Production of motif-specific and context-independent antibodies using peptide libraries as antigens

• US 6,982,318 B1
• Filed: 03/24/2000
• Issued: 01/03/2006
• Est. Priority Date: 09/04/1998
• Status: Expired due to Term

• Current Assignee: Fleet National Bank (Bank of America Corp.)
• Original Assignee: Cell Signaling Technology Incorporated
• Inventors: Comb, Michael J., Tan, Yi
• Primary Examiner(s): Canella, Karen A.
• Application Number:
  US09/535,364
• Time in Patent Office: 2,111 Days
• Field of Search: 530/387.1, 530/388.1, 530/389.1
• US Class Current: 530/387.1
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids

Abstract

A class of motif-specific, context-independent antibodies that bind conserved signal transduction motifs, such as kinase consensus substrate motifs and protein-protein binding motifs, containing one or more modified amino acids, such as a phosphorylated amino acid, is provided. The antibodies bind a plurality of peptides or proteins that contain the modified motif. Context-independent antibodies specific for single modified residues, such as phosphothreonine, are also provided. Methods for producing and using such antibodies are provided.

Claims

1. A motif-specific, context-independent antibody that specifically binds a recurring kinase consensus substrate motif or a protein-protein binding motif, said motif consisting of up to four invariant amino acids, at least one phosphorylated amino acid, and optionally, one or more variable/degenerate amino acid position(s), said antibody specifically binding said motif in a plurality of different peptides or proteins from an organism in which said motif recurs, wherein said antibody is not a site-specific antibody.

2. The antibody of claim 1, wherein said kinase consensus substrate motif is a MAPK consensus substrate motif[S] (PXS*P).

3. The antibody of claim 1, wherein said antibody is monoclonal.

4. The antibody of claim 1, wherein said antibody is polyclonal.

5. The antibody of claim 1, wherein said antibody is produced by a method comprising the steps of:

(a) constructing a combinatorial peptide library comprising (i) a fixed motif, wherein said fixed motif comprises said kinase consensus substrate motif or said protein-protein binding motif, and (ii) a plurality of degenerate amino acids surrounding said motif;

(b) immunizing a host with said peptide library; and

(c) isolating antisera from said host, and purifying said motif-specific, context-independent antibody from said antisera.

6. The antibody of claim 5, wherein said method further comprises the step of utilizing spleen cells from said host of step (b) to generate at least one monoclonal, motif-specific, context-independent antibody.

7. The antibody of claim 1, wherein said kinase consensus substrate motif is a DCK consensus substrate motif (PXT*/S*PXR).

8. The antibody of claim 1, wherein said kinase consensus substrate motif is a PKA consensus substrate motif (RRXT*).

9. The antibody of claim 1, wherein said kinase consensus substrate motif is a Akt consensus substrate motif (RXRXXT*).

10. The antibody of claim 1, wherein said kinase consensus substrate motif is a bulky ring-directed kinase consensus substrate motif ([F/Y][T*/S*] or [S/T*][F]).

11. The antibody of claim 1, wherein said kinase protein-protein binding motif is a 14-3-3 binding motif (RSXS*XP).