Measuring affinity constants of 1450 monoclonal antibodies to peptide targets with a microarray-based label-free assay platform

J Immunol Methods. 2015 Feb;417:86-96. doi: 10.1016/j.jim.2014.12.011. Epub 2014 Dec 20.


Monoclonal antibodies (mAbs) are major reagents for research and clinical diagnosis. For their inherently high specificities to intended antigen targets and thus low toxicity in general, they are pursued as one of the major classes of new drugs. Yet binding properties of most monoclonal antibodies are not well characterized in terms of affinity constants and how they vary with presentations and/or conformational isomers of antigens, buffer compositions, and temperature. We here report a microarray-based label-free assay platform for high-throughput measurements of monoclonal antibody affinity constants to antigens immobilized on solid surfaces. Using this platform we measured affinity constants of over 1410 rabbit monoclonal antibodies and 46 mouse monoclonal antibodies to peptide targets that are immobilized through a terminal cysteine residue to a glass surface. The experimentally measured affinity constants vary from 10 pM to 200 pM with the median value at 66 pM. We compare the results obtained from the microarray-based platform with those from a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).

Keywords: Affinity constants; Label-free detection; Microarray; Monoclonal antibody; Oblique-incidence reflectivity difference; Peptide.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Affinity / immunology*
  • Antigen-Antibody Reactions / immunology*
  • Antigens / immunology
  • High-Throughput Screening Assays / methods
  • Mice
  • Protein Array Analysis / methods
  • Rabbits
  • Surface Plasmon Resonance


  • Antibodies, Monoclonal
  • Antigens