DESCRIPTION

CD8b (T-cell surface glycoprotein CD8 beta chain) is an integral membrane glycoprotein that forms disulfide-linked heterodimers with CD8a (CD8 alpha chain). The CD8 alpha/beta heterodimer is the predominant CD8 complex expressed on the cell surface. CD8 is a transmembrane glycoprotein predominantly expressed on the surface of cytotoxic T cells and can also be found on natural killer cells, cortical thymocytes, and dendritic cells. CD8 serves both as an adhesion molecule for class I MHC molecules and as a coreceptor with the TCR for T cell activation. CD8 plays a role in T cell development and activation of mature T cells.

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 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	690.2	16.3	2.4
2	8	163.3	9.8	6.0
3	8	81.2	3.0	3.7

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	668.2	47.0	7.0
2	16	160.1	11.6	7.2
3	16	79.4	4.1	5.2

RECOVERY

The recovery of human CD8b spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:8	81	73-90
Cell lysate	1:16	97	94-101

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.

		Human serum(Sample Diluent PT 4B1)	Cell lysate(Sample Diluent PT 3-ef)
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	123	95
	Range (%)	116-130	88-102
1:8	Average% of Expected	110	98
	Range (%)	93-127	90-106
1:16	Average% of Expected	115	99
	Range (%)	114-116	92-106

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human CD8b produced at Sunlong Medical™.