DESCRIPTION

PD-L1 (programmed cell death ligand 1, also known as CD274 or B7-H1) is a 290 aa type I transmembrane protein. PD-L1 is expressed constitutively on T cells, B cells, DCs, macrophages, mesenchymal stem cells and cultured bone marrow-derived mast cells. In addition, PD-L1 is also expressed on many nonhematopoietic cell types, including vascular endothelial cells, epithelial cells, muscle cells, hepatocytes, pancreatic islet cells, astrocytes in the brain, placental syncytiotrophoblasts, and cells in cornea, iris-ciliary body and retina of eye. PD-L1 is frequently upregulated in a wide variety of solid tumors, including melanoma, ovarian, lung, glioblastoma, breast, and pancreatic cancers. PD-L1 and PD-L2 are two ligands of PD-1. Engagement of PD-1 by PDL1 or PD-L2 transduces a signal that inhibits T-cell proliferation, cytokine production, and cytolytic function. It is critical for the regulation of T cell function during tolerance, autoimmunity and infection. Besides the membrane-bound form, PD-L1 can also exist as a soluble form (sPD-L1) generated either by proteolytic cleavage of membrane-bound form or by translation of alternative spliced mRNA.

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the solution yellow. The color intensity is proportional to the quantity of bound protein, which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	546.3	13.4	2.5
2	8	127.0	0.5	0.4
3	8	65.2	0.7	1.1

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	569.8	26.7	4.7
2	16	129.2	2.8	2.2
3	16	67.2	2.4	3.6

RECOVERY

The recovery of human PD-L1/CD274 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:8	102	91-123
	1:16	92	79-103
Cell culture supernatant	1:8	118	115-119
	1:16	112	100-129
Urine	1:2	103	93-111
	1:4	110	108-112
Cell lysate	1:400	108	105-110
	1:800	112	108-114

LINEARITY

To assess the linearity of the assay, urine samples were spiked with high concentrations of human PD-L1/CD274 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Human plasma, cell culture supernatant and cell lysate samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The cell culture supernatant was initially diluted 1:2. The cell lysate was initially diluted 1:50.)

		Human plasma	Cell culturesupernatant	Urine	Cell lysate
1:2	Average% of Expected	100	100	113	100
	Range (%)	-	-	108-118	-
1:4	Average% of Expected	97	101	117	104
	Range (%)	95-99	100-102	116-118	103-105
1:8	Average% of Expected	93	104	126	106
	Range (%)	84-102	103-105	124-129	105-107
1:16	Average% of Expected	90	103	126	106
	Range (%)	80-101	102-104	121-131	105-106

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human PD-L1/CD274 One-Step ELISA Kit produced at Sunlong Medical™.