title: A high throughput electrochemiluminescent cell-binding assay for therapeutic anti-CD20 antibody selection.
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.
authors.affiliation: Assay and Automation Technology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Lu
authors.forename: Wai Lee
authors.initials: WL
authors.lastname: Wong
authors.forename: Y Gloria
authors.initials: YG
authors.lastname: Meng
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D018951:Antigens, CD20 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data*; D018628:Gene Dosage; D049449:Luminescence; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D011993:Recombinant Fusion Proteins