DESCRIPTION

PLAU(Urokinase-type plasminogen activator) is also named as uPA and belongs to the peptidase S1 family. uPA initiates a proteolytic cascade, which degrades extracellular matrix during tissue growth and remodelling. The secreted proform of uPA is activated after binding to a specific high affinity cell surface receptor. Secreted uPA is exposed to rapid inactivation in the pericellular space by plasminogen activator inhibitor-1 (PAI-1), which is stored in the extracellular matrix bound to vitronectin. It has 2 isoforms produced by alternative splicing. Defects in PLAU are the cause of Quebec platelet disorder (QPD).

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	1,090.9	55.6	5.1
2	8	268.7	10.7	4.0
3	8	133.4	5.1	3.8

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,080.0	44.0	4.1
2	16	266.5	8.2	3.1
3	16	133.3	4.1	3.1

RECOVERY

The recovery of human uPA/Urokinase 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	97	89-104
Cell culture supernatant	1:4	108	104-114
	1:8	115	109-120
Urine	1:320	114	99-127
	1:640	110	103-120
Cell lysate	1:320	82	80-85
	1:640	97	92-103

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 urine and cell lysate was initially diluted 1:40.)

		Human serum	Cell culturesupernatant	Urine	Cell lysate
1:2	Average% of Expected	100	100	100	100
	Range (%)	-	-	-	-
1:4	Average% of Expected	112	94	100	98
	Range (%)	106-119	83-104	99-101	92-104
1:8	Average% of Expected	109	98	96	115
	Range (%)	101-117	97-100	95-97	114-115
1:16	Average% of Expected	115	80	97	124
	Range (%)	113-117	77-83	96-98	119-129

CALIBRATION

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