Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of tissue-type and urokinase-type plasminogen activator, playing a major role in fibrinolysis . PAI-1 is mainly produced by the endothelium, but is also secreted by other tissue types, such as adipose tissue. It is normally present at low levels in plasma and tissue, but its expression and release increased in various disease states (such as a number of forms of cancer), as well as in obesity and the metabolic syndrome. PAI-1 is also involved in the pathophysiology of renal, pulmonary, cardiovascular, and metabolic diseases. Elevated local or systemic PAI-1 can also exacerbate such pathologic conditions.

This assay is a quantitative sandwich ELISA. The immunoplate is pre- coated with a mouse monoclonal antibody specific for human PAI-1. Standards and samples are pipetted into the wells and any human PAI-1 present is bound by the immobilized antibody. After washing away any unbound substances, a biotin labelled polyclonal antibody specific for human PAI-1 is added to the wells. After wash step to remove any unbound reagents, streptavidin-HRP conjugate (STP-HRP) is added. After the last wash step, an HRP substrate solution is added and colour develops in proportion to the amount of human PAI-1 bound initially. The assay is stopped and the optical density of the wells determined using a microplate reader. Since the increases in absorbance are directly proportional to the amount of captured human PAI-1, the unknown sample concentration can be interpolated from a reference curve included in each assay.

A. Typical Representation of Standard Curve

PAI-1 (ng/mL)	Absorbance (450 nm)	Blanked Absorbance
0	0.100	0
0.031	0.151	0.051
0.062	0.202	0.102
0.125	0.292	0.192
0.25	0.460	0.360
0.5	0.828	0.728
1.0	1.508	1.408
2.0	2.451	2.351

 

B. Sensitivity

The lowest level of PAI-1 that can be measured by this assay is 0.031 ng/mL.

 

C. Specificity

The antibodies used in this assay are specific to human PAI-1 and do not cross-react with mouse and rat PAI-1, and other cytokine or hormone molecules including human resistin, TNF, ANGPTL4, insulin, leptin and IL6.

 

D. Precision
Intra-assay Precision (Precision within an assay)

Sample	Mean (ng/mL)	SD (ng/mL)	CV (%)
1	7.51	0.159	2.12
2	2.32	0.070	3.02

Inter-assay Precision (Precision between assays)

Sample	Mean (ng/mL)	SD (ng/mL)	CV (%)
1	0.87	0.036	4.15
2	1.64	0.092	5.63
3	2.93	0.117	3.99
4	4.35	0.204	4.69

 

E. Recovery

Serum samples were spiked with different amounts of human PAI-1 and assayed. The measured concentration of the spiked sample in the assay was compared to the expected concentration. The average recovery was 98%.

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