An antibody-based biomarker discovery method by mass spectrometry sequencing of complementarity determining regions

Anal Bioanal Chem. 2011 Jan;399(3):1081-91. doi: 10.1007/s00216-010-4361-9. Epub 2010 Nov 24.

Abstract

Autoantibodies are increasingly used as biomarkers in the detection of autoimmune disorders and cancer. Disease specific antibodies are generally detected by their binding to specific antigens. As an alternative approach, we propose to identify specific complementarity determining regions (CDR) of IgG that relate to an autoimmune disorder or cancer instead of the specific antigen(s). In this manuscript, we tested the technical feasibility to detect and identify CDRs of specific antibodies by mass spectrometry. We used a commercial pooled IgG preparation as well as purified serum IgG fractions that were spiked with different amounts of a fully human monoclonal antibody (adalimumab). These samples were enzymatically digested and analyzed by nanoLC Orbitrap mass spectrometry. In these samples, we were able to identify peptides derived from the CDRs of adalimumab. These peptides could be detected at an amount of 110 attomole, 5 orders of magnitude lower than the total IgG concentration in these samples. Using higher energy collision induced dissociation (HCD) fragmentation and subsequent de novo sequencing, we could successfully identify 50% of the detectable CDR peptides of adalimumab. In addition, we demonstrated that an affinity purification with anti-dinitrophenol (DNP) monoclonal antibody enhanced anti-DNP derived CDR detection in a serum IgG background. In conclusion, specific CDR peptides could be detected and sequenced at relatively low levels (attomole-femtomole range) which should allow the detection of clinically relevant CDR peptides in patient samples.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adalimumab
  • Animals
  • Antibodies, Monoclonal / blood
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / immunology*
  • Antibodies, Monoclonal, Humanized
  • Biomarkers / blood
  • Complementarity Determining Regions / blood
  • Complementarity Determining Regions / chemistry*
  • Complementarity Determining Regions / immunology*
  • Humans
  • Immunoglobulin G / blood
  • Immunoglobulin G / chemistry
  • Immunoglobulin G / immunology
  • Mass Spectrometry
  • Mice
  • Peptides / blood
  • Peptides / chemistry
  • Peptides / immunology
  • Reference Values

Substances

  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Humanized
  • Biomarkers
  • Complementarity Determining Regions
  • Immunoglobulin G
  • Peptides
  • Adalimumab