CENTRO DI RIFERIMENTO ONCOLOGICO
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MONOCLONAL ANTIBODY - 215 anti BCR Monoclonal antibody, named 215, recognizing an epitope situated across the CDR1 and FWR2 domains of the k light chain of the B Cell Receptor. Epitope determination performed with the epitope excision approach (De Re V. et al. Leukemia 2006, 20, 1145–1154). Successfully used in Flow Cytometry toward DG75 and SH9 (spontaneous lymphoblastoid cell line, IgG+Vk3+, IgM−, EBV+) cells. 215 MAB has also been tested for its ability to mediate AntibodyDependent Cell-Mediated Cytotoxicity (ADCC). The results indicated that it induced a 25% of lysis in DG75 cells when incubated with peripheral blood mononuclear cells isolated from healthy donors. It is also reactive in ELISA assay and in Western Blot. Researchers have previously demonstrated that the B-cell receptor (BCR) repertoire expressed by type II mixed cryoglobulinemia (II-MC)- associated clonal B-cell proliferations and by HCV-associated non-Hodgkin’s lymphoma (NHL) is not random, with V1–69, V3–7, V4–59 variable heavy (VH)- and still more variable κ (VK)3–20 and VK3–15 light (VL)-chain genes the most represented. We produced this murine antibody, using an Idiotype prototype [a single-chain variable fragment (scFv) antibody], as antigen, which mimicked the VH/VL sequence of a tumor sample obtained from a patient with a B-cell lymphoma, a II-MC disorder, and an HCV infection (De Re, V. et al. 2000, Int. J Cancer 87: 211–216).
Antibody Overview 1. Clone name: 215 2. Specificity: human B Cell Receptor k light chain (VK3–20, VK3–15) 3. Species/Isotype: Murine (IgG1 isotype) 4. This antibody can be used for the following applications and corresponding conditions: _X_ Flow Cytometry _X_ ELISA: detection __x__ _X_ Western Blot: detection __x__ 5. What is the best positive tissue/cell line to use in testing this antibody? DG75 or SH9 cell line or recombinant protein 6. Publications: De Re V. et al. Annals of the New York Academy of Sciences, 1173: 152–160 A) Epitope Mapping by a Combination of Epitope Excision and MALDI-TOF MS. This approach that uses mass spectrometry for the identification of functional epitopes on antigens bound to antibodies. In epitope excision, a tryptic enzymatic digestion is done while the protein (VK3-20) is bound to the antibody. mAb prevents either proteolysis, or chemical modification of sites on the antigens that are situated in the antibody binding pocket. An important feature of this approach is that, because nondenaturing conditions are used, the antigen retains its native conformation so that conformational epitopes can be determined. B) Results of epitope excision.
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More Information Valli De Re, PhD Department of Molecular Oncology and Translational Medicine (DOMERT) vdere@cro.it +39-0434-659-672/675