DESCRIPTION

Fas ligand (Fas-L) and its receptor Fas (also known as CD95) are members of the tumor necrosis factor family, which interact to play a key role in initiating an apoptotic pathway and accounting for the molecular mechanism of activation-induced T-cell death. They may be linked to breast cancer progression. Fas can be a therapeutic target in rheumatoid arthritis as well. Serumsoluble Fas and soluble Fas-L levels were significantly higher in some rheumatic disease patients. It also has been reported that increased soluble CD95 (sFas/CD95) serum level correlates with poor prognosis in melanoma patients.

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 (ng/mL)	SD	CV%
1	8	10.10	0.58	5.74
2	8	2.61	0.09	3.45
3	8	1.28	0.04	3.13

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	10.34	0.64	6.19
2	16	2.58	0.14	5.43
3	16	1.26	0.07	5.56

RECOVERY

The recovery of mouse Fas/CD95 spiked to three different levels throughout the range of the assay in lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Lysate	1:320	93	87-100

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 lysate was initially diluted 1:100.)

		Lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	103
	Range (%)	102-104
1:8	Average% of Expected	111
	Range (%)	109-113
1:16	Average% of Expected	121
	Range (%)	120-122

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Mouse Fas/CD95 One-Step ELISA Kit produced at Sunlong Medical™.