DESCRIPTION

Glucocorticoid-induced TNFR-related protein (GITR), also known as CD357 or TNFRSF18, is a member of the tumor necrosis factor receptor (TNF-R) superfamily. GITR is expressed constitutively at high levels in T regulatory cells (Treg cells) and plays a key role in dominant immunological self-tolerance maintained by CD25+CD4+ regulatory T cells. It is expressed at low levels on resting responder T cells. The expression of GITR on T cells can be upregulated upon activation. GITR is activated by GITR ligand (GITRL) which is mainly expressed on APC. GITR-GITRL interactions could co-stimulate both responder T-cell functions and the suppressive functions of Treg cells.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with biotin) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with biotin also binds to the analyte. Streptavidin-HRP binds to the biotin. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns 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 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	20	165.7	2.2	1.3
2	20	663.9	13.3	2.0
3	20	2,812.0	99.6	3.5

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	169.7	7.2	4.3
2	24	694.1	19.8	2.8
3	24	2,957.6	107.8	3.6

RECOVERY

The recovery of human TNFRSF18 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:2	84	75-97
	1:4	82	74-89
Human plasma	1:2	88	75-112
	1:4	97	84-112
Cell culture supernatant	1:2	108	95-124
	1:4	112	97-124

LINEARITY

To assess the linearity of the assay, human serum, plasma and cell culture supernatant were spiked with high concentrations of TNFRSF18 in various matrices and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

Sample Type		Range (%)	Average% of Expected
Human serum(Sample Diluent PT 5)	1:2	76-105	87
	1:4	77-111	93
	1:8	92-127	108
	1:16	106-119	113
Human plasma(Sample Diluent PT 3-eg)	1:2	79-89	86
	1:4	87-107	98
	1:8	93-114	102
	1:16	88-123	103
Cell culture supernatant(Sample Diluent PT 1)	1:2	85-104	95
	1:4	89-111	100
	1:8	88-116	102
	1:16	88-118	103

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human GITR/TNFRSF18 Sandwich ELISA Kit produced at Sunlong Medical™.