PD-1 (Programmed Cell Death Protein 1; CD279) is a type I transmembrane protein belonging to the CD28/CTLA-4 family of immune receptors (1). These ‘coinhibitory’ receptors function as breaks for the adaptive immune response, serving as immune checkpoints that effector T cells must pass in order to exert their full functions (2). PD-1 is expressed on activated T cells, B cells, myeloid cells and on a subset of thymocytes. Ligation of PD-1 by PD-L1 (B7-H1; CD274) or PD-L2 (B7-DC; CD273) inhibits TCR-mediated T cell proliferation and production of IL-1, IL-4, IL-10 and IFN-γ. 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 (3). A novel approach to fight cancer is to target immune checkpoint proteins with antibody inhibitors, such as anti-PD-1 or anti-PD-L1 that function as a tumor suppressing factor via the modulation of immune cell-tumor cell interactions (4).
The soluble form of PD-1 (sPD-1) is expressed upon immune cells activation and can be secreted in the serum/plasma. Soluble checkpoints, such as sPD-1 or sPD-L1, are involved in positive or negative immune regulation and changes in their serum/plasma levels affect the development, prognosis and treatment of cancer (5). Increase of sPD-1 or sPD-L1 levels have been detected in patients with different types of cancer (6). This PD-1 (human) ELISA Kit is developed to detect specifically human soluble PD-1 in different biological fluids.
Sandwich type ELISA
Programmed Cell Death Protein 1, hPD-1, CD279
Principle of Method
This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human PD-1 in cell culture supernatants, serum and plasma (EDTA, heparin or citrate). A monoclonal antibody specific for PD-1 has been precoated onto the 96-well microtiter plate. Standards (STD) and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, PD-1 is recognized by the addition of a biotinylated monoclonal antibody specific for PD-1 (DET). After removal of excess biotinylated antibody, streptavidin-peroxidase (STREP-HRP) is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450nm after acidification and is directly proportional to the concentration of PD-1 in the samples.
Serum, Plasma or Cell Culture Supernatant
This ELISA is specific for the measurement of natural and recombinant human PD-1 [CD279].
Serum and Plasma : 0.25 µL (actual amount of sample loaded in well after dilution)
Calibration Range: 3.125 pg/ml – 200 pg/ml
Limit of Detection
Intra-assay (Within-Run) C.V.
Inter-assay (Run-to-Run) C.V.
References to Summary
1) The diverse functions of the PD1 inhibitory pathway: A.K. Sharpe & K.E. Pauken; Nat. Rev. Immunol. 18, 153 (2018)
(2) CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms: R.V. Parry, et al.; Mol. Cell. Biol. 25, 9543 (2005)
(3) Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice: H. Nishimura, et al.; Science 291, 319 (2001)
(4) PD-1 and PD-L1 Checkpoint Signaling Inhibition for Cancer Immunotherapy: Mechanism, Combinations, and Clinical Outcome: H.O. Alsaab, et al.; Front. Pharmacol. 8, 561 (2017)
(5) Hiding in Plain Sight: Soluble Immunomodulatory Receptors: L.N. Dahal, et al.; Trends Immunol. 39, 771 (2018)
(6) Soluble PD-L1 as a biomarker in malignant melanoma treated with checkpoint blockade: J. Zhou, et al.; Cancer Immunol. Res. 5, 480 (2017)