DESCRIPTION

Endoglin (ENG, CD105) is a homodimeric cell membrane glycoprotein of 180 kDa, composed of disulphide-linked subunits of 9095 kDa. Endoglin is a proliferation-associated and hypoxia-inducible protein mainly expressed on vascular endothelial cells. It acts as an accessory receptor for transforming growth factor beta (TFG-β) and is involved in vascular development and remodelling. The important role of Endoglin in angiogenesis and in tumor progression makes it an ideal target for antiangiogenic therapy and a good marker for tumor prognosis. The extracellular domain of membrane-bound Endoglin can be proteolytically cleaved, releasing a soluble form of Endoglin (sCD105). Increased levels of sCD105 are linked to the pathogenesis of severe vascular disease, and also correlate with poor prognosis in patients suffering from various types of cancer.

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 (pg/mL)	SD	CV%
1	8	1,054.7	45.0	4.3
2	8	268.1	6.9	2.6
3	8	136.4	4.3	3.2

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,035.1	40.3	3.9
2	16	262.2	9.1	3.5
3	16	133.5	4.5	3.4

RECOVERY

The recovery of human Endoglin/CD105 spiked to three different levels throughout the range of the assay was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:64	108	92-120
Cell culture suerpnatant	1:2	101	94-109
Lysate	1:1,600	97	91-106

LINEARITY

To assess the linearity of the assay, cell culture supernatant samples were spiked with high concenthumanion of human Endoglin/CD105 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Human plasma and lysate samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The human plasma was initially diluted 1:4. The lysate was initially diluted 1:200. )

		Human plasma	Cell culture supernatant	Lysate
1:2	Average% of Expected	100	109	100
	Range (%)	-	107-112	-
1:4	Average% of Expected	107	105	100
	Range (%)	106-108	104-106	99-101
1:8	Average% of Expected	114	113	102
	Range (%)	109-119	111-115	99-106
1:16	Average% of Expected	104	116	106
	Range (%)	84-124	109-123	99-113

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human Endoglin/CD105 One-Step ELISA Kit produced at Sunlong Medical™.