Western blot shows lysates of human tonsil tissue. PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human PD-1 Monoclonal Antibody (Catalog # MAB10864) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for PD-1 at approximately 45 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

PD‑1 in Human Tonsil.

PD-1 was detected in immersion fixed paraffin-embedded sections of human tonsil using Mouse Anti-Human PD-1 Monoclonal Antibody (Catalog # MAB10864) at 5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to lymphocytes. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

PD-L1/B7-H1 Binding to PD-1 Blocked by Human PD-1 Antibody.

In a functional ELISA, 0.09-0.72 µg/mL of this antibody (green line) will block 50% of the binding of 5 µg/mL of Recombinant Human PD-L1/B7-H1 Fc Chimera (orange line, Catalog # 156-B7) to immobilized Recombinant Human PD-1 His-tagged Protein (Catalog # 8986-PD) coated at 1 µg/mL (100 µL/well). At 5 µg/mL, this antibody will block >90% of the binding.

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).

Detection of PD-1 by Flow Cytometry

CMV-reactive CD4+ T cells are enriched in patients with CD4+ TEM cell expansion.CMV-reactive CD4+ T cells in patients with unresectable metastatic melanoma were assayed by in vitro stimulation with CMV lysates (blue boxes). Neutralising antibodies (red hatching) were used to detect T cells unable to respond to CMV antigens owing to expression of PD-1 or CTLA-4. T cell responses were quantified by flow cytometry analysis of cytokine expression. a Example data from a CMV IgG+ CD4+ TEM≥16% patient who developed hepatitis. Gating of live, singlet, IFN-gamma -producing CD4+ T cells is illustrated in Supplementary Fig. 11. b Frequencies of IFN-gamma -producing CD4+ T cells in patients categorised according to CMV IgG serostatus and baseline CD4+ TEM frequency (n = 42; two-way ANOVA with Tukey correction for multiple comparisons; n.s. = not significant). Boxplots represent the median, 25th and 75th percentiles, and Tukey whiskers. c Absolute numbers of circulating IFN-gamma -producing CD4+ T cells patients categorised according to CMV IgG serostatus and baseline CD4+ TEM frequency (n = 42; two-way ANOVA with Tukey correction for multiple comparisons; n.s. = not significant). Boxplots represent the median, 25th and 75th percentiles, and Tukey whiskers. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33664251), licensed under a CC-BY license. Not internally tested by R&D Systems.

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Applications for Human PD-1 Antibody

Application

Recommended Usage

Blockade of Receptor-ligand Interaction

In a functional ELISA, 0.09-0.72 µg/mL of this antibody will block 50% of the binding of 5 µg/mL of Recombinant Human PD-L1/B7-H1 Fc Chimera (Catalog # 156-B7) to immobilized Recombinant Human PD‑1 His-tagged Protein (Catalog # 8986-PD) coated at 1 µg/mL (100 µL/well). At 5 µg/mL, this antibody will block >90% of the binding.

ELISA

This antibody functions as an ELISA detection antibody when paired with Rabbit Anti-Human PD‑1 Monoclonal Antibody (Catalog # MAB10863).

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.

Immunohistochemistry

5-25 µg/mL
Sample: Immersion fixed paraffin-embedded sections of human tonsil

Western Blot

2 µg/mL
Sample: Human Tonsil Tissue

Formulation, Preparation, and Storage

Purification

Protein A or G purified from cell culture supernatant

Reconstitution

Reconstitute at 0.5 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.

12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.

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).

References

Ishida, Y. et al. (1992) EMBO J. 11:3887.
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

Long Name

Programmed Death-1

Alternate Names

CD279, PD1, PDCD1, SLEB2

Entrez Gene IDs

5133 (Human); 18566 (Mouse); 301626 (Rat); 100533201 (Porcine); 486213 (Canine); 102123659 (Cynomolgus Monkey)

Gene Symbol

PDCD1

UniProt

Q15116

Additional PD-1 Products

Product Documents for Human PD-1 Antibody

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COA

SDS

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Product Specific Notices for Human PD-1 Antibody

For research use only

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Human PD-1 Antibody

R&D Systems, part of Bio-Techne | Catalog # MAB10864

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Scientific Data Images for Human PD-1 Antibody

Detection of Human PD‑1 by Western Blot.