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mAbs Against HUMAN Protein

PD1 (PDCD1)

Antibody Validated
CONTACT INFORMATION:Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas
STATUS:Validated
TYPE:mouse anti human
CLONE NAME:NAT105
PROTEIN:Human full length PD1
PROTEIN WEB:http://www.ncbi.nlm.nih.gov/omim/600244
ANTIGEN USED:YT cell line
FUSION PARTNER:NS1/Ag4-1 (NS1) cells
ISOTYPE:IgG1
SPECIES REACTIVITY:human
PREPARATION AND STORAGE:Aliquot and store at 4C. Do not freeze
APP RECOMMENDED:IHQ-paraffin, IHQ-frozen, IF, WB, IP, Flow cytometry

DESCRIPTION

PD1 is a member of the extended CD28/CTLA-4 family of T cell regulators. It contains the immunoreceptor tyrosine-based inhibitory motif (ITIM) and plays a key role in peripheral tolerance and autoimmune disease. It is expressed by germinal center-associated T cells in reactive lymphoid tissue. Immune responses to foreign and self-antigens require specific and balanced responses to clear pathogens and tumors and yet maintain tolerance. Induction and maintenance of T cell tolerance requires PD1, and its ligand PD-L1 on nonhematopoietic cells can limit effector T cell responses and protect tissues from immune-mediated tissue damage. The PD1:PD-L pathway also has been usurped by microorganisms and tumors to attenuate antimicrobial or tumor immunity and facilitate chronic infection and tumor survival.

PUBLICATION DESCRIBING ANTIBODY CHARACTERIZATION/VALIDATION

Roncador, G., Verdes-Montenegro, J.F.G., Tedoldi, S., Paterson, J.C., Klapper, W., Ballabio, E., Maestre, L., Pileri, S., Hansmann, M.L., Piris, M.A., Mason, D.Y., Marafioti, T. Expression of two markers of germinal center T cells (SAP and PD-1) in angioimmunoblastic T-cell lymphoma. Haematologica. 2007 Aug;92(8):1059-66. http://www.haematologica.org/content/92/8/1059.full.pdf+html



REFERENCES

Roncador, G., Verdes-Montenegro, J.F.G., Tedoldi, S., Paterson, J.C., Klapper, W., Ballabio, E., Maestre, L., Pileri, S., Hansmann, M.L., Piris, M.A., Mason, D.Y., Marafioti, T. Expression of two markers of germinal center T cells (SAP and PD-1) in angioimmunoblastic T-cell lymphoma. Haematologica. 2007 Aug;92(8):1059-66.

Nam-Cha SH, Roncador G, Sanchez-Verde L, Montes-Moreno S, Acevedo A, Domínguez-Franjo P, Piris MA PD-1, a follicular T-cell marker useful for recognizing nodular lymphocyte-predominant Hodgkin lymphoma.
Am J Surg Pathol. 2008 Aug;32(8):1252-7

Rodríguez-Pinilla SM, Atienza L, Murillo C, Pérez-Rodríguez A, Montes-Moreno S, Roncador G, Pérez-Seoane C, Domínguez P, Camacho FI, Piris MA. Peripheral T-cell Lymphoma With Follicular T-cell Markers. Am J Surg Pathol. 2008. Dec;32(12):1787-99.

Rodríguez Pinilla SM, Roncador G, Rodríguez-Peralto JL, Mollejo M, García JF, Montes-Moreno S, Camacho FI, Ortiz P, Limeres-González MA, Torres A, Campo E, Navarro-Conde P, Piris MA. Primary cutaneous CD4+ small/medium-sized pleomorphic T-cell lymphoma expresses follicular T-cell markers. Am J Surg Pathol. 2009 Jan;33(1):81-90.

APPLICATIONS

IHC Techniques
IHC Techniques Clone Dilution Antibody concentration Antigen retrieval method Visualization kit +/- control Protein localization Positivity in other species Protocol
Frozen tissue and cytospins
Recommended Result obtained is satisfactory. The reagent can be use in this application
NAT105 1:4 supernatant None goat anti mouse HRP DAKO Tonsil / membrane
Paraffin tissue
Recommended Result obtained is satisfactory. The reagent can be use in this application
NAT105 1:50 purified 1mg/mi Tris-EDTA Novolink Tonsil / membrane Available
Immunofluorescence
Recommended Result obtained is satisfactory. The reagent can be use in this application
NAT105 1:30 purified 1mg/ml Tris-EDTA Goat anti mouse IgG1 Alexa Tonsil / membrane Available
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Anti-PD1 antibodies (NAT105 and R&D polyclonal) in HEK transfected cells.
Validation of NAT105 monoclonal antibody in transfected cells. Hek-BTLA transfected cells were used as negative control.
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PD1 (NAT105 and R&D antibodies) expression in YT cell line.
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Anti-PD1 (NAT105) antibody on frozen tonsil section.
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PD1 (NAT105 and R&D) expression on human paraffin sections
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Expression of PD1 (NAT105) antigen by lymphoid cells.
Double immunofluorescent staining shows that many of the PD1 (NAT105)-positive cells (red) in the germinal center cells co-express CD3 (green).
WB Techniques
WB Techniques Clone Dilution Antibody concentration +/- control Expected MW Observed Mw Positivity in other species Protocol
Western Blotting
Recommended Result obtained is satisfactory. The reagent can be use in this application
NAT105 1:2 supernatant YT cell line / Colo 205 cell line 32 KDa 47 kDa Available
Immunoprecipitation
Recommended Result obtained is satisfactory. The reagent can be use in this application
NAT105 supernatant YT cell line / Ramos cell line 32KDa 47KDa Available
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WB of anti-PD1 (NAT105 and R&D) antibodies.
Both antibodies are dettecting a 47kDa band.
Anti GAPDH was used as loading control.
Lane 1 Hek-PD1 transfected cells (20ug) (+)
Lane 2 Hek-PAX3 transfected cells (20ug) (-)
Lane 3 YT cell line (100ug) (+)
Lane 4 Hela cell line (100ug) (-)
Lane 5 Colo205 cell line (100ug) (-)
Lane 6 Human HL (100ug) (+)
Lane 7 Human HL (100ug) (+)
Lane 8 Human HL (100ug) (+)
Lane 9 Human tonsil (100ug) (+)
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NAT105 mAb can be use in IP technique
A. Immunoprecipitation of protein extracts from HEK-PD1, HEK-Bcl7b, YT and Ramos cell lines with NAT105C mAb (1ul/lane) followed by western blotting with the same antibody (neat supernatant).
B. Immunoprecipitation of protein extracts from HEK-PD1, HEK-Bcl7b, YT and Ramos cell lines with goat R&D antibody (1ul/lane) followed by western blotting with NAT105 antibody (neat supernatant).
FLOW CYTOMETRY
FLOW CYTOMETRY Clone Dilution +/- control Type of fluorocrom Protocol
Recommended Result obtained is satisfactory. The reagent can be use in this application
NAT 1:20 purified 1mg/ml Tonsil /
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NAT105 staining in blood and tonsillar lymphocytes
Column one. NAT105 (using, as second step, anti-mouse IgG1-PE) and CD4 staining in lymphocytes. Overlaid blue indicates staining in a parallel well with CD4 and anti-mouse iIg1-Pe in the absence of NAT105 (negative control). Column two. NAT105 and CXCR5 staining in CD3+ CD4+ lymphocytes. Column three. NAT105 and ICOS (PerCP) staining in CD3+ CD4+ Lymphocytes. Column four. Overlaid histograms of NAT105 staining in ICOS+CD57+ (blue line), ICOS+ CD57-(red line) and ICOS-CD57- (brown line) CXCR5+ CD45RO+CD4+ lymphocytes, and CXCR5- CD45RO- CD4+ lymphocytes (green line)
OTHERS
OTHERS Title Description Protocol
Recommended Result obtained is satisfactory. The reagent can be use in this application
Double PD1/KI67 immunoenzimatic staining Double immunoenzymatic labeling of p araffin sections was performed using a standard BOND system protocol with the Abs PD-1/KI67 (Leica, Milton Ke)
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Double PD1/KI67 immunoenzimatic staining
Double immunoenzymatic staining shows the expression of PD1 (red) and KI67 (nuclear, brown) and that PD1 cells lack the proliferation marker Ki-67.