Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach

MAbs. 2015;7(1):110-9. doi: 10.4161/19420862.2014.985919.


Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the Kon and Koff values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.

Keywords: KD; OIRD; OIRD, oblique-incidence reflectivity difference; mAb, monoclonal antibody; IP, immunoprecipitation; IHC, immunohistochemistry; ICC, immunocytochemistry; ChIP, chromatin immunoprecipitation; HuProt, human proteome microarray; ELISA, enzyme-linked immunosor; affinity; avidity; kinetics; monoclonal antibodies; protein microarrays.

Publication types

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

MeSH terms

  • Animals
  • Antibodies, Monoclonal, Murine-Derived / chemistry*
  • Antibodies, Monoclonal, Murine-Derived / immunology
  • Antibody Affinity*
  • Antibody Specificity*
  • Humans
  • Mice
  • Mice, Inbred BALB C
  • Protein Array Analysis / methods*


  • Antibodies, Monoclonal, Murine-Derived