Quantification of Surface Antigens and Quantitative Flow Cytometry

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1 5 Quantification of Surface Antigens and Quantitative Flow Cytometry Huai En Huang Chan, Iman Jilani, Richard Chang, and Maher Albitar Summary Measuring expression levels of cell surface antigens is important for the diagnosis of diseases such as B-cell chronic lymphocytic leukemia and the monitoring of targeted therapy, particularly antibody-based therapy. In some cases, the number of antigens that the therapeutic antibodies bind on the cell surface may reflect the efficacy of therapy. Thus, quantitating the number of molecules on the surface of cells before, during, and after therapy would provide important information for monitoring antibody-based therapy and potentially can be used to adjust dosing. We describe a quantitative flow cytometry approach to measuring levels of the CD20 surface antigen, the molecular target of rituximab. Key Words: CD20; surface antigen; quantitative flow cytometry; antibody therapy. 1. Introduction 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 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 apoptosis through complement fixation and antibody-dependent, cell-mediated cytotoxicity (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 65

2 66 Chan et al. 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 immunophenotyping 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. 2. Materials 2.1. Surface Marker Staining 1. Flow PBS: prepare with 5.6 g sodium phosphate dibasic anhydrous, g sodium chloride, 2.8 g albumin-bovine, and 4.0 g sodium azide. Add deionized water to 4 L and adjust to ph Red blood cell lysing reagent: 8.26 g ammonium chloride, 1.0 g potassium bicarbonate, and g tetrasodium EDTA. Add deionized water to 1 L and adjust to ph Isotype controls: IgG1 PE, IgG1 FITC, and IgG1 APC (BD Biosciences, San Jose, CA). 4. Surface markers: CD20-PE, CD19 FITC, and CD3 APC (BD Biosciences). 5. Sorvall Cell Washer 2 (Thermo Electron Corporation, Asheville, NC) Flow Cytometric Analysis 1. FACSCalibur flow cytometer (BD Biosciences). 2. QuantiBRITE PE beads (BD Biosciences). 3. Cell Quest Pro acquisition and analysis software (BD Biosciences). 4. FlowJO analysis software (Treestar, Ashland OR). 5. CaliBRITE 3 beads (BD Biosciences). 6. APC beads (BD Biosciences). 3. Methods This is a three-color immunophenotyping assay to assess the expression of CD20 on B-cells. We use QuantiBRITE PE beads to establish a fluorescence standard for each experiment. Each of the four bead populations has a calibrated mean number of bound PE molecules/bead. Samples are stained with antibodies that have PE conjugated at a 1:1 ratio. The number of bound PE-conjugated antibodies/cell in the sample is extrapolated using the PE beads as a standard. Thus, PE molecules bound per cell is equivalent to the number of antibodies bound to the cell. This in turn is equivalent to the number of molecules of target protein in each cell.

3 67 Fig. 1. Dot plots representing examples of CD20 expression in B-cells (CD19+) pre- (A) and post-treatment with rituximab (B,C). The antibody-binding capacity is 1367, 780, and 0 for A, B, and C, respectively.

4 68 Chan et al Instrument Setup 1. Calibration should be performed according to the manufacturer s protocol using CaliBRITE 3 beads with addition of APC beads every time the cytometer is used. 2. QuatiBRITE PE beads are reconstituted by addition of 1 ml flow PBS and vortexing. 3. CellQuest Pro software is launched and the appropriate acquisition template and instrument settings are selected events are captured Surface Marker Staining 1. Each sample is represented in two tubes: one stained with the isotype controls and the other stained with specific markers. 2. The appropriate volume of adjusted cells (equivalent to cells) is pipetted into each of the two mm flow tubes. 3. The control tube is incubated with 5 L of IgG1 FITC, IgG1 PerCP, and IgG1 APC. The markers tube is incubated with 10 L of CD19 FITC, CD20-PE, and CD3-APC. Incubations take place for 15 min at room temperature in the dark. 4. The samples are then incubated with 2 ml lysing reagent for 10 min at room temperature in the dark. 5. The samples are washed and resuspended in 500 L flow PBS for analysis Acquisition of Samples on Flow Cytometer 1. The samples are acquired up to 5000 events. An example of the results is shown in Fig Discussion We describe a simple and quantitative approach to the measurement of cell surface antigens using CD20 as an example. Though it is believed that effective treatment with rituximab will mask CD20 expression, quantitation of levels of CD20 may help in determining efficacy of therapy and give insight on the optimal dosage and scheduling of therapy. Patients without complete masking may not have received adequate dosing. This method can be modified to detect surface antigens in plasma and can be expanded to other antibody-based therapies such as alemtuzumab (anti-cd52; CamPath, Berlex, Montville, NJ) (14) and gemtuzumab ozogamicin (anti-cd33; Mylotarg, Wyeth Ayerst, Madison, NJ) (15). References 1. Stashenko, P., Nadler, L. M., Hardy, R., and Schlossman, S. F. (1980) Characterization of a human b lymphocyte-specific antigen. J. Immunol. 125, Andeson, K. C., Bates, M. P., Slaughenhoup, B. L., Pinkus, G. S., Schlossman, S. F., and Nadler, L. M. (1984) Expression of human B cell-associated antigens on leukemias and lymphomas: a model of human B cell differentiation. Blood 63,

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