DESCRIPTION

Cytotoxic and regulatory T-cell molecule (CRTAM, also known as CD355) has been characterized as an important activation marker for both CD8+ T cells and NK-T cells. Engagement of CRTAM promotes cytotoxic effects of NK cells towards Necl-2 expressing tumor cells. Coligation of TCR and CRTAM on CD8+ T cells causes interferon-c (IFN-c) secretion in vitro. Aside from the presence in cells of the immune system and its immunological functions, CRTAM is also involved in cell-cell adhesion due to its expression in epithelial cells and has a role in the transcytosis of neural stem cells across brain vascular endothelial 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 (ng/mL)	SD	CV%
1	8	4.88	0.25	5.03
2	8	1.22	0.05	3.94
3	8	0.38	0.02	4.01

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	4.78	0.29	6.09
2	16	1.22	0.04	3.65
3	16	0.38	0.02	4.08

RECOVERY

The recovery of mouse CRTAM/CD355 spiked to three different levels throughout the range of the assay in tissue lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Tissue lysate	1:256	83	82-84

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 1:32.)

		Tissue lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	89
	Range (%)	84-94
1:8	Average% of Expected	90
	Range (%)	88-93
1:16	Average% of Expected	89
	Range (%)	70-107

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Mouse CRTAM/CD355 Sandwich ELISA Kit produced at Sunlong Medical™.