mAbs Against HUMAN Protein
PD1 (PDCD1)
| STATUS: |  |
|---|---|
| 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 | 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 | 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 | 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 | 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.