Human PD-1 Antibody Summary
*Small pack size (-SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
Applications
This antibody functions as an ELISA capture antibody when paired with Mouse Anti-Human PD‑1 Monoclonal Antibody(Catalog # MAB10864).
This product is intended for assay development on various assay platforms requiring antibody pairs. We recommend the Human PD-1 DuoSet ELISA Kit (Catalog # DY1086) for convenient development of a sandwich ELISA.
Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.
Scientific Data
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Detection of Human PD‑1 by Western Blot. Western blot shows lysates of HEK293T human embryonic kidney cell line either mock transfected or transfected with human PD-1. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human PD-1 Monoclonal Antibody (Catalog # MAB10863) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). Specific bands were detected for PD-1 at approximately 40 kDa and 80 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
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Detection of PD‑1 in Human PBMCs by Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) either (A) treated with 5 ug/mL PHA for 5 days or (B) untreated, were stained with Rabbit Anti-Human PD-1 Monoclonal Antibody (Catalog # MAB10863) followed by Allophycocyanin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0111) and Mouse Anti-Human CD3 epsilon PE-conjugated Monoclonal Antibody (Catalog # FAB100P). Quadrant markers were set based on control antibody staining (Catalog # MAB1050). View our protocol for Staining Membrane-associated Proteins.
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Human PD‑1 ELISA Standard Curve. Recombinant Human PD-1 protein was serially diluted 2-fold and captured by Rabbit Anti-Human PD-1 Monoclonal Antibody (Catalog # MAB10863) coated on a Clear Polystyrene Microplate (Catalog # DY990). Mouse Anti-Human PD-1 Monoclonal Antibody(Catalog # MAB10864) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998) followed by Substrate Solution (Catalog # DY999) and stopping the enzymatic reaction with Stop Solution (Catalog # DY994).
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Preparation and Storage
Background: PD-1
Programmed Death-1 receptor (PD-1), also known as CD279, is type I transmembrane protein belonging to the CD28 family of immune regulatory receptors (1). Other members of this family include CD28, CTLA-4, ICOS, and BTLA (2-5). Mature human PD-1 consists of a 148 amino acid (aa) extracellular region (ECD) with one immunoglobulin-like V-type domain, a 24 aa transmembrane domain, and a 95 aa cytoplasmic region. The human PD-1 ECD shares 65% aa sequence identity with the mouse PD-1 ECD. The cytoplasmic tail contains two tyrosine residues that form the immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) that are important for mediating PD-1 signaling. PD-1 acts as a monomeric receptor and interacts in a 1:1 stoichiometric ratio with its ligands PD-L1 (B7-H1) and PD-L2 (B7-DC) (6, 7). PD‑1 is expressed on activated T cells, B cells, monocytes, and dendritic cells while PD-L1 expression is constitutive on the same cells and also on nonhematopoietic cells such as lung endothelial cells and hepatocytes (8, 9). Ligation of PD-L1 with PD-1 induces co-inhibitory signals on T cells promoting their apoptosis, anergy, and functional exhaustion (10). Thus, the PD-1: PD-L1 interaction is a key regulator of the threshold of immune response and peripheral immune tolerance (11). Finally, blockade of the PD-1: PD-L1 interaction by either antibodies or genetic manipulation accelerates tumor eradication and shows potential for improving cancer immunotherapy (12, 13, 14).
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- Sharpe, A.H. and G. J. Freeman (2002) Nat. Rev. Immunol. 2:116.
- Coyle, A. and J. Gutierrez-Ramos (2001) Nat. Immunol. 2:203.
- Nishimura, H. and T. Honjo (2001) Trends Immunol. 22:265.
- Watanabe, N et al. (2003) Nat. Immunol. 4:670.
- Zhang, X. et al. (2004) Immunity 20:337.
- Lázár-Molnár, E. et al. (2008) Proc. Natl. Acad. Sci. USA 105:10483.
- Nishimura, H et al. (1996) Int. Immunol. 8:773.
- Keir, M.E. et al. (2008) Annu. Rev. Immunol. 26:677.
- Butte, M.J. et al. (2007) Immunity 27:111.
- Okazaki, T. et al. (2013) Nat. Immunol. 14:1212.
- Iwai, Y. et al. (2002) Proc. Natl. Acad. Sci. USA 99: 12293.
- Nogrady, B. (2014) Nature 513:S10.
- Swaika, A. et al. (2015) Mol. Immunol. 67: 4
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A Western blot analysis was performed using lysates from the HEK293T human embryonic kidney cell line that had been transfected with human PD-1. A PVDF membrane was utilized and probed with the antibody at a concentration of 0.5 µg/mL. The bands corresponding to PD-1 were observed at around 70 kDa. The experimental conditions involved conducting the assay under reducing conditions.
