BD Simultest CD8/CD38

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1 Monoclonal Antibodies Detecting Human Antigens BD Simultest CD8/CD38 Catalog No Tests 20 µl/test RESEARCH APPLICATIONS DESCRIPTION Specificity Antigen distribution Research applications* include studies of: Human immunodeficiency virus (HIV) disease Diseases involving viral infection, such as those caused by Epstein-Barr virus (EBV) and Cytomegalovirus (CMV) 1,2 Transplantation The CD8 (Leu-2a) antigen is present on the human suppressor/cytotoxic T-lymphocyte subset 3,4 as well as on a subset of natural killer (NK) lymphocytes. 5 The CD8 antigenic determinant interacts with class I major histocompatibility complex (MHC) molecules resulting in increased adhesion between the CD8 + T lymphocytes and the target cells. 6-8 Binding of the CD8 antigen to class I MHC molecules enhances the activation of resting T lymphocytes. 6-8 CD8 recognizes an antigen expressed on the 32-kilodalton (kda) α-subunit of a disulfide-linked bimolecular complex. 9 The cytoplasmic domain of the α-subunit of the CD8 antigen is associated with the protein tyrosine kinase p56lck. 8,10 CD38 (Leu-17) recognizes an antigen that is an integral membrane glycoprotein, with a molecular weight of 45 kda, with a protein core of 35 kda. 11 The CD8 antigen is expressed on 19% to 48% of normal peripheral blood lymphocytes 12 and 60% to 85% of normal thymocytes. 3 The CD38 antigen is expressed on essentially all pre-b lymphocytes, plasma cells, and thymocytes. It is also present on activated-t lymphocytes, NK lymphocytes, monocytes, myeloblasts, and erythroblasts. 1,2,11,13,14,15-19 The antigen is expressed during the early stages of T- and B-lymphocyte differentiation, is lost during the intermediate stages of maturation, and then reappears during the final stages of maturation. 2,11,18,20 The antigen is also expressed in some cases of T- and B-acute lymphoblastic leukemia (ALL), Burkitt's lymphoma, multiple myeloma, and acute myeloid leukemia (AML). 17,21 CD8 + CD38 + T lymphocytes are involved in the immune response to viral infection. Elevated levels of CD8 + CD38 + lymphocytes are observed in HIV infection, and during the acute stages of EBV and CMV infection. 1,2 Increased numbers of CD8 + CD38 + T lymphocytes are also present in the period immediately following bone marrow transplantation. 22 In HIV infection, the coexpression of the CD8 and CD38 antigens, with a concomitant low percentage of CD4 + lymphocytes, is closely associated with disease * The published methods in the cited references have not been developed or tested by Becton Dickinson. 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 12/

2 Clones Composition PROCEDURE Method for BD Simultest immunofluorescence REPRESENTATIVE DATA progression. 14,23 Beginning at seroconversion, and during the first stages of HIV infection, the number of CD8 + T lymphocytes increases while the number of CD4 + T lymphocytes decreases. When the onset of acquired immune deficiency syndrome (AIDS) occurs, there is a decline in the absolute CD3 +, CD4 +, and CD8 + lymphocyte levels along with a further decrease in the CD4 + T lymphocyte percentage, but the relative percentage of CD8 + T lymphocytes continues to rise until, in the late stages of AIDS, the majority of the remaining T lymphocytes are CD8+. 1,2,14,15,16,23 Elevated levels of CD8 + CD38 + T lymphocytes become apparent at the time of seroconversion and continue to rise throughout the course of the disease, indicating persistent stimulation of the immune system by the virus. 2,14,16,23 CD8 + CD38 + T lymphocytes exhibit decreased activity of ecto-5'-nucleotidase (CD73 antigen), a maturation marker linked to T- and B-lymphocyte development, and appear to be activated and/or immature suppressor/cytotoxic-t lymphocytes. 2,16 The CD8 antibody, clone SK1, is derived from hybridization of NS-1 mouse myeloma cells with spleen cells from BALB/c mice immunized with human peripheral blood T lymphocytes. 4 The CD38 antibody, clone HB-7, is derived from hybridization of P3-X63-Ag8.653 mouse myeloma cells with spleen cells from BALB/c mice immunized with the BJAB cell line. 18 The CD8 antibody is composed of mouse IgG 1 heavy chains and kappa light chains. The CD38 antibody is composed of mouse IgG 1 heavy chains and kappa light chains. The BD Simultest reagent is supplied as a combination of CD8 (Leu-2a) FITC and CD38 (Leu-17) PE in 1.0 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. Peripheral Blood Mononuclear Cells (PBMCs): Add 20 µl of BD Simultest reagent to 10 6 PBMCs in 50 µl of medium containing 0.1% sodium azide. Mix thoroughly and incubate for 15 to 30 minutes in the dark at 2 C 8 C. Wash with 1X PBS with 0.1% sodium azide, add 0.5 ml of 1% paraformaldehyde, mix thoroughly, and analyze. If samples are not to be analyzed immediately, mix thoroughly just prior to analysis. See Becton Dickinson Procedure for Direct Immunofluorescence Staining of Cell Surfaces, Source Book Section 2.4. NOTE The occasional presence of cellular aggregates has been reported in PBMC preparations. For details, please refer to Becton Dickinson's technical update on The Escapee Phenomenon, Source Book Section 8.2. Lysed Whole Blood (LWB): Add 20 µl of BD Simultest reagent to 100 µl of whole blood in a staining tube. Mix thoroughly and incubate 15 to 30 minutes in the dark at room temperature (20 C 25 C). Add 2 ml of 1X BD FACS lysing solution (Cat. No ) at room temperature and vortex tube thoroughly. Incubate for no more than 10 to 12 minutes at room temperature in the dark. Wash cells with 1X PBS with 0.1% sodium azide, add 0.5 ml of 1% paraformaldehyde, mix thoroughly, and analyze. If samples are not to be analyzed immediately, mix thoroughly just prior to analysis. Refer to the BD FACS Lysing Solution package insert. Performed on peripheral blood leucocytes with scatter gates set on the lymphocyte fraction. BD Simultest reagents contain two fluorochromes, FITC and PE. Both fluorochromes can be excited at 488 nm, yet will emit light at different wavelengths. However, spectral overlap of these populations does occur. To obtain optimal results with BD Simultest reagents, both green (FITC) and red-orange (PE) signals must be electronically compensated to correct for spectral overlap. Refer to the Becton Dickinson Procedure for Two-Color Fluorescence Analysis Using BD Simultest Page 2

3 Figure 1 Two-parameter display of peripheral blood lymphocytes analyzed with a BD FACScan flow cytometer Peripheral Blood Mononuclear Cells Lysed Whole Blood CD38 PE CD38 PE CD8 FITC CD8 FITC HANDLING AND STORAGE WARNING CHARACTERIZATION WARRANTY 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 24,25 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. 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. Landay A, Ohlsson-Wilhelm B, Giorgi J. Application of flow cytometry to the study of HIV infection. AIDS. 1990;4: Salazar-Gonzalez JF, Moody DJ, Giorgi JV, Martinez-Maza O, Mitsuyasu RT, Fahey JL. Reduced ecto-5'- nucleotidase activity and enhanced OKT10 and HLA-DR expression on CD8 (T suppressor/cytotoxic) lymphocytes in the acquired immune deficiency syndrome: Evidence of CD8 cell immaturity. J Immunol. 1985;135: 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: Evans RL, Wall DW, Platsoucas CD, et al. Thymus-dependent membrane antigens in man: Inhibition of cell-mediated lympholysis by monoclonal antibodies to the TH2 antigen. Proc Natl Sci Acad USA. 1981;78: Lanier LL, Le AM, Phillips JH, Warner NL, Babcock GF. Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens. J Immunol. 1983;131: Page

4 6. Anderson P, Blue M-L, Morimoto C, Schlossman S. Cross-linking of T3 (CD3) with T4 (CD4) enhances the proliferation of resting T lymphocytes. J Immunol. 1987;139: Eichmann K, Johnson J, Falk I, Emmrich F. Effective activation of resting mouse T lymphocytes by crosslinking submitogenic concentrations of the T-cell antigen receptor with either Lyt-2 or L3T4. Eur J Immunol. 1987;17: Gallagher PF, Fazekas de St. Groth B, Miller JF. CD4 and CD8 molecules can physically associate with the same T-cell receptor. Proc Natl Acad Sci USA. 1989;86: Moebius U. Cluster report: CD8. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford: Oxford University Press;1989: Rudd CE, Burgess KE, Barber EK, Schlossman SF. Monoclonal antibodies to the CD4 and CD8 antigens precipitate variable amounts of CD4/CD8-associated p56 lck activity. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford: Oxford University Press;1989: Dörken B, Moller P, Pezzutto A, Schwartz-Albiez R, Molderhauer G. B-cell antigens: CD38. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford: Oxford University Press;1989: Reichert T, DeBruyère M, Denys V, et al. Lymphocyte subset reference ranges in adult Caucasians. Clin Immunol Immunopathol. 1991;60: Terstappen LWMM, Huang S, Safford M, Lansdorp PM, Loken MR. Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38 progenitor cells. Blood. 1991;77: Giorgi J, Hultin L. Lymphocyte subset alterations and immunophenotyping by flow cytometry in HIV disease. Clin Immunol Newslett. 1990;10(4): Nicholson JKA, Jones BM. Immunophenotyping by flow cytometry: Its use in HIV infection. Labmedica. 1989;6: / Prince HE, Arens L, Kleinman SH. CD4 and CD8 subsets defined by dual-color cytofluorometry which distinguish symptomatic from asymptomatic blood donors seropositive for human immunodeficiency virus. Diag Clin Immunol. 1987;5: Reinherz EL, Kung PC, Goldstein G, Levey RH, Schlossman SF. Discrete stages of human intrathymic differentiation: Analysis of normal thymocytes and leukemic lymphoblasts of T-cell lineage. Proc Natl Acad Sci USA. 1980;77: Tedder TF, Clement LT, Cooper MD. Discontinuous expression of a membrane antigen (HB-7) during B lymphocyte differentiation. Tissue Antigens. 1984;24: Terstappen LWMM, Hollander Z, Meiners H, Loken MR. Quantitative comparison of myeloid antigens on five lineages of mature peripheral blood cells. J Leuk Biol. 1990;48: Tedder TF, Crain MJ, Kubagawa H, Clement LT, Cooper MD. Evaluation of lymphocyte differentiation in primary and secondary immunodeficiency diseases. J Immunol. 1985;135: Pezzutto A, Behm F, Callard RE, et al. Flow cytometry analysis of the B-cell blind panel: Joint Report. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford: Oxford University Press;1989: Lum LG. The kinetics of immune reconstitution after human marrow transplantation. Blood. 1987;69: Giorgi JV, Detels R. T-cell subset alterations in HIV-infected homosexual men: NIAID multicenter AIDS cohort study. Clin Immunol Immunopathol. 1989;52: Pezzutto A, Behm F, Callard RE, et al. Flow cytometry analysis of the B-cell blind panel: Joint Report. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford: Oxford University Press;1989: Lum LG. The kinetics of immune reconstitution after human marrow transplantation. Blood. 1987;69: Giorgi JV, Detels R. T-cell subset alterations in HIV-infected homosexual men: NIAID multicenter AIDS cohort study. Clin Immunol Immunopathol. 1989;52: Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; CLSI document M29-A Page 4

5 25. 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 BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company BD Page