A novel, high-sensitivity and drug-tolerant sandwich immunoassay for the quantitative measurement of circulating proteins.
BACKGROUND
Accurate measurement of a total protein target (free plus bound) is essential to optimize dose selection for monoclonal antibody drugs. Herein, we describe a novel sandwich immunoassay format in which the biotherapeutic antibody itself serves as the primary detection antibody. A signal is then generated through the addition of a labeled secondary antibody that recognizes the biotherapeutic antibody. The secondary antibody is conjugated with ruthenium to facilitate electrochemiluminescent analysis.


RESULTS
Data are presented from the analysis of two protein biomarkers having disparate size and structure; a 4.5 kDa peptide and a 60 kDa protein. In both cases, validated, highly specific assays were developed and shown to be tolerant to elevated levels of the therapeutic monoclonal antibody in question.


CONCLUSIONS
Our novel format allows drug-tolerant measurement of soluble protein biomarkers targeted by monoclonal antibodies when only two independent epitopes for antibody binding are available and one is recognized by the therapeutic antibody.
Eli Lilly & Company, Indianapolis, IN 46285, USA. sloan_john_harrison@lilly.com
John H
JH
Sloan
Bradley L
BL
Ackermann
John W
JW
Carpenter
Huy T
HT
Nguyen
Kimberly
K
Stopa
Robert W
RW
Siegel
Robert J
RJ
Konrad
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D000882:Haplorhini; D006801:Humans; D007118:Immunoassay; D008163:Luminescent Measurements; D010455:Peptides / Q000097:blood* / Q000276:immunology; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000276:immunology; D012428:Ruthenium / Q000737:chemistry
D000906:Antibodies; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D010455:Peptides; D004364:Pharmaceutical Preparations; D011506:Proteins; D012428:Ruthenium