G01N 2333/4709 Amyloid plaque core protein
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 2800/2821 Alzheimer
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/6896 Neurological disorders, e.g. Alzheimer's disease
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function

Abstract

Soluble β-amyloid peptide (βAP) is measured in biological fluids at very low concentrations, typically in the range from 0.1 ng/ml to 10 ng/ml. The measurement of βAP concentrations in animals or conditioned medium from cultured cells can be used for drug screening, where test compounds are administered to the animals or exposed to the cultured cells and the accumulation of βAP in the animal or culture medium observed. It has been found that elevated levels of βAP in body fluids, such as blood and cerebrospinal fluid, is associated with the presence of a βAP-related condition in a patient, such as Alzheimer'"'"'s Disease. Methods for diagnosing and monitoring βAP-related conditions comprise measuring the levels of βAP in such body fluids from a patient.

Claims

1. A method for detecting a soluble β

-amyloid peptide (β

AP) in a fluid sample in the presence of β

-amyloid precursor protein (APP) or fragments thereof, said method comprising;

capturing soluble β

AP from the sample using a first binding substance under conditions in which the first specific binding substance binds to an epitope at a junction region on the β

AP and which is substantially free from cross-reactivity with APP and fragments thereof other than β

AP, wherein the junction region is centered at the site between β

AP amino acid residues 16 and 17 and is a target for normal proteolytic cleavage; and

detecting capture of the soluble β

AP using a labeled second binding substance which binds to an epitope on a second region of the soluble β

AP other than the epitope on the β

AP which is bound by the first binding substance.

2. A method as in claim 1, wherein the β

AP is intact β

AP.

3. A method as in claim 1, wherein the fluid sample is selected from the group consisting of culture medium, blood, CSF, urine, and peritoneal fluid.

4. A method as in claim 1, wherein the soluble β

AP is captured on a solid phase, and the capture is detected by exposing the solid phase to the labeled second binding substance and thereafter detecting the presence of the label on the solid phase.

5. A method for detecting a soluble β

-amyloid peptide (β

AP) in a fluid sample, said method comprising;

exposing the sample to a first binding substance specific for an epitope at a junction region on soluble β

AP disposed between amino acid residues 13 to 28; and

detecting binding between the first binding substance and the soluble β

AP under conditions where the first binding substance binds to the β

AP without substantial cross-reactivity with β

-amyloid precursor protein (APP) and fragments thereof other than β

AP.

6. A method as in claim 5, wherein the fluid sample is selected from the group consisting of culture medium, blood, CSF, urine, and peritoneal fluid.

7. A method as in claim 5, wherein the first binding substance is an antibody raised against a peptide consisting of amino acid residues 13 to 28 of β

AP.

8. A method as in claim 7, wherein the antibody is a monoclonal antibody.

9. A method as in claim 5, wherein binding of the first binding substance and the soluble β

AP is detected by separating bound complexes of the binding substance and β

AP, exposing the separated bound complexes to a labeled second binding substance specific for the N-terminal region of β

AP, and detecting the presence of label on the bound complexes.

10. A method as in claim 9, wherein the second binding substance is an antibody raised against a peptide consisting of amino acid residues 1-16 of β

AP.

11. A method as in claim 10, wherein the antibody is a monoclonal antibody.

12. A method for detecting a soluble β

-amyloid peptide (β

AP) in a fluid sample which may contain β

AP and β

AP fragments as well as soluble fragments of β

-amyloid precursor protein (APP) other than β

AP, said method comprising;

exposing the fluid sample to a first binding substance under conditions in which the first binding substance will bind to an epitope on soluble β

AP and β

AP fragments but will not bind to epitopes on APP fragments which may be present in the sample; and

detecting binding between the first binding substance and the soluble β

AP and β

AP fragments.

13. A method as in claim 12, wherein the fluid sample is selected from the group consisting of culture medium, blood, CSF, urine, and peritoneal fluid.

14. A method as in claim 12, wherein the first binding substance binds specifically to intact β

AP.

15. A method as in claim 12, wherein the first binding substance binds specifically to an approximately 3 kD fragment of β

AP.

16. A method as in claim 12, wherein the first binding substance is immobilized on a solid phase, and binding is detected by exposing the solid phase to a labeled second binding substance and thereafter detecting the presence of the label on the solid phase.