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

• US 6,441,140 B1
• Filed: 09/04/1998
• Issued: 08/27/2002
• 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): Ponnaluri, Padmashri
• Application Number:
  US09/148,712
• Time in Patent Office: 1,453 Days
• Field of Search: 530/388.23, 530/388.1, 530/387.9, 530/387.1, 530/300, 530/331, 530/333, 435/7.1, 435/4, 435/DIG.34, 435/DIG.35
• US Class Current: 506/9
• 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

The present invention is related to a method for producing motif-specific, context-independent antibodies which are specific to at least one modified fixed amino acid residue in the context of variable surrounding amino acid or peptide sequences. The method is particularly useful in producing antibodies which recognize phosphorylated serine, threonine, and tyrosine, or acetylated lysine, as well as other modified amino acid-containing motifs of one or more amino acids.

Claims

1. A method for producing a motif-specific, context-independent antibody that recognizes a plurality of peptides or proteins within a genome that contain the motif, said method comprising the steps of:

(a) constructing a combinatorial peptide library comprising (i) a fixed motif, comprises a kinase consensus substrate motif or a protein-protein binding motif and wherein said fixed motif comprises at least one phosphorylated amino acid selected from the group consisting of phosphothreonine, phosphoserine, and phosphotyrosine, 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, said antibody recognizing a plurality of peptides or a proteins with a genome that contain said motif.

2. The method of claim 1, further comprising the step of utilizing spleen cells from the host of step (b) to generate at least one monoclonal, motif-specific, context-independent antibody.

3. The method of claim 1, wherein said kinase consensus substrate motif is selected from the group consisting MAPK consensus substrate motifs and CDK consensus substrate motifs, and wherein said protein binding motif is a 14-3-3 binding motif.

5. The method of claim 1 or 4, wherein said peptide library is from 6 to 14 amino acids long.

4. A method for producing a motif-specific, context-independent antibody that recognizes a plurality of peptides or proteins within a genome that contain the motif, said method comprising the steps of:

(a) constructing a combinatorial peptide library comprising (I) a fixed motif, wherein said fixed motif consists of a single phosphothreonine, phosphoserine or phosphotyrosine, 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, said antibody recognizing a plurality of peptides or proteins with a genome that contain said motif.