PD-1 Antibody [Unconjugated]

Western blot shows lysate of human thymus tissue. PVDF membrane was probed with 2 µg/mL of Goat Anti-Human PD‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1086) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). Specific bands were detected for PD‑1 at approximately 40-50 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Human peripheral blood mononuclear cells (PBMCs) either (A) untreated or (B) treated with 5 μg/mL PHA overnight were stained with Goat Anti-Human PD‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1086) followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107) and Mouse Anti-Human CD3 epsilon APC‑conjugated Monoclonal Antibody (Catalog # FAB100A). Quadrant markers were set based on control antibody staining (Catalog # F0107). View our protocol for Staining Membrane-associated Proteins.

Western blot shows lysates of HEK293 human embryonic kidney cell line either mock transfected or transfected with human PD-1. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human PD‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1086) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). Specific bands were detected for PD‑1 at approximately 40-80 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

PD‑1 was detected in immersion fixed paraffin-embedded sections of human lymph node using Goat Anti-Human PD‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1086) at 3 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to lymphocytes. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

• Reactivity: Hu
• Applications: WB, Flow, IHC, B/N, CyTOF-ready, ELISA(Cap)
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated
• Concentration: LYOPH

PD-1 Antibody [Unconjugated] Summary

• Immunogen: Mouse myeloma cell line NS0-derived recombinant human PD-1
  Leu25-Gln167
  Accession # Q8IX89
• Specificity: Detects human PD-1 in ELISAs and Western blots. In sandwich ELISAs, less than 2% cross-reactivity with recombinant mouse PD‑1 and less than 0.2% cross-reactivity with recombinant human (rh) CD28, rhICOS, and rhCTLA‑4 is observed.
• Details of Functionality: ActivityAssaywCitation not found None None None None None
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Gene: PDCD1
• Endotoxin Note: <0.10 EU per 1 μg of the antibody by the LAL method.

Applications/Dilutions

• Dilutions:
  • Western Blot 0.5-2 ug/mL
  • Flow Cytometry 0.25 ug/10^6 cells
  • Immunohistochemistry 5-15 ug/mL
  • Blockade of Receptor-ligand Interaction
  • CyTOF-ready
  • ELISA Capture (Matched Antibody Pair) 0.2-0.8 ug/mL
  • ELISA Detection (Matched Antibody Pair) 0.1-0.4 ug/mL
  • ELISA Standard (Matched Pair)
• Application Notes: ELISA Capture: Human PD-1 Antibody (Catalog # AF1086)
  ELISA Detection: Human PD-1 Biotinylated Antibody (Catalog # BAF1086)
  Standard: Recombinant Human PD-1 Fc Chimera (Catalog # 1086-PD)

Packaging, Storage & Formulations

• 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.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for PD-1 Antibody [Unconjugated]

• CD279 antigen
• CD279
• hPD-1
• PD-1
• PD1hPD-l
• PDCD1
• programmed cell death 1
• programmed cell death protein 1
• Protein PD-1
• SLEB2

Background

Programmed Death-1 (PD-1) is a type I transmembrane protein belonging to the CD28/CTLA-4 family of immunoreceptors that mediate signals for regulating immune responses (1). Members of the CD28/CTLA-4 family have been shown to either promote T cell activation (CD28 and ICOS) or down-regulate T cell activation (CTLA-4 and PD-1) (2). PD-1 is expressed on activated T cells, B cells, myeloid cells, and on a subset of thymocytes. In vitro, ligation of PD-1 inhibits TCR-mediated T-cell proliferation and production of IL-1, IL-4, IL-10, and IFN-gamma. In addition, PD-1 ligation also inhibits BCR mediated signaling. PD-1 deficient mice have a defect in peripheral tolerance and spontaneously develop autoimmune diseases (2, 3).

Two B7 family proteins, PD-L1 (also called B7-H1) and PD-L2 (also known as B7-DC), have been identified as PD-1 ligands. Unlike other B7 family proteins, both
PD‑L1 and PD-L2 are expressed in a wide variety of normal tissues including heart, placenta, and activated spleens (4). The wide expression of PD-L1 and PD-L2 and the inhibitor effects on PD-1 ligation indicate that PD-1 might be involved in the regulation of peripheral tolerance and may help prevent autoimmune diseases (2).

The human PD-1 gene encodes a 288 amino acid (aa) protein with a putative 20 aa signal peptide, a 148 aa extracellular region with one immunoglobulin-like V-type domain, a 24 aa transmembrane domain, and a 95 aa cytoplasmic region. The cytoplasmic tail contains two tyrosine residues that form the immuno-receptor
tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) that are important in mediating PD-1 signaling. Mouse and human PD-1 share approximately 60% aa sequence identity (4).

1. Ishida, Y. et al. (1992) EMBO J. 11:3887.
2. Nishimura, H. and T. Honjo (2001) Trends in Immunol. 22:265.
3. Latchman, Y. et al. (2001) Nature Immun. 2:261.
4. Carreno, B.M. and M. Collins (2002) Annu. Rev. Immunol. 20:29.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for PD-1 Antibody (AF1086)(17)

Publications using AF1086

• W Fan, AJ Demers, Y Wan, Q Li Altered Ratio of T Follicular Helper Cells to T Follicular Regulatory Cells Correlates with Autoreactive Antibody Response in Simian Immunodeficiency Virus-Infected Rhesus Macaques J. Immunol., 2018;0(0):. 2018 [PMID: 29610141] (Flow,, Primate)
• MA Rahman, KM McKinnon, TS Karpova, DA Ball, DJ Venzon, W Fan, G Kang, Q Li, M Robert-Gur Associations of Simian Immunodeficiency Virus (SIV)-Specific Follicular CD8+T Cells with Other Follicular T Cells Suggest Complex Contributions to SIV Viremia Control J. Immunol., 2018;0(0):. 2018 [PMID: 29507105] (IHC,, Primate)
• D Amodio, N Cotugno, G Macchiarul, S Rocca, Y Dimopoulos, MR Castrucci, R De Vito, FM Tucci, AB McDermott, S Narpala, P Rossi, RA Koup, P Palma, C Petrovas Quantitative Multiplexed Imaging Analysis Reveals a Strong Association between Immunogen-Specific B Cell Responses and Tonsillar Germinal Center Immune Dynamics in Children after Influenza Vaccination J. Immunol., 2017;0(0):. 2017 [PMID: 29237774] (IHC,, Human)
• Y Liu, Y Cheng, Y Xu, Z Wang, X Du, C Li, J Peng, L Gao, X Liang, C Ma Increased expression of programmed cell death protein 1 on NK cells inhibits NK-cell-mediated anti-tumor function and indicates poor prognosis in digestive cancers Oncogene, 2017;0(0):. 2017 [PMID: 28692048] (Functional Assay,, Human)
• ELV Silveira, KA Rogers, S Gumber, P Amancha, P Xiao, SM Woollard, SN Byrareddy, MM Teixeira, F Villinger Immune Cell Dynamics in Rhesus Macaques Infected with a Brazilian Strain of Zika Virus J. Immunol., 2017;0(0):. 2017 [PMID: 28667164] (IHC,, Primate - Macaca mulatta (Rhesus Macaque))
• P Martinez-M, K Tran, J Guenaga, G Lindgren, M Àdori, Y Feng, GE Phad, N Vázquez Be, S Bale, J Ingale, V Dubrovskay, S O'Dell, L Pramanik, M Spångberg, M Corcoran, K Loré, JR Mascola, RT Wyatt, GB Karlsson H Particulate Array of Well-Ordered HIV Clade C Env Trimers Elicits Neutralizing Antibodies that Display a Unique V2 Cap Approach Immunity, 2017;46(5):804-817.e7. 2017 [PMID: 28514687] (IHC,, Primate)
• Joseph M Obeid PD-L1, PD-L2 and PD-1 expression in metastatic melanoma: Correlation with tumor-infiltrating immune cells and clinical outcome Oncoimmunology, 2016;5(11):e1235107. 2016 [PMID: 27999753] (IHC-P,, Human)
• PT Nghiem, S Bhatia, EJ Lipson, RR Kudchadkar, NJ Miller, L Annamalai, S Berry, EK Chartash, A Daud, SP Fling, PA Friedlande, HM Kluger, HE Kohrt, L Lundgren, K Margolin, A Mitchell, T Olencki, DM Pardoll, SA Reddy, EM Shantha, WH Sharfman, E Sharon, She PD-1 Blockade with Pembrolizumab in Advanced Merkel-Cell Carcinoma N Engl J Med, 2016;0(0):. 2016 [PMID: 27093365] (IHC - Paraffin embedded,, Human)
• Vargas-Inchaustegui D, Demers A, Shaw J, Kang G, Ball D, Tuero I, Musich T, Mohanram V, Demberg T, Karpova T, Li Q, Robert-Guroff M Vaccine Induction of Lymph Node-Resident Simian Immunodeficiency Virus Env-Specific T Follicular Helper Cells in Rhesus Macaques. J Immunol, 2016;196(4):1700-10. 2016 [PMID: 26773147] (IHC Fluorescence,, Primate - Macaca mulatta (Rhesus Macaque))
• Zhang Z, Cheng L, Zhao J, Li G, Zhang L, Chen W, Nie W, Reszka-Blanco N, Wang F, Su L Plasmacytoid dendritic cells promote HIV-1-induced group 3 innate lymphoid cell depletion. J Clin Invest, 2015;125(9):3692-703. 2015 [PMID: 26301812] (IHC Paraffin-embedded,, Mouse)
• Melendreras S, Martinez-Camblor P, Menendez A, Bravo-Mendoza C, Gonzalez-Vidal A, Coto E, Diaz-Corte C, Ruiz-Ortega M, Lopez-Larrea C, Suarez-Alvarez B Soluble co-signaling molecules predict long-term graft outcome in kidney-transplanted patients. PLoS ONE, 2014;9(12):e113396. 2014 [PMID: 25478957] (ELISA Development,, Human)
• Hong J, Amancha P, Rogers K, Courtney C, Havenar-Daughton C, Crotty S, Ansari A, Villinger F Early lymphoid responses and germinal center formation correlate with lower viral load set points and better prognosis of simian immunodeficiency virus infection. J Immunol, 2014;193(2):797-806. 2014 [PMID: 24907346] (IF,, Primate - Macaca mulatta (Rhesus Macaque))
• CD4 T follicular helper cell dynamics during SIV infection. J. Clin. Invest., 2012;122(9):3281-94. 2012 [PMID: 22922258] (IHC Paraffin-embedded,, Primate - Macaca mulatta (Rhesus Macaque))
• Schatton T, Schutte U, Frank NY, Zhan Q, Hoerning A, Robles SC, Zhou J, Hodi FS, Spagnoli GC, Murphy GF, Frank MH Modulation of T-cell activation by malignant melanoma initiating cells. Cancer Res., 2010;70(2):697-708. 2010 [PMID: 20068175] (IF,, Human)
• Estes JD, Gordon SN, Zeng M, Chahroudi AM, Dunham RM, Staprans SI, Reilly CS, Silvestri G, Haase AT Early resolution of acute immune activation and induction of PD-1 in SIV-infected sooty mangabeys distinguishes nonpathogenic from pathogenic infection in rhesus macaques. J. Immunol., 2008;180(10):6798-807. 2008 [PMID: 18453600] (IHC Paraffin-embedded,, Primate - Cercocebus torquatus (Sooty Mangabey))
• Onlamoon N, Rogers K, Mayne AE, Pattanapanyasat K, Mori K, Villinger F, Ansari AA Soluble PD-1 rescues the proliferative response of simian immunodeficiency virus-specific CD4 and CD8 T cells during chronic infection. Immunology, 2008;124(2):277-93. 2008 [PMID: 18266718] (Neut,, Primate - Macaca mulatta (Rhesus Macaque))
• Wan B, Nie H, Liu A, Feng G, He D, Xu R, Zhang Q, Dong C, Zhang JZ Aberrant regulation of synovial T cell activation by soluble costimulatory molecules in rheumatoid arthritis. J. Immunol., 2006;177(12):8844-50. 2006 [PMID: 17142787] (ELISA Development,, Human)

Review for PD-1 Antibody (AF1086) (1)

Average Rating: 4

(Based on 1 review)

We have 1 review tested in 1 species: Human.

reviewed by: Christopher England IF Human 07/09/2016

Summary

• Application: Immunofluorescence
• Sample Tested: Mouse salivary gland (one injected with human T cells and one with no human T cells)
• Species: Human
• Lot: ICA0714071

Blocking

• Blocking Details: 10% Normal Donkey Serum, 0.25% Triton-X in PBS, 1 hr at room temperature

Primary Anitbody

• Dilution Ratio: 1:200, 12 h, 4 degree Celcius, PBS w/0.25% Triton-X

Secondary Antibody

• Secondary Description: Donkey anti-Goat DyLight 488
• Secondary Manufacturer Cat#: NBP-1-72843
• Secondary Concentration: 1:4000

Details

• Detection Notes: Confocal microscopy, Nikon A1R with 488-nm laser
• Fixation Details: Frozen Sections, 4% Paraformaldehyde in PBS for 30 min at room temperature
• Wash Description: PBS, 15 min, 3-times after primary and 3-times after secondary

Comments

• Comments: The antibody worked well. Since goat-derived antibodies are not the best, we were happy to see that this one worked well.

Bioinformatics

• Gene Symbol: PDCD1
• Entrez:
  • 5133(Human)
  • 18566(Mouse)
  • 301626(Rat)
  • 486213(Canine)
  • 102123659(Cynomolgus Monkey)
• Uniprot:
  • Q8IX89()