A high throughput electrochemiluminescent cell-binding assay for therapeutic anti-CD20 antibody selection

J Immunol Methods. 2006 Jul 31;314(1-2):74-9. doi: 10.1016/j.jim.2006.05.011. Epub 2006 Jun 19.

Abstract

A cell-based ELISA using suspension WIL2 cells in 96-well format was previously developed for measuring relative binding affinities of humanized anti-CD20 variants. We further developed a new cell-binding assay that uses high binding capacity carbon electrode plates for rapid attachment of suspension WIL2 cells and electrochemiluminescence for detection. Compared to the cell-based ELISA, which requires centrifugation for the manual wash steps, significant improvement in assay throughput was achieved by using a microplate washer. The assay can be performed on both 96- and 384-well plates with a standard curve range of 2.74-2000 ng/ml, which is wider than the range of 15.6-1000 ng/ml for the cell-based ELISA. Using CD20 expressing CHO cell clones, surface expression of >or=33,000 CD20 molecules was sufficient to obtain a dose-response curve in 384-well format. Relative affinities of 15 humanized variants correlated well (r(2)=0.94) between electrochemiluminescent cell-binding assay and cell-based ELISA. A competitive assay format, using mouse anti-CD20 antibody as the tracer, with a dose-response range of 27.4-20,000 ng/ml was also developed. The new cell-binding assay method can be used to efficiently support humanization process for selection of anti-CD20 antibody drug candidates and to characterize antibody binding to other cell surface proteins.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / immunology
  • Antibody Specificity*
  • Antigens, CD20 / chemistry*
  • Antigens, CD20 / genetics
  • Antigens, CD20 / metabolism
  • CHO Cells
  • Clone Cells
  • Cricetinae
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Enzyme-Linked Immunosorbent Assay / statistics & numerical data*
  • Gene Dosage
  • Luminescence*
  • Mice
  • Recombinant Fusion Proteins / chemistry

Substances

  • Antibodies, Monoclonal
  • Antigens, CD20
  • Recombinant Fusion Proteins