BD Oncomark. Lambda/Kappa/CD5/CD10/CD19/CD45. Catalog No Tests 20 µl/test

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1 Monoclonal Antibodies Detecting Human Antigens BD Oncomark Lambda/Kappa/CD5/CD10/CD19/CD45 Catalog No Tests 20 µl/test RESEARCH APPLICATIONS Research applications include: Studies of B-cell biology DESCRIPTION Specificity Anti-Lambda is specific for lambda light chains of human immunoglobulins. 1 Antigen distribution Anti-Kappa is specific for kappa light chains of human immunoglobulins. CD5 recognizes a human T-lymphocyte antigen, with a molecular weight of 67 kilodaltons (kda). 2 CD10 (Anti-CALLA) recognizes a human common acute lymphoblastic leukemia antigen (CALLA), with a molecular weight of 100 kda. 3,4 The CD10 antigen is identical to human membrane associated neutral endopeptidase (NEP; EC ), also known as enkephalinase. 5 CD19 (SJ25C1) recognizes a 90-kDa antigen that is present on human B lymphocytes. 6,7 CD45 (Anti HLe-1) recognizes human leucocyte antigens, with a molecular weight of 180 to 220 kda, that are members of the T200 family. 8 Immunoglobulins bearing lambda light chains are present on approximately 40% of normal B lymphocytes and on Igλ + neoplastic cells In serum, Anti-Lambda reacts with immunoglobulins bearing lambda light chains as well as free lambda light chains. Immunoglobulins bearing kappa light chains are present on approximately 60% of normal B lymphocytes and on Igκ + neoplastic cells In serum, Anti-Kappa reacts with immunoglobulins bearing kappa light chains as well as free kappa light chains. The CD5 antigen is present on approximately 70% of normal peripheral blood lymphocytes and on virtually all T lymphocytes in thymus and peripheral blood The CD5 antibody reacts with most cells in T-lymphocyte areas of spleen and lymph node and with many T-cell leukemias and lymphomas It also reacts with a distinct subset of normal B lymphocytes, 22 occasional cells in B-lymphocyte areas of spleen and lymph node, 19 and most Ig + B chronic lymphoblastic leukemia (CLL) cells Some lymphomas also express the CD5 antigen. 20 The CD10 antigen is found on lymphocytes from acute B-lymphoid leukemia samples. 26,27 The antigen is also present on a wide variety of normal and neoplastic cell types including renal epithelium, fibroblasts, granulocytes, and some lymphoma, melanoma, and glioma cell lines. 5 For Research Use Only. Not for use in diagnostic or therapeutic procedures. Becton, Dickinson and Company BD Biosciences 2350 Qume Drive San Jose, CA USA bdbiosciences.com ResearchApplications@bd.com 01/

2 Clones Composition PROCEDURE The CD19 antigen is present on approximately 7% to 23% of human peripheral blood lymphocytes 28 and on splenocytes. 29 The CD19 antigen is present on human B lymphocytes at most stages of maturation. 7,30 CD19 does not react with resting or activated T lymphocytes, granulocytes, or monocytes. 7,30 The CD45 antigen is present on all human leucocytes, including lymphocytes, monocytes, granulocytes, eosinophils, and basophils in peripheral blood and has a role in signal transduction, modifying signals from other surface molecules. 8 The CD45 antibody has been reported to react weakly with mature circulating erythrocytes and platelets. 8,31 Anti-Lambda, clone , * is derived from hybridization of P3-X63-Ag8.653 mouse myeloma cells with cells from BALB C/J mice immunized with human IgA 1 -λ myeloma protein. Anti-Kappa, clone TB28-2,* is derived from hybridization of P3-X63-Ag8.653 mouse myeloma cells with cells from CB6 (BC57b x BALB/c) mice immunized with human IgG-κ myeloma protein. CD5, clone L17F12, 16 is derived from hybridization of NS-1/Ag4 mouse myeloma cells with spleen cells from BALB/c mice immunized with human T-acute lymphoblastic leukemia cells. CD10, clone HI10a, 4 is derived from the hybridization of P3-63-Ag8.653 mouse myeloma cells with spleen cells of BALB/c mice immunized with blasts from a patient with acute CALLA leukemia. CD19, clone SJ25C1, 7 is derived from the hybridization of Sp2/0 mouse cells with spleen cells from BALB/c mice immunized with NALM1 + NALM16 cells. CD45, clone 2D1, 8 is derived from hybridization of NS-1 mouse myeloma cells with spleen cells from BALB/c mice immunized with human peripheral blood mononuclear cells (PBMCs). Anti-Kappa, CD19, and CD45 are each composed of mouse IgG 1 heavy chains and kappa light chains. Anti-Lambda is composed of mouse IgG 1 heavy chains and lambda light chains. CD5 and CD10 are each composed of mouse IgG 2a heavy chains and kappa light chains. This BD Oncomark reagent is supplied as a combination of Anti-Lambda FITC, Anti-Kappa PE, CD5 PerCP-Cy 5.5, CD10 PE-Cy 7, CD19 APC, and CD45 APC-Cy7 in 1 ml of phosphate-buffered saline (PBS) containing gelatin and 0.1% sodium azide. Visit our website (bdbiosciences.com) or contact your local BD representative for the lyse/wash protocol for direct immunofluorescence. To avoid serum interference when using this reagent: 1. Prewash the whole blood sample using at least 25 volumes of excess 1X PBS with 0.1% sodium azide (For example, 48 ml of 1X PBS with sodium azide per 2 ml of whole blood to be washed) and mix well. 2. Pellet cells by centrifugation. 3. Resuspend in 1X PBS with 0.1% sodium azide to the original volume. NOTE Spectral overlap values for PE-Cy7 and APC-Cy7 conjugates can vary from lot to lot. It is important to check these values on a known sample when using a new lot of reagents. * This clone has not been submitted to any previous workshop on human leucocyte differentiation antigens Page 2

3 CAUTION Some APC-Cy7 conjugates, and to a lesser extent PE-Cy7 and APC-H7 conjugates, show changes in their emission spectra with prolonged exposure to paraformaldehyde or light. For overnight storage of stained cells, wash and resuspend in buffer without paraformaldehyde after 1 hour of fixation.we recommend that you analyze fixed samples within four hours. REPRESENTATIVE DATA Performed on whole blood stained and lysed using BD FACS lysing solution (Cat. No ). Figure 1 Representative data analyzed with a BD FACS brand flow cytometer 50 FSC-A 250 granulocytes monocytes lymphocytes 10 2 CD45 APC-Cy CD CD19 APC CD10 PE-Cy CD5 PerCP-Cy CD5 + CD19 Q3 CD5 + CD19 + Q Anti-Kappa PE 10 5 Anti-Kappa + Q3 Q2 Anti-Lambda CD19 APC CD20 APC-Cy HANDLING AND STORAGE WARNING CHARACTERIZATION Store vials at 2 C 8 C. Conjugated forms should not be frozen. Protect from exposure to light. Each reagent is stable until the expiration date shown on the bottle label when stored as directed. All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 32,33 and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves. To ensure consistently high-quality reagents, each lot of antibody is tested for conformance with characteristics of a standard reagent. Representative flow cytometric data is included in this data sheet. Page

4 WARRANTY Unless otherwise indicated in any applicable BD general conditions of sale for non-us customers, the following warranty applies to the purchase of these products. THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT. BD S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT. REFERENCES 1. Kubagawa H, Gaithings WE, Levitt D, Kearney JF, Cooper MD. Immunoglobulin isotype expression of normal pre-b cells as determined by immunofluorescence. J Clin Immunol. 1982;2: Knowles RW. Immunochemical analysis of the T-cell specific antigens. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human T Lymphocytes. New York, NY: Springer- Verlag; 1986;1: Greaves MF. Monoclonal antibodies as probes for leukemic heterogeneity and hematopoietic differentiation. In: Knapp W, ed. Leukemia Markers. New York, NY: Academic Press; 1981: Zola H. CD10 Workshop Panel report. In: Schlossman SF, Boumsell L, Gilks W, et al, eds. Leucocyte Typing V: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1995: Letarte M, Vera S, Tran R, et al. Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase. J Exp Med. 1988;168: Moldenhauer G, Dörken B, Schwartz R, Pezzutto A, Knops J, Hammerling GJ. Analysis of ten B lymphocyte-specific workshop monoclonal antibodies. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human B Lymphocytes. New York, NY: Springer-Verlag; 1986;2: Nadler LM. B Cell/Leukemia Panel Workshop: summary and comments. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human B Lymphocytes. New York, NY: Springer- Verlag; 1986;2: Schwinzer R. Cluster report: CD45/CD45R. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989: Chung J, Gong G, Huh J, Khang SK, Ro JY. Flow cytometric immunophenotyping in fine-needle aspiration of lymph nodes. J Korean Med Sci. 1999;14: Zardawi IM, Jain S, Bennett G. Flow-cytometric algorithm on fine-needle aspirates for the clinical workup of patients with lymphadenopathy. Diagn Cytopathol. 1998;19: Davidson B, Risberg B, Berner A, Smeland EB, Torlakovic E. Evaluation of lymphoid cell populations in cytology specimens using flow cytometry and polymerase chain reaction. Diagn Mol Pathol. 1999;8: Braylan RC, Benson NA, Iturraspe J. Analysis of lymphomas by flow cytometry: current and emerging strategies. Ann NY Acad Sci. 1993;677: Johnson A, Cavallin-Stahl E, Akerman M. Flow cytometric light chain analysis of peripheral blood lymphocytes in patients with non-hodgkin's lymphoma. Br J Cancer. 1985;52(2): Letwin BW, Wallace PK, Muirhead KA, Hensler GL, Kashatus WH, Horan PK. An improved clonal excess assay using flow cytometry and B-cell gating. Blood. 1990;75: Geary WA, Frierson HF, Innes DJ, Normansell DE. Quantitative criteria for clonality in the diagnosis of B-cell non-hodgkin's lymphoma by flow cytometry. Mod Pathol. 1993;6: Engleman EG, Warnke R, Fox RI, Dilley J, Benike CJ, Levy R. Studies of a human T lymphocyte antigen recognized by a monoclonal antibody. Proc Natl Acad Sci USA. 1981;78: Ledbetter JA, Evans RL, Lipinski M, Cunningham-Rundles C, Good RA, Herzenberg LA. Evolutionary conservation of surface molecules that distinguish T-lymphocyte helper/inducer and T cytotoxic/suppressor subpopulations in mouse and man. J Exp Med. 1981;153: Ledbetter JA, Frankel AE, Herzenberg LA. Human Leu T-cell differentiation antigens: quantitative expression on normal lymphoid cells and cell lines. In: Hämmerling G, Hämmerling U, Kearney J, eds. Monoclonal Antibodies and T Cell Hybridomas: Perspectives and Technical Notes. New York, NY: Elsevier/North Holland; 1981: Page 4

5 19. Warnke RA, Levy R. Detection of T and B antigens with hybridoma monoclonal antibodies: a biotinavidin-horseradish peroxidase method. J Histochem Cytochem. 1980;28: Warnke R, Miller R, Grogan T, Pederson M, Dilley J, Levy R. Immunologic phenotype in 30 patients with diffuse large-cell lymphoma. N Eng J Med. 1980;303: Zipf RF, Fox R, Dilley J, Levy R. Definition of the high risk ALL patient by immunologic phenotyping with monoclonal antibodies. Cancer Res. 1981;41: Gadol N, Ault KA. Phenotypic and functional characterization of human Leu-1 (CD5) B cells. Immunol Rev. 1986;93: Royston I, Majda JA, Baird SM, Meserve BL, Griffiths JC. Human T-cell antigens defined by monoclonal antibodies: the 65,000-dalton antigen of T cells (T65) is also found on chronic lymphocytic leukemia cells bearing surface immunoglobulin. J Immunol. 1980;125: Dono M, Cerruti G, Zupo S. The CD5 + B-cell. Int J Biochem Cell Biol. 2004;36: D'Arena G, Di Renzo N, Brugiatelli M, Vigliotti ML, Keating MJ. Biological and clinical heterogeneity of B-cell chronic lymphocytic leukemia. Leuk Lymphoma. 2003;44: Le Bien TW, McCormack RT. The common acute lymphoblastic leukemia antigen (CD10): emancipation from a functional enigma. Blood. 1989;73: Morabito F, Mangiola M, Rapezzi D, et al. Expression of CD10 by B-chronic lymphocytic leukemia cells undergoing apoptosis in vivo and in vitro. Haematologica. 2003;88: Reichert T, DeBruyère M, Deneys V, et al. Lymphocyte subset reference ranges in adult Caucasians. Clin Immunol Immunopath. 1991;60: Tedder T, Zhou L-J, Engel P. The CD19/CD21 signal transduction complex of B lymphocytes. Immunol Today. 1994;15: Loken MR, Shah VO, Dattilio KL, Civin CI. Flow cytometric analysis of human bone marrow. II. Normal B-lymphocyte development. Blood. 1987;70: Jackson A. Basic phenotyping of lymphocytes: selection and testing of reagents and interpretation of data. Clin Immunol Newslett. 1990;10: Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; CLSI document M29-A Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR. 1988;37: PATENTS AND TRADEMARKS APC-Cy7: US Patent Number 5,714,386 Cy is a trademark of GE Healthcare. This product is subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and is made and sold under license from GE Healthcare. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one, return this material, unopened, to BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, and any money paid for the material will be refunded. BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company BD Page