Methods for detecting antibodies

• US 9,250,233 B2
• Filed: 03/27/2009
• Issued: 02/02/2016
• Est. Priority Date: 03/27/2008
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Kipps, Thomas J., Messmer, Bradley T., Sanchez, Ana B., Kummel, Andrew C., Ruidiaz, Manuel
• Primary Examiner(s): Counts, Gary W
• Application Number:
  US12/934,624
• Publication Number:
  US 20110124020A1
• Time in Patent Office: 2,503 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: G01N 33/537 with separation of immune complex from unbound antigen or antibody
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/577 involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens; G01N33/53 - G01N33/576 take precedence
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Abstract

Methods for detection of any antibody utilizing a standardized approach applicable to any antibody which provides highly specific assays specific for individual or multiple antibodies. The methods enable improved pharmacokinetic analysis during development and clinical use of antibody-based therapies as well as determination of diagnostic and/or prognostic factors.

Claims

1. A method for detecting an antibody which is not complexed to an epitope of a target protein, said antibody being in a biological sample obtained from a subject which is suspected of containing such antibody, the method comprising:

a) synthesizing one or more peptides having a length of about 5-15 amino acids comprising a mimetope recognized by the antibody;

wherein the one or more peptides are attached directly on a solid support and wherein said mimetope comprises a linear sequence of amino acids which is different than a linear sequence of amino acids in said epitope of the target protein recognized by said antibody;

b) contacting the peptide with said biological sample obtained from said subject;

c) allowing binding of the one or more peptides with the antibody not complexed to an antigen to form an antibody-peptide complex; and

d) detecting the antibody-peptide complex in said biological sample obtained from a subject.

2. The method of claim 1, wherein detection of the antibody-peptide complex is at antibody concentrations from about 50 ng/ml to about 500 ng/ml.

3. The method of claim 1, wherein detection of the antibody-peptide complex is at antibody concentrations from about 10 ng/ml to about 500 ng/ml.

4. The method of claim 1, wherein the mimetope is identified from a phage-displayed peptide library.

5. The method of claim 1, wherein the mimetope binds to the antigen binding site of the antibody.

6. The method of claim 1, wherein detection of the antibody-peptide complex is performed by detection of a detectable label on the antibody or the peptide.

7. The method of claim 5, wherein detection of the antibody-peptide complex is determined by Western blot analysis, flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competition immunoassay, dual antibody sandwich assay, chemiluminescent assay, bioluminescent assay, fluorescent assay, or agglutination assay.

8. The method of claim 1, wherein the antibody is alemtuzumab.

9. The method of claim 8, wherein the one or more peptides comprise one or more amino acid sequences as set forth in SEQ ID NOs:

1-3.

10. The method of claim 1, wherein the antibody is bevacizumab.

11. The method of claim 10, wherein the one or more peptides comprise one or more amino acid sequences as set forth in SEQ ID NOs:

11-14.

12. The method of claim 1, wherein the antibody is rituximab.

13. The method of claim 12, wherein the one or more peptides comprise one or more amino acid sequences as set forth in SEQ ID NOs:

4-5.

14. The method of claim 1, wherein the antibody is trastuzumab.

15. The method of claim 14, wherein the one or more peptides comprise one or more amino acid sequences as set forth in SEQ ID NO:

15.

16. The method of claim 1, wherein the antibody is zanolimumab.

17. The method of claim 16, wherein the one or more peptides comprise one or more amino acid sequences as set forth in SEQ ID NOs:

16-19.

18. The method of claim 1, wherein the biological sample is a fluid.

19. The method of claim 1 wherein the antibody is a monoclonal antibody.

20. The method of claim 1, wherein the solid support is a bead.

21. The method of claim 20, wherein the bead is magnetic.

22. The method of claim 20, wherein the bead comprises magnetic nanoparticles.

23. The method of claim 22, wherein the magnetic nanoparticles comprise iron oxide (FeO).

24. The method of claim 22, wherein the bead has a particle size of about 1 μ

m to 50 μ

m.

25. The method of claim 24, wherein the bead has a particle size of about 10 μ

m.

26. The method of claim 22, wherein the bead further comprises a fluorophore.

27. The method of claim 1, wherein said subject is a human subject.

28. The method of claim 1, wherein said antibody peptide complex is detected at antibody concentrations below 500 ng/ml of said biological sample.