High-Density Antigen Microarrays for the Assessment of Antibody Selectivity and Off-Target Binding

Methods Mol Biol. 2018:1785:231-238. doi: 10.1007/978-1-4939-7841-0_15.


With the increasing availability of collections of antibodies, their evaluation in terms of binding selectivity becomes an important but challenging task. Planar antigen microarrays are very suitable tools to address this task and provide a powerful proteomics platform for the characterization of the binding selectivity of antibodies toward thousands of antigens in parallel. In this chapter, we describe our in-house developed procedures for the generation of high-density planar antigen microarrays with over 21,000 features. We also provide the details of the assay protocol, which we routinely use for the assessment of binding selectivity of the polyclonal antibodies generated within the Human Protein Atlas.

Keywords: Affinity proteomics; Antibody selectivity; Antigen microarrays; Protein microarrays.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / immunology
  • Antibody Specificity / immunology*
  • Antigens / genetics
  • Antigens / immunology*
  • Humans
  • Protein Array Analysis / methods*
  • Protein Binding / immunology
  • Proteomics / methods*


  • Antibodies
  • Antigens