Methods for generating phosphorylation site-specific immunological reagents

• US 6,309,863 B1
• Filed: 01/25/1999
• Issued: 10/30/2001
• Est. Priority Date: 01/25/1999
• Status: Expired due to Term

• Current Assignee: Brookhaven Science Associates, LLC (Government of the United States of America), The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: Brookhaven Science Associates, LLC (Government of the United States of America)
• Inventors: Sakaguchi, Kazuyasu, Appella, Ettore, Anderson, Carl W.
• Primary Examiner(s): SAUNDERS, DAVID A
• Application Number:
  US09/236,415
• Time in Patent Office: 1,009 Days
• Field of Search: 435/70.21, 436/547, 436/548, 530/387.9, 530/413, 530/388.1, 530/388.2, 530/389.1
• US Class Current: 435/70.21
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/32 against translation products of oncogenes

Abstract

The present invention provides methods for generating phosphorylation site-specific immunological reagents. More specifically, a phosphopeptide mimetic is incorporated into a polypeptide in place of a phosphorylated amino acid. The polypeptide is used as antigen by standard methods to generate either monoclonal or polyclonal antibodies which cross-react with the naturally phosphorylated polypeptide. The phosphopeptide mimetic preferably contains a non-hydrolyzable linkage from the appropriate carbon atom of the amino acid residue to a phosphate group. A preferred linkage is a CF₂ group. Such a linkage is used to generate the phosphoserine mimetic F₂Pab, which is incorporated into a polypeptide sequence derived from p53 to produce antibodies which recognize a specific phosphorylation state of p53. A CF₂ group linkage is also used to produce the phosphothreonine mimetic F₂Pmb, and to produce the phosphotyrosine mimetic, F₂Pmp.

Claims

1. A method for isolating polyclonal antibodies which bind specifically to a phosphorylated polypeptide which is phosphorylated at a particular amino acid, comprising:

a) providing a synthetic polypeptide comprising a mimetic of the particular amino acid which is phosphorylated, the synthetic polypeptide being formulated in a physiologically acceptable carrier;

b) immunizing an animal with the synthetic polypeptide of step a);

c) collecting and preparing serum or ascitic fluid from the immunized animal of step b);

d) screening for the presence of antibodies specific for the phosphorylated polypeptide; and

e) when necessary, further enriching for antibodies specific for the phosphorylated polypeptide through conventional methods.

2. The method of claim 1 wherein binding of the polyclonal antibodies to the phosphorylated polypeptide is dependent on amino acid sequences which flank the particular amino acid which is phosphorylated.

3. The method of claim 1 wherein binding of the polyclonal antibodies to the phosphorylated polypeptide is independent of amino acid sequences which flank the particular amino acid which is phosphorylated.

4. The method of claim 1 wherein antibodies specific for the phosphorylated polypeptide are further enriched by affinity purification.

5. The method of claim 1 wherein antibodies specific for the phosphorylated polypeptide are further enriched by depleting co-mingled antibodies which exhibit undesired binding activities.

6. The method of claim 1 wherein the animal is further immunized with a natural phosphopeptide-conjugate comprised of an appropriate amino acid sequence, as a boost.

7. The method of claim 1 wherein the synthetic polypeptide of step a) is modified prior to immunization step b).

8. The method of claim 7 wherein the synthetic polypeptide is modified through coupling to a carrier protein selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin and purified protein derivative of tuberculin (PPD), using any bifunctional reagent.

9. The method of claim 1 wherein the particular amino acid which is phosphorylated is selected from the group consisting of serine, threonine and tyrosine.

10. The method of claim 1 wherein the mimetic contains a non-hydrolyzable linkage from a carbon atom to a phosphate group.

11. The method of claim 10 wherein the non-hydrolyzable linkage comprises a CF₂ group.

12. The method of claim 10 wherein F₂Pab is a mimetic for phopsphoserine.

13. The method of claim 10 wherein F₂Pmp is a mimetic for phosphotyrosine.

14. The method of claim 10 wherein F₂Pmb is a mimetic for phosphothreonine.

15. The method of claim 1 wherein the synthetic polypeptide of step a) comprises an amino acid sequence derived from amino acid sequences of p53.

16. The method of claim 15 wherein the synthetic polypeptide is as specified in SEQ ID NO:

1.

17. The method of claim 15 wherein the synthetic polypeptide is selected from the group consisting of SEQ ID NOS:

2, 3, 4, 5, 6, 7, 8, 9, and 10.

18. A method for isolating monoclonal antibodies which bind specifically to a phosphorylated polypeptide which is phosphorylated at a particular amino acid, comprising:

a) providing a synthetic polypeptide comprising a mimetic of the particular amino acid which is phosphorylated, the synthetic polypeptide being formulated in a physiologically acceptable carrier;

b) immunizing an animal with the synthetic polypeptide of step a);

c) collecting B-cells from the animal of step b)

d) generating hybridomas by fusing the B-cells of step c) with myeloma cells;

e) screening the hybridomas of step d) for antibodies which bind specifically to the phosphorylated polypeptide;

f) isolating and propagating hybridomas identified in step e); and

g) isolating monoclonal antibodies from the isolated and propagated hybridomas of step f).

19. The method of claim 18 wherein binding of the monoclonal antibodies to the phosphorylated polypeptide is dependent on amino acid sequences which flank the particular amino acid which is phosphorylated.

20. The method of claim 18 wherein binding of the monoclonal antibodies to the phosphorylated polypeptide is independent of amino acid sequences which flank the particular amino acid which is phosphorylated.

21. The method of claim 18 wherein the animal is further immunized with a natural phosphopeptide-conjugate comprised of an appropriate amino acid sequence, as a boost.

22. The method of claim 18 wherein the synthetic polypeptide of step a) is modified prior to the immunization step b).

23. The method of claim 22 wherein the synthetic polypeptide is modified through coupling to a carrier protein selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin and purified protein derivative of tuberculin (PPD), using any bifunctional reagent.

24. The method of claim 18 wherein the particular amino acid which is phosphorylated is selected from the group consisting of serine, threonine and tyrosine.

25. The method of claim 18 wherein the mimetic contains a non-hydrolyzable linkage from a carbon atom to a phosphate group.

26. The method of claim 25 wherein the non-hydrolyzable linkage comprises a CF₂ group.

27. The method of claim 25 wherein F₂Pab is a mimetic for phopsphoserine.

28. The method of claim 25 wherein F₂Pmp is a mimetic for phosphotyrosine.

29. The method of claim 25 wherein F₂Pmb is a mimetic for phosphothreonine.

30. The method of claim 18 wherein the synthetic polypeptide of step a) comprises an amino acid sequence derived from amino acid sequences of p53.

31. The method of claim 30 wherein the synthetic polypeptide is as specified in SEQ ID NO:

1.

32. The method of claim 30 wherein the synthetic polypeptide is selected from the group consisting of SEQ ID NOS:

2, 3, 4, 5, 6, 7, 8, 9, and 10.