Polypeptide marker for Alzheimer's disease and its use for diagnosis

• US 4,666,829 A
• Filed: 05/15/1985
• Issued: 05/19/1987
• Est. Priority Date: 05/15/1985
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: University of California
• Inventors: Glenner, George G., Wong, Caine W.
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Wieder, Stephen C.
• Application Number:
  US06/734,660
• Time in Patent Office: 734 Days
• Field of Search: 530/324, 530/328, 530/344, 530/806, 530/808, 530/810, 935/95, 935/78, 935/103, 935/110, 935/78, 435/7, 435/6, 435/68, 435/172.2, 435/948, 424/88, 436/548, 436/63, 436/501, 436/504, 436/503, 536/27
• US Class Current: 435/6.16
• CPC Class Codes: C07K 14/4711 Alzheimer's disease; Amyloid plaque core protein
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: G01N 2474/20 Immunohistochemistry assay
• CPC Class Codes: G01N 2800/2821 Alzheimer
• CPC Class Codes: G01N 33/6896 Neurological disorders, e.g. Alzheimer's disease
• CPC Class Codes: Y10S 435/961 including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins

Abstract

A novel polypeptide, Alzheimer'"'"'s Amyloid Polypeptide (AAP), is provided in substantially purified form which is isolated from amyloid deposits in patients with Alzheimer'"'"'s Disease. The polypeptide has the following amino acid sequence: ##STR1## The polypeptide, or fragments thereof, may be used to produce antibodies useful in a diagnostic test for Alzheimer'"'"'s Disease. Nucleotide probes corresponding to portions of the polypeptide are also useful for diagnostic purposes.

Claims

1. A substantially purified polypeptide isolated from patients with Alzheimer'"'"'s Disease, Alzheimer'"'"'s Amyloid Polypeptide, having the following amino acid sequence:

##STR6##

2. A composition comprising the substantially purified polypeptide of claim 1, solubilized in a basic guanidine solution.

3. A composition comprising the substantially purified polypeptide of claim 2, wherein the basic guanidine solution is 6M guanidine-HCl, having a pH of about 8.0.

20. A method of detecting individuals possessing a nucleotide sequence coding for the polypeptide of claim 1, comprising the steps of:

a. providing a labelled nucleotide probe capable of selectively hybridizing with a portion of the nucleotide sequence coding for the substantially purified polypeptide isolated from patients with Alzheimer'"'"'s Disease, Alzheimer'"'"'s Amyloid Polypeptide, having the following amino acid sequence;

##STR11## b. exposing the nucleotide probe to a tissue sample from a patient;

c. monitoring the reaction of step b. for nucleic acid hybridization.

21. The method of claim 20, wherein said label is selected from the group consisting of ³² P, ³ H, and ¹⁴ C.

4. A reagent for use in the diagnosis of Alzheimer'"'"'s Disease in a human patient, comprising antibodies obtained by means of an immune response to exposure to the substantially purified polypeptide isolated from patients with Alzheimer'"'"'s Disease, Alzheimer'"'"'s Amyloid Polypeptide, having the following amino acid sequence:

##STR7##

5. The diagnostic reagent of claim 4, wherein the antibodies are polyclonal.

6. The diagnostic reagent of claim 4 wherein the immune response is produced in a mammal selected from the group consisting of rabbits, goats and mice.

7. The diagnostic reagent of claim 4 wherein the antibodies are monoclonal.

8. The diagnostic reagent of claim 4 wherein the immune response is produced in murine-murine hybridoma cells.

9. A diagnostic method for determining the presence of Alzheimer'"'"'s Disease in a human patient by detecting the presence of Alzheimer'"'"'s Amyloid Polypeptide in the patient through an immunoassay, comprising the steps of:

a. combining a sample of the human patient'"'"'s body fluid suspected of containing Alzheimer'"'"'s Amyloid Polypeptide with antibodies specific to Alzheimer'"'"'s Amyloid Polypeptide, having the following amino acid sequence;

##STR8## b. monitoring the combination of step a. to determine whether said antibodies have bound to said Alzheimer'"'"'s Amyloid Polypeptide in an immunological reaction, thereby indicating that said patient has Alzheimer'"'"'s Disease.

10. The diagnostic method of claim 9, wherein said combining step further includes adding a known quantity of labelled Alzheimer'"'"'s Amyloid Polypeptide, whereby a competitive immunoassay is established .

11. The diagnostic method of claim 10, wherein said label is capable of emitting radiation.

12. The diagnostic method of claim 11, wherein said label is ¹²⁵ I.

13. A diagnostic method for determining the presence of Alzheimer'"'"'s Disease in a human patient by histological localization of Alzheimer'"'"'s Amyloid Polypeptide, comprising the steps of:

a. combining a sample of the human patient'"'"'s tissue with antibodies specific to Alzheimer'"'"'s Amyloid Polypeptide, having the following amino acid sequence;

##STR9## b. monitoring the combination of step a. to determine whether said antibodies have bound to said Alzheimer'"'"'s Amyloid Polypeptide in an immunological reaction, thereby indicating that said patient has Alzheimer'"'"'s Disease.

14. The diagnostic method of claim 13, wherein said combining step further includes adding a known quantity of labelled Alzheimer'"'"'s Amyloid Polypeptide, whereby a competitive immunoassay is established.

15. The diagnostic method of claim 13, wherein said label is capable of emitting radiation.

16. The diagnostic method of claim 13, wherein said label is ¹²⁵ I.

17. The diagnostic method of claim 14, wherein said label is a component of an enzymatic reaction.

18. A labelled nucleotide probe, comprising a sequence of nucleic acid substantially complementary to the nucleotide sequence coding for the substantially purified polypeptide isolated from patients with Alzheimer'"'"'s Disease, Alzheimer'"'"'s Amyloid Polypeptide, having the following amino acid sequence:

##STR10##

19. The nucleotide probe of claim 18, wherein the nucleotide sequence coding for said polypeptide is DNA.

22. An immunogenic peptide fragment of a substantially purified polypeptide isolated from patients with Alzheimer'"'"'s Disease, Alzheimer'"'"'s Amyloid Polypeptide, comprising:

a linear sequence of amino acids corresponding to any subsequence of the following amino acid sequence;

##STR12## said linear sequence being immunogenic so as to induce the production of active antibodies specific to Alzheimer'"'"'s Amyloid Polypeptide.

23. An immunogenic peptide fragment of substantially purified polypeptide isolated from patients with Alzheimer'"'"'s Disease, Alzheimer'"'"'s Amyloid Polypeptide, as recited in claim 22, wherein the peptide fragment is about 10 amino acids in length.

24. An immunogenic peptide fragment of a substantially purified polypeptide isolated from patients with Alzheimer'"'"'s Disease, Alzheimer'"'"'s Amyloid Polypeptide, as recited in claim 23, within the linear sequence of amino acids is as follows:

space="preserve" listing-type="equation">H.sub.2 N-ASP-ALA-GLU-PHE-ARG-HIS-ASP-SER-GLY-TYR-COOH.

25. A composition comprising the peptide fragment of a substantially purified polypeptide isolated from patients with Alzheimer'"'"'s Disease, Alzheimer'"'"'s Amyloid Polypeptide, as recited in claim 22, solubilized in a basic guanidine solution.

26. A composition comprising the peptide fragment of a substantially purified polypeptide isolated from patients with Alzheimer'"'"'s Disease, Alzheimer'"'"'s Amyloid Polypeptide, as recited in claim 22, coupled to an immunogenic carrier.

27. A reagent for use in the diagnosis of Alzheimer'"'"'s Disease in a human patient, comprising antibodies obtained by means of an immune response to exposure to the peptide fragment as recited in claim 22.

28. The diagnostic reagent of claim 27, wherein the antibodies are polyclonal.

29. The diagnostic reagent of claim 27 wherein the immune response is produced in a mammal selected from the group consisting of rabbits, goats and mice.

30. The diagnostic reagent of claim 27, wherein the antibodies are monoclonal.

31. The diagnostic reagent of claim 27, wherein the immune response is produced in murine-murine hybridoma cells.

32. A diagnostic method for determining the presence of Alzheimer'"'"'s Disease in a human patient, comprising the steps of:

a. combining a sample of body fluid from the patient with the antibodies obtained by means of an immune response to a peptide fragment as recited in claim 22

b. monitoring the combination of step a. to determine whether said antibodies have bound to said Alzheimer'"'"'s Amyloid Polypeptide in an immunological reaction, thereby indicating that said patient has Alzheimer'"'"'s Disease.

33. The diagnostic method of claim 32, wherein said combining step further includes adding a known quantity of the peptide fragment as recited in claim 32, which peptide fragment has been labelled.

34. The diagnostic method of claim 33, wherein said label is capable of emitting radiation.

35. The diagnostic method of claim 34, wherein said label is ¹²⁵ I.

36. The diagnostic method of claim 33, wherein said label is a component of an enzymatic reaction.

37. A diagnostic method for determining the presence of Alzheimer'"'"'s Disease in a human patient by detecting the presence of Alzheimer'"'"'s Amyloid Polypeptide in the patient through an immunoassay, comprising the steps of:

a. combining a sample of the patient'"'"'s tissue suspected of containing Alzheimer'"'"'s Amyloid Polypeptide with antibodies specific to a peptide fragment as recited in claim 22

b. monitoring the combination of step a. to determine whether said antibodies have bound to said Alzheimer'"'"'s Amyloid Polypeptide in an immunological reaction, thereby indicating that said patient has Alzheimer'"'"'s Disease.

38. The diagnostic method of claim 37, wherein said combining step further includes adding a known quantity of the immunogenic peptide fragment recited in claim 22, whereby a competitive immunoassay is established.

39. The diagnostic method of claim 38, wherein said label is capable of emitting radiation.

40. The diagnostic method of claim 39, wherein said label is ¹²⁵ I.

41. The diagnostic method of claim 38, wherein said label is a component of an enzymatic reaction.