Anti-CD47 Antibody, clone C5/D5

Leukocyte surface antigen CD47 (UniProt Q08722; also known as Antigenic surface determinant protein OA3, CD47, IAP, Integrin-associated protein, Protein MER6) is encoded by the CD47 (also known as MER6) gene (Gene ID 861) in human. Originally defined as a tumor-specific marker for ovarian carcinoma, CD47 a multitransmembrane glycoprotein widely expressed on all hematopoietic cells and most other cell types. CD47 plays a role in αvβ3-mediated cell adhesion and Ca2+ influx, as well as in platelet activation via interaction with thrombospondin-1. In addtion, CD47 modulates peripheral mononuclear (PMN) neutrophil migration across endothelial and epithelial monolayers. The rate of fMLP-stimulated PMN migration is shown to be decreased by CD47 functional blockage upon neutralizaing antibody treatment, which can be partially reversed by PI3K inhibition. Following fMLP stimulation and after PMN transmigration, secondary granules-derived CD47 molecules are shown to accumulate on plasma membrane, resulting in increased cell surface CD47 immunoreactivity. CD47 is produced with a signal peptide sequence (a.a. 1-18) that is removed posttranslationally to yiled the 5-transmembrane (a.a. 142-162, 177-197, 208-228, 236-256, 269-289) protein with its N-terminal end (a.a. 19-141), composed almost entirely of a V-type Ig-like domain (a.a. 19-127), exposed extracellularly and its C-terminal end (a.a. 290-323) at the cytoplasmic side.

Clone C5/D5 (C5D5) neutralized CD47-dependent cellular signaling by targeting human CD47 extracellular domain (Liu, Y., et al. (2001). J. Biol. Chem. 276(43). In contrast to clone PF3.1 (Cat. No. MABF958), clone C5/D5 is suitable for neutralizing CD47-dependent cellular signaling, but not suitable for immunoblotting, while both clones can stain cell surface CD47.

Epitope: extracellular domain

T84 human intestinal epithelial cell membrane preparation (Parkos, C.A., et al. (1996). J. Cell Biol. 132(3):437-450).

ELISA Analysis: A representative lot detected affinity purified CD47 by standard ELISA, as well as enhanced surface CD47 immunoreactivity on T84 & HT29 (subclone Cl 19.A) monolayer by "cell ELISA" following IFN- stimulation (Parkos, C.A., et al. (1996). J. Cell Biol. 132(3):437-450).

Flow Cytometry Analysis: A representative lot detected the expression of exogenously transfected human CD47 on the surface of CaCO2 cells (Liu, Y., et al. (2001). J. Biol. Chem. 276(43):40156-40166).

Flow Cytometry Analysis: Representative lots detected human polymorphonuclear (PMN) neutrophils surface CD47 immunoreactivity, which was upregulated after fMLP stimulation (Liu, Y., et al. (2001). J. Biol. Chem. 276(43):40156-40166; Parkos, C.A., et al. (1996). J. Cell Biol. 132(3):437-450).

Immunoaffinity Purification: A representative lot was immobilized on resins and employed for affinity purification of CD47 from HT29 (subclone Cl 19.A) lysate (Parkos, C.A., et al. (1996). J. Cell Biol. 132(3):437-450).

Immunocytochemistry Analysis: A representative lot immunostained the basolateral surfaces of methanol-fixed CaCO2 monolayers expressing exogenously transfected human CD47 by fluorescent immunocytochemistry (Liu, Y., et al. (2001). J. Biol. Chem. 276(43):40156-40166).

Immunocytochemistry Analysis: A representative lot immunostained the basolateral membrane of paraformaldehyde-fixed T84 human intestinal epithelial cell monolayer by fluorescent immunocytochemistry (Parkos, C.A., et al. (1996). J. Cell Biol. 132(3):437-450).

Immunofluorescence Analysis: A representative lot immunostained the colonic crypts and lamina propria leukocytes at the basal and lateral, but not apical, aspects of the epithelium in paraformaldehyde-fixed frozen human colon tissue sections by fluorescent immunohistochemistry (Parkos, C.A., et al. (1996). J. Cell Biol. 132(3):437-450).

Immunoprecipitation Analysis: A representative lot immunoprecipitated ~60-65 kDa glycosylated CD47 from T84 human intestinal epithelial cell lysate. Target band size reduced to ~35 kDa following deglycosylation by N glycosidase F treatment (Parkos, C.A., et al. (1996). J. Cell Biol. 132(3):437-450).

Neutralizing Analysis: Representative lots caused a delay of human polymorphonuclear (PMN) neutrophils migration across polarized monolayers of T84 human intestinal epithelial cells in a bidirectional fashion without affecting PMN neutrophils or T84 adhesion. Tyrosine phosphorylation inhibitor genistein prevented delay by clone C5/D5 (Liu, Y., et al. (2001). J. Biol. Chem. 276(43):40156-40166; Parkos, C.A., et al. (1996). J. Cell Biol. 132(3):437-450).

Research Category
Inflammation & Immunology

This mouse monoclonal Anti-CD47, clone C5/D5 Antibody, Cat. No. MABF959 is validated for use in ELISA, Flow Cytometry, Immunoaffinity Purification, Immunocytochemistry, Immunofluorescence, Immunoprecipitation, and Neutralization studies.

Evaluated by Flow Cytometry in human PBMCs.

Flow Cytometry Analysis: 0.4 µL of this antibody detected CD47 surface expression on the gated lymphocytes population among one million human PBMCs.

33.47/30.00/31.45/32.10 kDa (mature isoform OA3-323/OA3-293/OA3-305/OA3-312) calculated. ~60-65 kDa (glycosylated) and ~35 kDa (deglycosylated) reported (Parkos, C.A., et al. (1996). J. Cell Biol. 132(3):437-450).

Format: Purified

Protein G purified.

Purified mouse IgG1 in PBS without preservatives.

Stable for 1 year at -20°C from date of receipt.
Handling Recommendations: Upon receipt and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.

Concentration: Please refer to lot specific datasheet.

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.