DESCRIPTION

B7-H2, also known as ICOSLG or GL50, is a type I transmembrane glycoprotein belonging to the B7 ligand family. B7-H2/ICOSLG is extensively expressed on professional antigen-presenting cells including B cells, macrophages, and dendritic cells, as well as non-lymphoid cells including mesenchymal stem cells, endothelial cells, fibroblasts, and tumor cells. It is a specific ligand for the T-cell-specific cell surface receptor ICOS and acts as a co-stimulatory molecule. The interaction of B7-H2/ICOSLG and ICOS plays important roles in the activation, proliferation, differentiation, and cytokine production of T cells as well as in the antibody secretion from B cells during secondary immune responses.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. 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	2,272.6	115.0	5.1
2	20	494.7	16.2	3.3
3	20	111.3	3.9	3.5

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	2,097.9	101.2	4.8
2	24	487.7	17.0	3.5
3	24	115.8	5.7	5.0

RECOVERY

The recovery of mouse B7-H2/ICOSLG spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:20	97	88-106
	1:40	95	92-103
Cell culture supernatant	1:2	109	102-120
	1:4	106	96-118

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The mouse serum was initially diluted 1:2.5.)

		Mouse serum	Cell culture supernatant
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	110	101
	Range (%)	106-114	98-104
1:8	Average% of Expected	114	111
	Range (%)	113-114	100-123
1:16	Average% of Expected	104	96
	Range (%)	103-105	95-97

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Mouse B7-H2/ICOSLG Sandwich ELISA Kit produced at Sunlong Medical™.