Nori Human PD1 ELISA Kit Summary

Alternative names for PD1: Programmed cell death protein 1, PD-1, CD279, cluster of differentiation 279, PCD1, PDCD1

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Programmed cell death protein 1 ( PD-1) also known as CD279 (cluster of differentiation 279) encoded by PCD1 gene,^[1] is a protein on the surface of T and B cells that has a role in regulating the immune system‘s response to the cells by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells.^[2] PD-1 is an immune checkpoint and guards against autoimmunity through two mechanisms. First, it promotes apoptosis of antigen-specific T-cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells.^[3] The PD-1 is a cell surface receptor that binds two ligands, PD-L1 and PD-L2. PD-L1 is upregulated on macrophages and dendritic cells (DC) in response to LPS and GM-CSF treatment.^[4]  PD-1 and its ligands negatively regulate immune responses. PD-1 knockout mice have been shown to develop lupus-like glomerulonephritis and dilated cardiomyopathy on the C57BL/6 and BALB/c backgrounds, respectively.^[5]  Blocking of PD-1 leads to a reduction in cerebral amyloid-β plaques and improves cognitive performance in mice.^[6] Immune blockade of PD-1 evoked an IFN-γ dependent immune response that recruited monocyte-derived macrophages to the brain that were then capable of clearing the amyloid-β plaques from the tissue. Expression of PD-L1 on tumor cells inhibits anti-tumor activity through engagement of PD-1 on effector T cells.^[7] Triggering PD-1, expressed on monocytes and up-regulated upon monocytes activation, by its ligand PD-L1 induces IL-10 production which inhibits CD4 T-cell function.^[8] Overexpression of PD1 on CD8+ T cells is one of the indicators of T-cell exhaustion (e.g. in chronic infection or cancer).^[2][9]

References

1. Ishida Y, et al. (1992). The EMBO Journal. 11 (11): 3887–95.
2. Francisco LM, et al. (2010). Immunological Reviews. 236: 219–42.
3. Fife BT, Pauken KE (2011). Annals of the New York Academy of Sciences. 1217 (1): 45–59.
4. Iwai Yet al. (2002). Proc Natl Acad Sci USA. 99 (19): 12293–7.
5. Nishimura H, et al. (2001). Science. 291 (5502): 319–22.
6. Kurnellas MP, et al. (2013). Science Translational Medicine. 5 (179): 179ra42.
7. Nishimura H, et al. (1999). Immunity. 11 (2): 141–51.
8. Pauken KE, Wherry EJ (2015). Trends in Immunology. 36 (4): 265–76.
9. Wang X, et al. (2016). OncoTargets and Therapy. 9: 5023–39.