One of the most well-characterized therapeutic monoclonal antibodies is rituximab (Rituxan®, Genentech, South San Francisco, CA; Biogen Idec, Cambridge, MA), a chimeric anti-CD20 antibody. CD20 is a 33-36-kDa transmembrane phosphoprotein expressed on the surface of mature B-cells and the majority of immature B-cells (1,2). It is involved in proliferation, most likely by regulating transmembrane calcium conductance (3). Rituximab induces apop-tosis through complement fixation and antibody-dependent, cell-mediated cyto-toxicity (4-6). Immunotherapy with rituximab has been successful both alone and in combination with chemotherapy in treating B-cell lymphoproliferative diseases (7-10) with high response rates in various non-Hodgkin's lymphomas and chronic B-Chronic Lymphocytic Leukemia (CLL) (10-12).

Quantitative flow cytometry can be applied to measure cell surface antigens such as CD20. Measuring levels of surface antigens would provide valuable

From: Methods in Molecular Biology, vol. 378: Monoclonal Antibodies: Methods and Protocols Edited by: M. Albitar © Humana Press Inc., Totowa, NJ

insight to optimal dosing and scheduling of therapy. It has been observed that CD20 antigen cannot be detected by flow cytometry on the surface of B-cells in patients treated with rituximab because of the masking effect of rituximab on CD20 itself (13). Hence, monitoring levels of CD20 delineates the efficacy of rituximab therapy. We describe a three-color flow cytometry immuno-phenotyping procedure to assess the expression of CD20 on specific cellular populations such as B-cells. CD20 is labeled with phycoerythrin (PE) in a 1:1 fluorescence-to-protein ratio. This methodology would be beneficial for monitoring response in patients treated with rituximab and can also be applied to other cell surface markers.

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