Establishment of an Anti-CD20 Monoclonal Antibody (C20Mab-60) for Immunohistochemical Analyses

Sensitive and specific monoclonal antibodies (mAbs) are needed for detecting CD20. This antigen, one of several B lymphocyte antigens, is a target for every application used in the diagnosis of B cell lymphoma. Many anti-CD20 mAbs have been established, although applications of these antibodies are limited. This study aims to establish sensitive and specific anti-CD20 mAbs suitable for broad application, such as flow cytometry, Western blotting, and immunohistochemical analyses. Using the Cell-Based Immunization and Screening (CBIS) method, all procedures were performed by utilizing CD20-stable transfectants, and a clone, C20Mab-60 (IgG2a, kappa), was developed. In flow cytometry, C20Mab-60 detected overexpression of CD20 in LN229 cell and endogenous CD20 in BALL-1 (a human B cell leukemia cell line) but did not react with CD20-knockout BALL-1 (BINDS-24), indicating specificity for CD20. In Western blotting, C20Mab-60 detected CD20-overexpressing Chinese hamster ovary-K1, BALL-1, and Raji (a human Burkitt's lymphoma cell line) displaying both sensitivity and specificity. Furthermore, B cell but not T cell lymphomas were strongly stained with C20Mab-60 in immunohistochemical analyses. C20Mab-60, which was developed by CBIS method, is shown to be useful for the detection of cells expressing CD20 in lymphoma tissues by flow cytometry, Western blotting, and immunohistochemical analyses.