Simple quantitative live cell and anti-idiotypic antibody based ELISA for humanized antibody directed to cell surface protein CD20.
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.
Hong
Kyu
K
Assay and Automation Technology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Presta
Leonard G
LG
Lu
Yanmei
Y
Penn
Ashley
A
Adams
Camellia
C
Chuntharapai
Anan
A
Yang
Jihong
J
Wong
Wai Lee
WL
Meng
Y Gloria
YG
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001327:Autoimmune Diseases / Q000097:blood / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D008228:Lymphoma, Non-Hodgkin / Q000097:blood / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D000069283:Rituximab; D044967:Serum / Q000737:chemistry; D014161:Transduction, Genetic
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000069283:Rituximab