DESCRIPTION

uPAR/PLAUR is a highly glycosylated GPI-anchored membrane protein. In addition to the membrane-anchored form, uPAR is released from the plasma membrane by cleavage of the GPI anchor and can be found as a soluble form (suPAR). uPAR contains three homologous domains (D1-D3) of which the N-terminal one (D1) represents the uPA-binding domain. After binding to uPAR, uPA cleaves plasminogen, generating the active protease plasmin which is involved in a wide variety of physiologic and pathologic processes. In addition to regulating proteolysis, uPAR has important function in cell adhesion, migration and proliferation. Studies reveal that uPAR expression is elevated during inflammation and tissue remodelling and in many human cancers, in which it frequently indicates poor prognosis. suPAR has been detected in plasma, and increased plasma concentrations of suPAR have been found in patients with some advanced cancers.

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 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	20	1,081.1	43.5	4.0
2	20	240.8	12.1	5.0
3	20	12.7	0.5	4.2

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	1,009.0	80.1	7.9
2	24	223.2	20.4	9.1
3	24	49.8	4.6	9.3

RECOVERY

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

Sample Type		Average% of Expected	Range (%)
Human serum	1:8	91	76-113
	1:16	100	84-122
Cell culture supernatant	1:4	95	80-108
	1:8	104	93-120
Urine	1:16	93	74-108
	1:32	98	84-120

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 human serum was initially diluted 1:2. The cell culture supernatant was initially diluted 1:2. The urine was initially diluted 1:2.)

		Human serum	Cell culture supernatant	Urine
1:2	Average% of Expected	100	100	100
	Range (%)	-	-	-
1:4	Average% of Expected	116	97	106
	Range (%)	105-127	96-98	103-109
1:8	Average% of Expected	102	94	100
	Range (%)	84-120	93-96	92-108
1:16	Average% of Expected	101	98	104
	Range (%)	78-125	98-99	91-116

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human uPAR/PLAUR Sandwich ELISA Kit produced at Sunlong Medical™.