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 death-ligand 1 (PD-L1) also known as CD274 or B7-H1 is a protein that is encoded by the CD274 gene. PD-L1 is a 40kDa type 1 transmembrane protein that may play a major role in suppressing the adaptive arm of immune system during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states such as hepatitis. PD-L1 binds to its receptor, PD-1, found on activated T cells, B cells, and myeloid cells, to modulate activation or inhibition. Said et al. showed that PD-1, up-regulated on activated CD4 T-cells, can bind to PD-L1 expressed on monocytes and induces IL-10 production.^[1] The binding of PD-L1 to PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM) motif ^[2]. Engagement of PD-L1 with PD-1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. The mechanism involves inhibition of ZAP70 phosphorylation and its association with CD3ζ.^[3] PD-1 signaling attenuates PKC-θ activation loop phosphorylation, necessary for the activation of transcription factors NF-κB and AP-1, and for production of IL-2. PD-L1 binding to PD-1 also contributes to ligand-induced TCR down-modulation during antigen presentation to naive T cells, by inducing the up-regulation of the E3 ubiquitin ligase CBL-b.^[4] Upon IFN-γ stimulation, PD-L1 is expressed on T cells, NK cells, macrophages, myeloid DCs, B cells, epithelial cells, and vascular endothelial cells.^[5] The PD-L1 gene promoter region has a response element to IRF-1.^[6] Type I interferons can also upregulate PD-L1 on murine hepatocytes, monocytes, DCs, and tumor cells.^[7] It appears that upregulation of PD-L1 may allow cancers to evade the host immune system. An analysis of 196 tumor specimens from patients with renal cell carcinoma found that high tumor expression of PD-L1 was associated with increased tumor aggressiveness and a 4.5-fold increased risk of death.^[8] In normal tissue, feedback between transcription factors like STAT3 and NF-κB restricts the immune response to protect host tissue and limit inflammation. In cancer, loss of feedback restriction between transcription factors can lead to increased local PD-L1 expression.

References

1. Said EA, et al. (2010). Nature Medicine. 16 (4): 452–9.
2. Chemnitz JM, et al. (2004). Journal of Immunology. 173 (2): 945–54.
3. Sheppard KA, et al. (2004). FEBS Letters. 574 (1–3): 37–41.
4. Karwacz K, et al. (2011). EMBO Molecular Medicine. 3 (10): 581–92.
5. Flies DB, Chen L (2007). Journal of Immunotherapy. 30 (3): 251–60.
6. Lee SJ, et al. (2006). FEBS Letters. 580 (3): 755–62.
7. Yamazaki T, et al. (2002). Journal of Immunology. 169 (10): 5538–45.
8. Thompson RH, et al. (2004). Proc Natl Acad Sci USA. 101 (49): 17174–9.