DESCRIPTION

TRAIL, also known as TNFSF10, is a member of the TNF superfamily of proteins. TRAIL is a type II transmembrane protein with a TNF homology domain. The soluble form is generated by proteolytic cleavage of the membrane-bound form and forms a trimeric ligand. TRAIL is expressed in various immune cells, including T-cells, B-cells, macrophages, natural killer (NK) cells, and dendritic cells. Its expression is upregulated in activated immune cells upon cytokine stimulation. TRAIL can activate both pro-apoptotic and pro-survival pathways depending on the cell type and context. Binding to DR4 and DR5 can trigger the extrinsic apoptosis pathway, leading to caspase activation and cell death. However, it can also activate the NF-κB pathway, which promotes cell survival and proliferation. One of the key functions of TRAIL is its ability to selectively induce apoptosis in transformed cells, such as cancer cells, while sparing normal cells. This makes it a potential target for cancer therapy. TRAIL plays a role in immune regulation and maintaining immune homeostasis.

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	405.8	10.7	2.6
2	8	93.9	1.5	1.6
3	8	43.9	1.9	4.3

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	374.5	40.0	10.7
2	16	86.4	8.8	10.2
3	16	40.0	4.6	11.4

RECOVERY

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

Sample Type		Average% of Expected	Range (%)
Human serum	1:4	84	81-87
Cell culture supernatant	1:2	89	86-91
Saliva	1:64	95	86-104

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 cell culture supernatant and saliva was initially diluted 1:4. )

		Human serum	Cell culture supernatant	Saliva
1:2	Average% of Expected	100	100	100
	Range (%)	-	-	-
1:4	Average% of Expected	115	95	109
	Range (%)	101-128	89-101	107-112
1:8	Average% of Expected	99	77	107
	Range (%)	81-117	72-82	103-110
1:16	Average% of Expected	-	88	106
	Range (%)	-	85-91	103-108

CALIBRATION

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