DESCRIPTION

Fas (CD95/APO-1) is a transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) receptor superfamily. It can mediate apoptosis by ligation with an agonistic anti-Fas antibody or Fas ligand. Stimulation of Fas results in the aggregation of its intracellular death domains, leading to the formation of the death-inducing signaling complex (DISC). FAS-mediated apoptosis may have a role in the induction of peripheral tolerance, in the antigen-stimulated suicide of mature T-cells, or both.

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	6.12	0.35	5.72
2	8	1.61	0.03	1.86
3	8	0.86	0.04	4.65

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	6.19	0.37	5.98
2	16	1.63	0.07	4.29
3	16	0.86	0.05	5.81

RECOVERY

The recovery of human Fas/CD95 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:8	94	92-96
	1:16	99	94-103
Cell culture supernatant	1:2	114	108-124
	1:4	121	116-125

LINEARITY

To assess the linearity of the assay, cell culture supernatant samples were spiked with high concentrations of human Fas/CD95 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Human plasma samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Human plasma	Cell culture supernatant
1:2	Average% of Expected	100	125
	Range (%)	-	118-132
1:4	Average% of Expected	93	111
	Range (%)	92-94	106-117
1:8	Average% of Expected	94	108
	Range (%)	84-104	103-113
1:16	Average% of Expected	107	110
	Range (%)	88-126	105-114

CALIBRATION

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