Specification Please note DAKO Sheet Some information s Lot dependent Monoclonal mouse Anti-Human CD62P, P-Selectin, Clone 1E3 Code No, M 7199 Lot 038. Edition 06.12. 00 Antigen synonyms Platelet activation-dependent granule membrane protein(PADGEM)or GMP-140 Introduction The CD62P antigen a 140 kDa transmembrane glycoprotein present on megakaryocytes, activated platelets and activated endothelial cells. In unstimulated platelets and endothelial cells CD62P is stored in the membranes of secretory granules and shows no surface immunostaining with anti-CD62P. When platelets and endothelial cells are activated by agonists such as thrombin, the granules rapidly fuse with the plasma membrane, which will express CD62P on the surface(1-3). CD62P is a receptor for neutrophils and monocytes when it is expressed on activated platelets and endothelium (4) CD62P is a member of the small selectin family of cellular adhesion molecules which also includes CD62E(E-selectin) and CD62L (L-selectin)(5, 6). Its structure, similar to the other members of the selectin family, consists of an N-terminal lectin-like domain of c type(6)followed by an epidermal growth factor-like domain, nine short consensus repeats (SCR)related to those in complement-binding proteins, a transmembrane region and a short cytoplasmic tail which bears no homology to the CD62E and CD62L cytoplasmic domains (3). The major CD62P ligand on neutrophils is the P-Selectin Glycoprotein igand 1, CD162(PSGL-1)(5, 7) Presentation Monoclonal mouse antibody supplied in liquid form as tissue culture supernatant(RPMI 1640 medium containing fetal calf serum)dialysed against 0.05 mol/L Tris/HCL, pH 7.2 containing 15 mmol/L NaN Mouse lg concentration: 170 mg/L Isotype: IgG2a, kappa tal protein concentration 11.7 Storage Clone 1E3.(8 immunogen Fixed thrombin-activated human platelets Specificity/reactivity Anti-CD62P, 1E3, was included in the Sixth International Workshop and Conference on Human Leucocyte Differentiation Antigens( Kobe 1996), and studies by a number of The antibody stains megakaryocytes, activated platelets and activated endothelium. Other normal cells do not react with the antibody Anti-CD62P, 1E3, is useful for assessing platelet activation in acute coronary syndromes by flow cytometry (10)
Specification Sheet Please note Some information is Lot dependent. Monoclonal Mouse Anti-Human CD62P, P-Selectin, Clone 1E3 Code No. M 7199 Lot 038. Edition 06.12.00 Antigen synonyms Platelet activation-dependent granule membrane protein (PADGEM) or GMP-140. Introduction The CD62P antigen is a 140 kDa transmembrane glycoprotein present on megakaryocytes, activated platelets and activated endothelial cells. In unstimulated platelets and endothelial cells CD62P is stored in the membranes of secretory granules and shows no surface immunostaining with anti-CD62P. When platelets and endothelial cells are activated by agonists such as thrombin, the granules rapidly fuse with the plasma membrane, which will express CD62P on the surface (1-3). CD62P is a receptor for neutrophils and monocytes when it is expressed on activated platelets and endothelium (4). CD62P is a member of the small selectin family of cellular adhesion molecules which also includes CD62E (E-selectin) and CD62L (L-selectin) (5,6). Its structure, similar to the other members of the selectin family, consists of an N-terminal lectin-like domain of Ctype (6) followed by an epidermal growth factor-like domain, nine short consensus repeats (SCR) related to those in complement-binding proteins, a transmembrane region and a short cytoplasmic tail which bears no homology to the CD62E and CD62L cytoplasmic domains (3). The major CD62P ligand on neutrophils is the P-Selectin Glycoprotein Ligand 1, CD162 (PSGL-1) (5,7). Presentation Monoclonal mouse antibody supplied in liquid form as tissue culture supernatant (RPMI 1640 medium containing fetal calf serum) dialysed against 0.05 mol/L Tris/HCl, pH 7.2 containing 15 mmol/L NaN3. Mouse Ig concentration: 170 mg/L. Isotype: IgG2a, kappa. Total protein concentration: 11.7 g/L. Storage 2-8 °C. Clone 1E3. (8). Immunogen Fixed thrombin-activated human platelets. Specificity/reactivity Anti-CD62P, 1E3, was included in the Sixth International Workshop and Conference on Human Leucocyte Differentiation Antigens (Kobe 1996), and studies by a number of laboratories confirmed its reactivity with the CD62P antigen (9). The antibody stains megakaryocytes, activated platelets and activated endothelium. Other normal cells do not react with the antibody. Anti-CD62P, 1E3, is useful for assessing platelet activation in acute coronary syndromes by flow cytometry (10). (2)
Staining procedures Formalin-fixed and paraffin-embedded sections Can be used on formalin-fixed, paraffin-embedded tissue sections. Antigen retrieval, such as by heating in DAKO Target Retrieval Solution, code No. S 1700 is mandatory. The slides should not be allowed to dry out during this treatment or during the following immunohistochemical staining procedure For tissue sections sensitive staining techniques are recommended such as the LSAB@+or the EnVision M+ system The antibody may be used at a dilution of 1: 25-1: 50 with the EnVision TM+ system when tested on formalin-fixed, paraffin-embedded sections of human tonsil Frozen sections and cell smears Can be used for labelling acetone-fixed cryostat sections or fixed cell smears. The antibody may be used at a dilution of 1: 25-1: 50 in the APAAP technique when tested on acetone-fixed This is a guideline only, an optimal dilution should be determined by the individual laboratory (1) McEver RP, Martin MN. A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. J Biol Chem 1984; 259: 9799-80 (2) Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton DF. A platelet alpha-granule membrane O)is expressed on the plasma membrane after activation. J Cell Bi (3) Johnston Gl, Cook RG, McEver RP Cloning of GMP-140, a granule membane protein of platelets ar ins involved in cell adhesion and inflammation. Cell 198956:103344 4) McEver RP GMP-140: a receptor for neutrophils and monocytes on activated platelets and endothelium J Cell Biochem 1991: 45: 156-61 interactions. J Biol Chem 1995: 270: 11025-8 (6) Lasky LA. Selectin-carbohydrate interactions and the initiation of the inflammatory response. Annu Rev lochen199564:113-39. (7) Rosen SD, Bertozzi CR. Two selectins converge on sulphate Leukocyte adhesion. Curr Biol 1996: 6: 261 (8) Carmody MW, Ault KA, Mitchell JG, Rote NS, Ah-Kau NG Production of monoclonal antibodies specific for platelet activation antigens and their use in evaluating platelet function. Hybridoma 1990: 9: 631-41 (9) de Haas M, von dem Borne AEG. PL8. CD62P workshop panel report. In: Kishimoto T et al., eds Leucocyte Typing VI. White Cell Differentiation Antigens. New York-London: Garland Publishing, Inc 1997:6634. (10)Becker RC, Tracy RP, Bovill EG, Mann KG, Ault K. The clinical use of flow cytometry for assessing latelet activation in acute coronary syndromes. TIMI-lll Thrombosis and Anticoagulation Group. Conor Artery Dis1994:5:33945 M7199/JU06.1200
- 2 - Staining procedures Formalin-fixed and paraffin-embedded sections Can be used on formalin-fixed, paraffin-embedded tissue sections. Antigen retrieval, such as by heating in DAKO Target Retrieval Solution, code No. S 1700 is mandatory. The slides should not be allowed to dry out during this treatment or during the following immunohistochemical staining procedure. For tissue sections sensitive staining techniques are recommended, such as the LSAB+ or the EnVision™+ system. The antibody may be used at a dilution of 1:25-1:50 with the EnVision™+ system when tested on formalin-fixed, paraffin-embedded sections of human tonsil. Frozen sections and cell smears Can be used for labelling acetone-fixed cryostat sections or fixed cell smears. The antibody may be used at a dilution of 1:25-1:50 in the APAAP technique when tested on acetone-fixed sections. This is a guideline only; an optimal dilution should be determined by the individual laboratory. References (1) McEver RP, Martin MN. A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. J Biol Chem 1984;259:9799-804. (2) Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton DF. A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol 1985;101:880-6. (3) Johnston GI, Cook RG, McEver RP. Cloning of GMP-140, a granule membane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell 1989;56:1033-44. (4) McEver RP. GMP-140: a receptor for neutrophils and monocytes on activated platelets and endothelium. J Cell Biochem 1991;45:156-61. (5) McEver RP, Moore KL, Cummings RD. Leukocyte trafficking mediated by selectin-carbohydrate interactions. J Biol Chem 1995;270:11025-8. (6) Lasky LA. Selectin-carbohydrate interactions and the initiation of the inflammatory response. Annu Rev Biochem 1995;64:113-39. (7) Rosen SD, Bertozzi CR. Two selectins converge on sulphate. Leukocyte adhesion. Curr Biol 1996;6:261- 4. (8) Carmody MW, Ault KA, Mitchell JG, Rote NS, Ah-Kau NG. Production of monoclonal antibodies specific for platelet activation antigens and their use in evaluating platelet function. Hybridoma 1990;9:631-41. (9) de Haas M, von dem Borne AEG. PL8. CD62P workshop panel report. In: Kishimoto T et al., eds. Leucocyte Typing VI. White Cell Differentiation Antigens. New York - London: Garland Publishing, Inc., 1997:663-4. (10) Becker RC, Tracy RP, Bovill EG, Mann KG, Ault K. The clinical use of flow cytometry for assessing platelet activation in acute coronary syndromes. TIMI-III Thrombosis and Anticoagulation Group. Coron Artery Dis 1994;5:339-45. M 7199/TJU/06.12.00