Methods for detecting antibodies
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.
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 μm to 50 μm.
25. The method of claim 24 , wherein the bead has a particle size of about 10 μm.
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.