DESCRIPTION

Mouse CD8a (cluster of differentiation 8a, formerly known as Lyt-2) is a type I transmembrane glycoprotein primarily expressed on the surface of cytotoxic T lymphocytes (CTLs) and a subset of dendritic cells. As a co-receptor for the TCR, its extracellular Iglike domain binds to the α3 region of MHC class I molecules, while its intracellular tail associates with the Lck kinase, thereby enhancing antigen recognition sensitivity and signal transduction efficiency in T cells. CD8a exists as either an αα homodimer or an αβ heterodimer and is a critical molecule for thymic positive selection and peripheral CTL activation. Studies in knockout mice reveal impaired CTL development and defective antiviral/antitumor immunity, establishing CD8a as a central marker and target for T-cell subset identification, adoptive immunotherapy, and vaccine evaluation.

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	1,303.0	20.2	1.6
2	8	331.0	4.3	1.3
3	8	161.2	2.8	1.7

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,344.8	52.5	3.9
2	16	347.7	20.2	5.8
3	16	171.2	10.8	6.3

RECOVERY

The recovery of mouse CD8a spiked to three different levels throughout the range of the assay in tissue lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Tissue lysate	1:1,600	101	93-110

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 tissue lysate was initially diluted 1:100.)

		Tissue lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	105
	Range (%)	102-107
1:8	Average% of Expected	109
	Range (%)	105-112
1:16	Average% of Expected	101
	Range (%)	100-101

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Mouse CD8a One-Step ELISA Kit produced at Sunlong Medical™.