DESCRIPTION

TSG101 (tumor susceptibility gene 101 protein) is a multi-structural domain protein that is widely expressed in a variety of cells and is involved in a variety of intracellular processes. It plays a key role in endoplasmic reticulum sorting, membrane receptor degradation, and the final stages of cytokinesis. TSG101 was initially defined as a tumor suppressor gene, but in some epithelial tumor cells, it exhibits tumor-promoting effects. In addition, TSG101 is involved in exosome secretion, positive regulation of ubiquitin-dependent endocytosis, and viral outgrowth. It also plays an important role in cell cycle regulation, cell survival, and maintenance of genome stability.

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	5.28	0.15	2.81
2	8	1.32	0.11	8.10
3	8	0.56	0.06	10.78

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	5.30	0.35	6.55
2	16	1.33	0.14	10.70
3	16	0.55	0.05	8.50

RECOVERY

The recovery of human TSG101 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:16	106	94-115
Cell lysate	1:512	90	70-106

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 lysate was initially diluted 1:32.)

		Human serum(Sample Diluent PT 3)	Cell lysate(Sample Diluent PT 4B1)
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	99	103
	Range (%)	90-104	101-105
1:8	Average% of Expected	92	112
	Range (%)	82-102	108-116
1:16	Average% of Expected	74	123
	Range (%)	72-76	119-128

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human TSG101 produced at Sunlong Medical™.