Simple quantitative live cell and anti-idiotypic antibody based ELISA for humanized antibody directed to cell surface protein CD20

• Published in: Journal of immunological methods 2004-11, Vol.294 (1), p.189-197
• Main Authors: Hong, Kyu, Presta, Leonard G., Lu, Yanmei, Penn, Ashley, Adams, Camellia, Chuntharapai, Anan, Yang, Jihong, Wong, Wai Lee, Meng, Y. Gloria
• Format: Article
• Language: English
• Subjects: Animals; Anti-idiotypic antibody; Antibodies, Anti-Idiotypic - chemistry; Antibodies, Anti-Idiotypic - immunology; Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - therapeutic use; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20 - genetics; Antigens, CD20 - immunology; Antigens, CD20 - metabolism; Autoimmune Diseases - blood; Autoimmune Diseases - drug therapy; Autoimmune Diseases - immunology; Autoimmune Diseases - pathology; Binding affinity; Biological and medical sciences; CHO cell; CHO Cells; Clone Cells; Cricetinae; ELISA; Enzyme-Linked Immunosorbent Assay - methods; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Gene Expression; Humanized anti-CD20; Humans; Lymphoma, Non-Hodgkin - blood; Lymphoma, Non-Hodgkin - drug therapy; Lymphoma, Non-Hodgkin - immunology; Lymphoma, Non-Hodgkin - pathology; Membrane Proteins - genetics; Membrane Proteins - immunology; Mice; Molecular immunology; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - therapeutic use; Rituximab; Serum - chemistry; Techniques; Transduction, Genetic; WIL2 cell
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2004.09.003

Rituxan, a chimeric anti-CD20 antibody, has been used for treating non-Hodgkin's lymphoma and some autoimmune diseases. However, a humanized anti-CD20 antibody is desirable for long-term treatment of autoimmune diseases. CD20 is an integral membrane protein with a small intervening extracellular loop. Lacking a native soluble CD20 protein, we developed a simple cell-based enzyme-linked immunosorbent assay (ELISA) using live WIL2 cells in a 96-well format to measure relative binding affinity to support the humanization process. Although WIL2 cells grow in suspension and require centrifugation during the wash steps, the assay was quantitative and reproducible. We also demonstrated that cloned adherent transfected Chinese hamster ovary (CHO) cells could be used to improve assay throughput. For clinical studies requiring quantification of the humanized antibody in serum, we used an alternate approach and developed a high throughput ELISA using an anti-idiotypic antibody as a surrogate antigen for capture and an anti-idiotypic antibody for detection to overcome serum effects. These assay strategies may be applied for characterization of other antibodies directed to multitransmembrane proteins.