Determining antigen specificity of a monoclonal antibody using genome-scale CRISPR-Cas9 knockout library

J Immunol Methods. 2016 Dec;439:8-14. doi: 10.1016/j.jim.2016.09.006. Epub 2016 Sep 21.


An essential step in monoclonal antibody (mAb) development is the characterization and final identification of the specific target antigen and its epitope. Antibody validation is rather straightforward when immunization is carried out with peptide or purified protein, but is more difficult when whole cells or other complex antigens are used for the immunization. Determining antigen specificity of a mAb is further complicated, when reactivity of an antibody is not detected in Western blotting and/or immunoprecipitation assay. In addition to protein-based methods used for antibody characterization, a number of gene-based techniques, such as cDNA expression or short-interfering RNA (siRNA) knockdown have been applied for validation of antibodies with restricted reactivities. Earlier we have generated, characterized, but not identified the BF4 mAb that specifically stains viral biofilms on the surface of the Human T-lymphotropic Virus Type I (HTLV-1) infected T cells. In this study, using the recently developed genome-scale CRISPR-Cas9 knockout (GeCKO) library vectors, we have established the CEM T- and the Raji B cell lines with pooled libraries. After immunofluorescent staining of these cells, negative cell sorting, and guide-RNA (gRNA) sequencing, we have identified BF4 as an anti-CD82 mAb. A deep sequence analysis of GeCKO library transferred to the cells shows that the chance to succeed in the selection of antibody-negative cells and, therefore, to identify a mAb depends on the quality of cell library preparation. We believe that the described method is applicable for identification of many other hybridomas and represents a good alternative to the current protein- and gene-based methods used for mAb validation.

Keywords: Antigen specificity; CRISPR/Cas9; Cell sorting; Knockout library; Monoclonal antibody.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / immunology*
  • Antibodies, Monoclonal / metabolism
  • Antibody Specificity*
  • CRISPR-Cas Systems*
  • Cell Line
  • Cell Separation / methods
  • Epitopes*
  • Flow Cytometry
  • Gene Editing / methods*
  • Gene Knockout Techniques*
  • Gene Library*
  • High-Throughput Nucleotide Sequencing
  • Human T-lymphotropic virus 1 / genetics
  • Human T-lymphotropic virus 1 / immunology*
  • Humans
  • Hybridomas
  • Kangai-1 Protein / genetics
  • Kangai-1 Protein / immunology*
  • Kangai-1 Protein / metabolism
  • Leukocyte Common Antigens / genetics
  • Leukocyte Common Antigens / immunology
  • Leukocyte Common Antigens / metabolism
  • Transfection


  • Antibodies, Monoclonal
  • CD82 protein, human
  • Epitopes
  • Kangai-1 Protein
  • Leukocyte Common Antigens
  • PTPRC protein, human