Bernard A, Boumsell L, Hill C. Joint report of the first international workshop on
human leucocyte differentiation ... Chen JH, Prince H, Buck D, et al. Leu-23: an ...
Monoclonal Antibodies Detecting Human Antigens APPLICATIONS
DESCRIPTION Specificity
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BD FastImmune Anti-Hu–IFN-γ FITC/CD69 PE/CD4 PerCP-Cy5.5/CD3 APC
Catalog No. 337184
50 Tests
BD FastImmune™ Anti-Hu-IFN-γ/CD69/CD4/CD3* 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 HIV,6,7 herpes viruses,1 and tumor antigens.8 Anti-Human Interferon-γ (Anti-Hu–IFN-γ) recognizes a 20- to 25-kilodalton (kd) glycoprotein.9 CD69 recognizes a very early human activation antigen. The CD69 antigen is a surface homodimer formed by the association of 28- and 32-kd chains that are held together by disulfide bridges.10 The CD411,12 antigen, Mr 55 kd,13 is present on T-helper/inducer lymphocytes and monocytes.14,15 CD3 reacts with the epsilon chain of the CD3 antigen/T-cell antigen receptor (TCR) complex.16 This complex is composed of at least six proteins that range in molecular weight from 20 to 30 kd.17 The antigen recognized by CD3 antibodies is noncovalently associated with either α/β or γ/δ TCR (70 to 90 kd).18
Antigen Distribution
IFN-γ is produced, upon activation, by most CD8+ T lymphocytes, by the TH1 and TH0 subsets of CD4+ T lymphocytes, and by natural killer (NK) lymphocytes.9,19-21 IFN-γ is a multifunctional immunomodulator with anti-tumor and anti-viral activity.22,23 IFN-γ is a pleiotropic cytokine instrumental in the regulation of immune and inflammatory processes,24-26 and it has a role in the differentiation and function of monocytes.26 The CD69 antigen is present on activated T, B, and NK lymphocytes10 and platelets.27 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).28-30 CD69 and phorbol ester are comitogenic for T lymphocytes.30 In thymus, the CD69 antigen is constitutively expressed on the bright CD3+ subset.31 The CD4 antigen is present on the helper/inducer T-lymphocyte subset, such as CD3+CD4+, that comprises 28% to 58%32 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+).33 The CD4 antigen is the receptor for the human immunodeficiency virus (HIV).34 Some CD4 antibodies, * Use of this product can fall under one or more claims of the following patents: US 5,445,939, 5,656,446, 5,834,689; Europe 319,543; Canada 1,296,622; Australia 615,880; Japan 2,769,156. † 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.
BD Biosciences 2350 Qume Drive San Jose, CA 95131-1807 Tel 877.232.8995; Fax 408.954.2347 www.bdbiosciences.com 23-7188-00 08/04
BD Biosciences
including CD4, inhibit HIV binding to CD4+ cells.35 Subjects infected with HIV were found to exhibit a continuous loss of CD4+ lymphocytes and a relative increase in the CD8 (Leu-2a)+ lymphocyte subset.36-38 The CD3 antigen is present on 61% to 85% of normal peripheral blood lymphocytes.32 Clones
Anti-Hu–IFN-γ, clone 25723.11, is derived from the hybridization of mouse P3X-63Ag8.653 myeloma cells with lymph node cells from BALB/c mice immunized with recombinant human IFN-γ. CD69, clone L78, is derived from hybridization of mouse Sp2/0-Ag14 myeloma cells with lymph node cells from BALB/c mice immunized with a CD8+ alloantigen-directed cytotoxic T-lymphocyte (CTL) cell line.39 CD4, clone SK3, is derived from hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immununized with human peripheral blood T lymphocytes. CD3, clone SK7, is derived from hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immunized with human thymocytes.
Composition
Anti-Hu–IFN-γ is composed of mouse IgG2b heavy chains and kappa light chains. CD69, CD4, and CD3 are each composed of mouse IgG1 heavy chains and kappa light chains. The BD FastImmune Anti-Hu–IFN-γ/CD69/CD4/CD3 reagent is supplied as a combination of IFN-γ FITC, CD69 PE*, CD4 PerCP-Cy5.5*, and CD3 APC* in 1.0 mL of phosphate-buffered saline (PBS) containing bovine serum albumin (BSA), betalactoglobulin, and 0.1% sodium azide.
DIRECT INTRACELLULAR IMMUNOFLUORESCENCE Product/Amount for Staining
BD FastImmune Anti-Hu–IFN-γ FITC/CD69 PE/CD4 PerCP-Cy5.5/CD3 APC Cat. No. 337184 20 µL/test
Method for Intracellular Cytokine Detection
For complete activation and staining protocol and the appropriate application note, refer to our website (www.bdbiosciences.com) or contact your local BD representative. Abbreviated Intracellular Staining Procedure Add 1 mL of 1X BD FACS™ lysing solution* (Cat. No. 349202) to 100 µL of activated heparinized whole blood. Incubate 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. 340973). Vortex and incubate for 10 minutes at room temperature. Wash by adding PBS containing 0.5% BSA and 0.1% NaN3, and centrifuge for 5 minutes. Add 20 µL of BD FastImmune Anti-Hu–IFN-γ FITC/CD69 PE/CD4 PerCP-Cy5.5/CD3 APC. Vortex and incubate for 30 minutes at room temperature in the dark. Repeat wash step; resuspend cells in 1% paraformaldehyde in PBS.
* Patents—PE and APC: US 4,520,110; 4,859,582; 5,055,556; Europe 76,695; Canada. 1,179,942 PerCP: US 4,876,190 Cy5.5: US 5,268,486; 5,486,616; 5,569,587; 5,569,766; 5,627,027 BD FACS lysing solution: US 4,654,312; 4,902,613; 5,098,849
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104
104
Performed on SEB-activated whole blood. Laser excitation is at 488 nm and 635 nm. 1000
Representative Data
3.21% R3
0
FSC
CD69 PE
100
0
R1
100
SSC
CD4 PerCP-Cy5.5
R2
1000
100
CD3 APC
104
100
Anti–IFN-γ FITC
104
Figure 1 Analyzed on a BD FACS™ brand flow cytometer
HANDLING AND STORAGE
Store vials at 2° to 8°C. Conjugated forms should not be frozen and should be protected from prolonged exposure to light. Each reagent is stable for the period shown on the bottle label when stored as directed.
WARNING
Reagents contain sodium azide. Sodium azide is harmful if swallowed. Keep out of reach of children. Keep away from food, drink, and animal feedingstuff. Wear suitable protective clothing. If swallowed, seek medical advice immediately and show this container or label. Contact with acids liberates very toxic gas. Azide compounds should be flushed with large volumes of water during disposal to avoid deposits in lead or copper plumbing where explosive conditions can develop.
CHARACTERIZATION
To ensure consistently high-quality reagents, each lot of monoclonal antibody is tested for conformance with characteristics of a standard reagent. Representative flow cytometric data are included in this data sheet.
WARRANTY
The products sold hereunder are warranted only to conform to the quantity and contents stated on the label at the time of delivery to the customer. There are no warranties, expressed or implied, that extend beyond the description on the label of the product. 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, 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;181:859-866. 2. 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;4:953-956. 3. Nomura LE, Walker JM, Maecker HT. Optimization of whole blood antigen-specific cytokine assays for CD4+ T cells. Cytometry. 2000;40:60-68. 4. Suni MA, Picker LJ, Maino VC. Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry. J Immunol Methods. 1998;212:89-98. 5. 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;161:5284-5295. 6. 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;5:518-525. 7. 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;99:1739-1750. 8. Lee PP, Yee C, Savage PA, et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med. 1999;5:677-685.
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9. 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; 1995:3-24. 10. 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; 1989:428-432. 11. 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; 1984:9-108. 12. 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 Acad Sci USA. 1981;78:544-548. 13. 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;153:310-323. 14. 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;153:193-198. 15. Kotzin BK, Benike CJ, Engleman EG. Induction of immunoglobulin secreting cells in the allogeneic mixed leukocyte reaction: regulation by helper and suppressor lymphocyte subsets in man. J Immunol. 1981;127:931-935. 16. van Dongen JJM, Krissansen GW, Wolvers-Tettero ILM, et al. Cytoplasmic expression of the CD3 antigen as a diagnostic marker for immature T-cell malignancies. Blood. 1988;71:603-612. 17. Brenner MB, Groh V, Porcelli SA, et al. Structure and distribution of the human γ/δ T-cell receptor. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989:1049-1053. 18. Clevers H, Alarcón B, Wileman T, Terhorst C. The T cell receptor/CD3 complex: a dynamic protein ensemble. Annu Rev Immunol. 1988;6:629-662. 19. 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;141:849-855. 20. 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;182:1357-1367. 21. Street N, Mosmann T. Functional diversity of T lymphocytes due to secretion of different cytokine patterns. FASEB J. 1991;5:171-176. 22. Hardy KJ, Sawada T. Human γ interferon strongly upregulates its own gene expression in peripheral blood lymphocytes. J Exp Med. 1989;170:1021-1026. 23. Johnson HM, Bazer FW, Szente BE, Jarpe MA. How interferons fight disease. Scientific American. 1994:64-75. 24. 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;23:2740-2745. 25. Romagnani S, Del Prete G, Maggi E, et al. Human TH1 and TH2 subsets. Int Arch Allergy Immunol. 1992;99:242-245. 26. Powrie F, Coffman RL. Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunol Today. 1993;14:270-274. 27. 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;172:701-707. 28. Chen JH, Prince H, Buck D, et al. Leu-23: an early activation antigen on human lymphocytes. Fed Proc. 1988;2:A1214. 29. Testi R, Philips J, Lanier LL. Constitutive expression of a phosphorylated activation (Leu-23) by CD3 bright thymocytes. J Immunol. 1988;141:2257. 30. Testi R, Philips JH, 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;142:1854.
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31. Testi R, Philips J, Lanier LL. T cell activation via Leu-23 (CD69). J Immunol. 1989;143:1123-1128. 32. Reichert T, DeBruyère M, Deneys V, et al. Lymphocyte subset reference ranges in adult Caucasians. Clin Immunol Immunopath. 1991;60:190-208. 33. Wood GS, Warner NL, Warnke RA. Anti–Leu-3/T4 antibodies react with cells of monocyte/macrophage and Langerhans lineage. J Immunol. 1983;131:212-216. 34. 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;312:763-767. 35. Sattentau QJ, Dalgleish AG, Weiss RA, Beverley PCL. Epitopes of the CD4 antigen and HIV infection. Science. 1986;234:1120-1123. 36. 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 Dis. 1985;151:555-559. 37. 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 two-color immunofluorescence flow cytometry. J Exp Med. 1988;154:157-172. 38. 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;38:161-177. 39. Lanier LL, Buck DW, Rhodes L, et al. Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 activation antigen. J Exp Med. 1988;167:1572.
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