Labelling and selection of molecules

• US 6,180,336 B1
• Filed: 06/17/1998
• Issued: 01/30/2001
• Est. Priority Date: 07/08/1996
• Status: Expired due to Term

• Current Assignee: Cambridge Silicon Radio Limited (Qualcomm, Inc.)
• Original Assignee: Cambridge Silicon Radio Limited (Qualcomm, Inc.)
• Inventors: Vaughan, Tristan John, Osbourn, Jane Katharine, Derbyshire, Elaine Joy, Johnson, Kevin Stuart, McCafferty, John Gerald
• Primary Examiner(s): Caputa, Anthony C.
• Assistant Examiner(s): Brumback, Brenda G.
• Application Number:
  US09/098,244
• Time in Patent Office: 958 Days
• Field of Search: 435/5, 435/7.1, 435/7.8, 435/7.9
• US Class Current: 435/5
• 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: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types
• CPC Class Codes: C07K 14/523 Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
• CPC Class Codes: C07K 14/70542 CD106
• CPC Class Codes: C07K 14/70596 Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• 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 16/065 Purification, fragmentation
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/2854 against selectins, e.g. CD62
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/3007 Carcino-embryonic Antigens
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/4266 against anti-tumor receptor Ig
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates

Abstract

A method of labelling molecules which includes providing in a common medium a label molecule, a marker ligand able to bind a member of a specific binding pair, such as an antigen, a sbp member, an enzyme able to catalyse binding of the label molecule to other molecules, the enzyme being associated with the marker ligand; causing or allowing binding of the marker ligand to the sbp member; and causing or allowing binding of the label molecule to other molecules in the vicinity of the marker ligand bound to the sbp member. The marker ligand may be an antibody or any specific binding molecule, such as a chemokine or cytokine. A complementary member of the specific binding pair may be included, e.g. an antibody, or a diverse population of such sbp members, e.g. antibodies, may be included within which those which bind the counterpart sbp member, e.g. antigen, may be labelled and subsequently isolated for manipulation and/or use. Suitable labels include biotin-tyramine with signal transfer being catalysed by hydrogen peroxidase. Cells, virus particles and other moieties may be labelled, for identification or obtention of proteins which interact or are in close proximity with a particular sbp member, or of cells of interest, or for enhancement of labelling, e.g. for cell sorting.

Claims

1. A method of labeling proteins, peptides or ligands, the method comprising providing in a common medium:

label molecules;

a marker ligand able to bind a member of a specific binding pair (sbp), which sbp has a first sbp member and a second sbp member, and wherein said marker ligand is able to bind said second sbp member;

a said second sbp member;

an enzyme able to catalyze binding of said label molecules to proteins, peptides or ligands, said enzyme being conjugated to said marker ligand;

causing or allowing binding of said marker ligand to said second sbp member; and

causing or allowing binding of said label molecules to proteins, peptides or ligands in the vicinity of said ligand bound to said second sbp member

wherein said proteins, peptides or ligands in the vicinity of said marker ligand bound to said second sbp member are on a cell surface, on a cell membrane, or in solution.

2. A method according to claim 1 wherein said proteins, peptides or ligands to which said label molecule binds comprise a sbp member (“

first sbp member”

) which binds said second sbp member.

3. A method according to claim 2 wherein said marker ligand comprises a non-human antibody antigen binding domain.

4. A method according to claim 3 wherein said first sbp member comprises a human antibody antigen binding domain.

5. A method according to claim 1 wherein said proteins, peptides or ligands to which said label molecule binds comprise a sbp member (“

first sbp member”

) which binds a molecule in the vicinity of said second sbp member.

6. A method according to claim 5 wherein said molecule in the vicinity of said second sbp member is on a cell surface.

7. A method according to claim 5 wherein said molecule in the vicinity of said second sbp member is complexed with said second sbp member.

8. A method according to claim 1, claim 2, claim 5, claim 6 or claim 7 wherein the amount of labeling of in the vicinity of said first marker ligand bound to said second sbp member is determined.

9. A method according to claim 2 or claim 5 wherein said first sbp member is displayed on the surface of a virus particle.

10. A method according to claim 2 or claim 5 wherein a diverse population of first sbp members is provided in the common medium, comprising a first sbp member able to bind said second sbp member.

11. A method according to claim 2 or claim 3 wherein said first sbp member comprises an antibody antigen binding domain.

12. A method according to claim 11 wherein said first sbp member comprises a scFv, Fab, Fv, dAb, Fd or diabody molecule.

13. A method according to claim 1, claim 2 or claim 5 wherein said marker ligand comprises a cytokine or chemokine.

14. A method according to claim 1, claim 2 or claim 5 wherein said marker ligand comprises an antibody antigen binding domain.

15. A method according to claim 1, claim 2 or claim 5 wherein the label comprises an activatible binding molecule.

16. A method according to claim 15 wherein the activatible binding molecule is tyramine.

17. A method according to claim 1, claim 2 or claim 5 wherein the enzyme is hydrogen peroxidase.

18. A method according to claim 1, claim 2 or claim 5 wherein the enzyme is conjugated to the marker ligand.

19. A method according to claim 18 wherein the enzyme is conjugated to the marker ligand via a peptidyl linkage.

20. A method according to claim 19 wherein the enzyme is conjugated to the marker ligand via a peptide or polypeptide linker.

21. A method according to claim 18 wherein the enzyme is conjugated to the marker ligand via a member of a specific binding pair.

22. A method according to claim 18 wherein the enzyme is conjugated to the marker ligand via a spacer.

23. A method according to claim 22 wherein the spacer is a chemical linker, polymer, peptide, polypeptide, rigid bead, phage molecule, or other particle.

24. A method according to claim 2 or claim 5 wherein a first sbp member labeled in accordance with the method is employed as a second ligand (“

second marker ligand”

) in a further performance of said method.

25. A method according to claim 1 wherein said second sbp member is displayed on the surface of a virus particle.

26. A method according to claim 1 wherein a diverse population of said second sbp members is provided in the common medium, including a second sbp member able to bind said marker ligand.

27. A method according to claim 1 or claim 26 wherein the second sbp member is a peptide.