BD FastImmune. Catalog No Tests 20 µl/test

Transcription

1 Monoclonal Antibodies Detecting Human Antigens BD FastImmune Anti-Hu IFN-γ/CD69/CD4 Catalog No Tests 20 µl/test RESEARCH APPLICATIONS BD FastImmune Anti-Hu IFN-γ/CD69/CD4 is designed for the detection of intracellular cytokines and expression of the activation marker CD69 in antigenactivated CD4 + T lymphocytes in whole blood. Applications include studies of T-cell responses to antigens such as cytomegalovirus (CMV), 1 5 Human immunodeficiency virus (HIV), 6,7 herpes viruses, 1 and tumor antigens. 8 DESCRIPTION Specificity Anti-Human Interferon-γ (Anti-Hu IFN-γ) recognizes a 20- to 25-kilodalton (kda) glycoprotein. 9 CD69 recognizes a very early human activation antigen. The CD69 antigen is a surface homodimer formed by the association of 28-kDa and 32-kDa chains that are held together by disulfide bridges. 10 The CD4 11,12 antigen, with a molecular weight of 55 kda, 13 is present on T-helper/inducer lymphocytes and monocytes. 14,15 Antigen distribution IFN-γ is produced, upon activation, by most CD8 + T lymphocytes, by the T H 1 and T H 0 subsets of CD4 + T lymphocytes, and by natural killer (NK) lymphocytes. 9,16 18 IFN-γ is a multifunctional immunomodulator with anti-tumor and anti-viral activity. 19,20 IFN-γ is a pleiotropic cytokine instrumental in the regulation of immune and inflammatory processes, and it has a role in the differentiation and function of monocytes. 23 The CD69 antigen is present on activated T, B, NK lymphocytes 10 and platelets. 24 In normal peripheral blood, a variable percentage of lymphocytes express the CD69 antigen.* Upon activation, CD69 antigen expression increases on lymphocytes; peak expression generally occurs within 18 hours, preceding the appearance of HLA-DR, interleukin-2 (IL-2) receptor (CD25 antigen), and transferrin receptor (CD71 antigen) CD69 and phorbol ester are comitogenic for T lymphocytes. 27 In thymus, the CD69 antigen is constitutively expressed on the bright CD3 + subset. 28 The CD4 antigen is present on the helper/inducer T-lymphocyte subset, such as CD3 + CD4 +, that comprises 28% to 58% 29 of normal peripheral blood lymphocytes. 13,15 It is also present on 80% to 95% of normal thymocytes. 13,15 The CD4 antigen is present in low density on the cell surface of monocytes and in the cytoplasm of monocytes and macrophages (CD3 CD4 + ). 30 The CD4 antigen is the receptor for the HIV. 31 Some CD4 antibodies, including CD4, inhibit HIV binding to CD4 + cells. 32 Subjects infected with HIV were found to exhibit a continuous loss * Due to the variable density of the antigen, the number of positive events can vary depending upon the brightness of the fluorochrome and the sensitivity of the instrument. 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 Method for Intracellular Cytokine Detection REPRESENTATIVE DATA of CD4 + lymphocytes and a relative increase in the CD8 (Leu-2a) + lymphocyte subset Anti-Hu IFN-γ, clone , is derived from the hybridization of P3X-63-Ag8.653 mouse myeloma cells with lymph node cells from BALB/c mice immunized with recombinant human IFN-γ. CD69, clone L78, is derived from hybridization of Sp2/0-Ag14 mouse myeloma cells with lymph node cells from BALB/c mice immunized with a CD8 + alloantigen-directed cytotoxic T-lymphocyte (CTL) cell line. 36 CD4, clone SK3, is derived from hybridization of NS-1mouse myeloma cells with spleen cells from BALB/c mice immunized with human peripheral blood T lymphocytes. Anti-Hu IFN-γ is composed of mouse IgG 2b heavy chains and kappa light chains. CD69 and CD4 are each composed of mouse IgG 1 heavy chains and kappa light chains. The BD FastImmune Anti-Hu IFN-γ/CD69/CD4 reagent is supplied as a combination of IFN-γ FITC, CD69 PE, and CD4 PerCP-Cy 5.5 in 1.0 ml of phosphate-buffered saline (PBS) containing bovine serum albumin (BSA), betalactoglobulin, and 0.1% sodium azide (NaN 3 ). Visit our website (bdbiosciences.com) or contact your local BD representative for the lyse/wash protocol for direct immunofluorescence. For complete activation and staining protocol and the appropriate application note, visit our website (bdbiosciences.com) or contact your local BD representative. Abbreviated Intracellular Staining Procedure: Add 1 ml of 1X BD FACS lysing solution (Cat. No ) to 100 µl of activated heparinized whole blood. Incubate for 10 minutes at room temperature. Centrifuge at 500 x g for 5 minutes; decant the supernatant. Add 0.5 ml of 1X BD FACS Permeabilizing Solution 2 (Cat. No ). Vortex and incubate for 10 minutes at room temperature. Wash by adding PBS containing 0.5% BSA and 0.1% NaN 3, and centrifuge for 5 minutes. Add 20 µl of Anti-Hu IFN-γ FITC/CD69 PE/CD4 PerCP-Cy5.5. Vortex and incubate for 30 minutes at room temperature in the dark. Repeat wash step. Resuspend cells in 1% paraformaldehyde in PBS. Flow cytometric analysis was performed on CMV-activated whole blood. Laser excitation was at 488 nm. Figure 1 Representative data analyzed on a BD FACS brand flow cytometer 0 SSC R CD4 PerCP-Cy SSC R2 0 FSC 10 0 CD69 PE Anti IFN-γ FITC 10 4 HANDLING AND STORAGE WARNING 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 37,38 and dispose of with Page 2

3 proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves. CHARACTERIZATION WARRANTY 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. Asanuma H, Sharp M, Maecker HT, Maino VC, Arvin AM. Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus determined by intracellular detection of cytokine expression. J Infec Dis. 2000;181(3): Komanduri KV, Viswanathan MN, Wieder ED, et al. Restoration of cytomegalovirus-specific CD4 + T- lymphocyte responses after ganciclovir and highly active antiretroviral therapy in individuals infected with HIV-1. Nat Med. 1998;4: Nomura LE, Walker JM, Maecker HT. Optimization of whole blood antigen-specific cytokine assays for CD4 + T-cells. Cytometry. 2000;40(1): Suni MA, Picker LJ, Maino VC. Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry. J Immunol Methods. 1998;212: Waldrop SL, Davis KA, Maino VC, Picker LJ. Normal human CD4 + memory T cells display broad heterogeneity in their activation threshold for cytokine synthesis. J Immunol. 1998;161: Pitcher CJ, Quittner C, Peterson DM, et al. HIV-1 specific CD4 + T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression. Nat Med. 1999;5: Waldrop SL, Pitcher CJ, Peterson DM, Maino VC, Picker LJ. Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency. J Clin Invest. 1997;99: Lee PP, Yee C, Savage PA, et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Ned. 1999;5: Aggarwal BB, Puri RK. Common and uncommon features of cytokines and cytokine receptors: an overview. In: Human Cytokines: Their Role in Disease and Therapy. Cambridge, MA: Blackwell Science; 1995: Schwarting R, Biedobitek G, Stein H. Cluster report: CD69. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989: Bernard A, Boumsell L, Hill C. Joint report of the first international workshop on human leucocyte differentiation antigens by the investigators of the participating laboratories. In: Bernard A, Boumsell L, Dausset J, Milstein C, Schlossman SF, eds. Leucocyte Typing. New York, NY: Springer-Verlag; 1984: Evans RL, Wall DW, Platsoucas CD, et al. Thymus-dependent membrane antigens in man: inhibition of cell-mediated lympholysis by monoclonal antibodies to the T H 2 antigen. 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: Engleman EG, Benike CJ, Glickman E, Evans RL. Antibodies to membrane structures that distinguish suppressor/cytotoxic and helper T lymphocyte subpopulations block the mixed leukocyte reaction in man. J Exp Med. 1981;153: Page

4 15. Kotzin B, Benike C, Engleman E. Induction of immunoglobulin secreting cells in the allogeneic mixed leukocyte reaction: Regulation by helper and suppressor lymphocyte subsets in man. J Immunol. 1981;127: Paliard X, Malefijt RDW, Yssel H, et al. Simultaneous production of IL-1, IL-4, and IFH-γ by activated human CD4 + and CD8 + T cell clones. J Immunol. 1988;141: Openshaw P, Murphy EE, Hosken NA, et al. Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations. J Exp Med. 1995;182: Street N, Mosmann T. Functional diversity of T lymphocytes due to secretion of different cytokine patterns. The FASEB Journal. 1991;5: Hardy KJ, Sawada T. Human γ interferon strongly upregulates its own gene expression in peripheral blood lymphocytes. J Exp Med. 1989;170: Johnson HM, Bazer FW, Szente BE, Jarpe MA. How interferons fight disease. Scientific American. 1994: ElGhazali GEB, Paulie S, Andersson G, et al. Number of interleukin-4 and interferon-γ secreting human T cells reactive with tetanus toxoid and the mycobacterial antigen PPD or phytohemagglutinin: distinct response profiles depending on the type of antigen used for activation. Eur J Immunol. 1993;23: Romagnani S, Del Prete G, Maggi E, et al. Human T H 1 and T H 2 subsets. Int Arch Allergy Immunol. 1992;99: Powrie F, Coffman RL. Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunol Today. 1993;14: Testi R, Pulcinelli F, Frati L, Gazzaniga P, Santoni A. CD69 is expressed on platelets and mediates platelet activation and aggregation. J Exp Med. 1990;172: Chen JH, Prince H, Buck D, et al. Leu-23: an early activation antigen on human lymphocytes. Fed Proc. 1988;2:A Testi R, Philips J, Lanier LL. Constitutive expression of a phosphorylated activation (Leu-23) by CD3 bright thymocytes. J Immunol. 1988;141: Testi R, Philips J, Lanier LL. Leu-23 induction as an early marker for functional CD3/T cell antigen receptor triggering: requirement for receptor cross-linking, prolonged elevation of intracellular (Ca ++ ), and stimulation of protein kinase C. J Immunol. 1989;142: Testi R, Philips J, Lanier LL. T cell activation via Leu-23 (CD69). J Immunol. 1989;143: Reichert T, DeBruyère M, Deneys V, et al. Lymphocyte subset reference ranges in adult Caucasians. Clin Immunol Immunopath. 1991;60: Wood GS, Warner NL, Warnke RA. Anti Leu-3/T4 antibodies react with cells of monocyte/macrophage and Langerhans lineage. J Immunol. 1983;131: Dalgleish AG, Beverley PCL, Clapham PR, Crawford DH, Greaves MF, Weiss RA. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature. 1984;312: Sattentau QJ, Dalgleish AG, Weiss RA, Beverley PCL. Epitopes of the CD4 antigen and HIV infection. Science. 1986;234: Lewis DE, Puck JM, Babcock GF, Rich R. Disproportionate expansion of a minor T cell subset in patients with lymphadenopathy syndrome and acquired immunodeficiency syndrome. J Infect Disease. 1985;151: Ohno T, Kanoh T, Suzuki T, et al. Comparative analysis of lymphocyte phenotypes between carriers of human immunodeficiency virus (HIV) and adult patients with primary immunodeficiency using twocolor immunofluorescence flow cytometry. J Exp Med. 1988;154: Stites DP, Casavant CH, McHugh TM, et al. Flow cytometric analysis of lymphocyte phenotypes in AIDS using monoclonal antibodies and simultaneous dual immunofluorescence. Clin Immunol Immunopathol. 1986;38: Lanier LL, Buck DW, Rhodes L, et al. Interleukin 2 activation of natural killer cells rapidly induces the expression and phyosophorylation of the Leu-23 activation antigen. J Exp Med. 1988;167: 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 38. 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 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