96-well microplate

PD-L1 (CD274) Human ELISA

KT1018
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Principle of Method

This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human PD-L1 in cell culture supernatants, serum and plasma (EDTA, heparin or citrate). A monoclonal antibody specific for PD-L1 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-L1 is recognized by the addition of a biotinylated monoclonal antibody specific for PD-L1 (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-L1 in the samples.

Description

PD-L1, (also known as CD274 or B7-H1), is an immuno-coinhibitory member of the B7 family and functions as an immune checkpoint via its interaction with its receptors PD-1 (CD279) and CD80 (also known as B7.1) (1). PD-L1 is expressed by hematopoietic cells such as T, B and dendritic cells and by parenchymal cells in response to cytokine (i.e., IFN-γ) induction. 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 and also inhibits BCR mediated signaling (2). PD-L1 binding to CD80 inhibits T cells proliferation. 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-L1 (sPD-L1) 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-L1 or sPD-1 levels have been detected in patients with different types of cancer (6). This PD-L1 (human) ELISA Kit is developed to detect specifically human soluble PD-L1 in different biological fluids.

Type

Sandwich type ELISA

Other names

Programmed Cell Death Protein 1 Ligand 1, PDCD1 ligand 1, hPD-L1, B7 homolog 1, B7-H1

Sample Types

Serum, Plasma or Cell Culture Supernatant

Species crossreactivity

This ELISA is specific for the measurement of natural and recombinant human PD-L1 [CD274]. 

Sample Volume

Serum and Plasma : 0.25 µL (actual amount of sample loaded in well after dilution)

Standard Curve

PD-L1 Standard Curve

 

Calibration Range: 2.343 pg/ml – 150 pg/ml

Limit of Detection

0.8 pg/ml

Intra-assay (Within-Run) C.V.

4.77%

Inter-assay (Run-to-Run) C.V.

3.47%

Resources

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)