Measurement of plasminogen activator inhibitor 1 in biologic fluids with a murine monoclonal antibody-based enzyme-linked immunosorbent assay

Abstract

An enzyme-linked immunosorbent assay for plasminogen activator inhibitor-1 (PAI-1) in biologic fluids was developed on the basis of two murine monoclonal antibodies raised against PAI-1 purified from HT- 1080 fibrosarcoma cells. The lower limit of sensitivity of the assay in plasma is 2 ng/mL. The assay is 12 times less sensitive toward the PAI- 1/human tissue-type plasminogen activator (t-PA) complex as compared with free PAI-1. The intraassay, interassay, and interdilution coefficients of variation are 5.2%, 8.0%, and 7.1%, respectively. The level of PAI-1 in platelet-poor plasma of healthy subjects is 18 +/- 10 ng/mL (mean +/- SD, n = 45). In platelet-rich plasma after freezing and thawing, 92% of PAI-1 antigen is released from platelets, whereas only 8% is found in the corresponding platelet-poor plasma. In platelet-poor plasma from healthy subjects, a linear correlation (r = 0.80) was found between PAI activity and PAI-1 antigen. In plasma approximately two thirds of the PAI-1 antigen was functionally active, whereas only 5% of the PAI-1 antigen released from platelets was active. During pregnancy a progressive increase of PAI-1 antigen levels up to three- to sixfold the control value was observed. In plasma of patients with recurrent deep vein thrombosis, PAI-1 levels were 44 +/- 20 ng/mL (mean +/- SD, n = 7), during a clinically silent phase. Four of these patients had a level above 38 ng/mL (mean +/- 2 SD of normal). The present assay, based on stable and reproducible reagents, allows the specific determination of PAI-1 antigen in biologic fluids. It may facilitate interlaboratory comparisons and be useful for further investigations of the role of PAI-1 in clinical conditions associated with impaired fibrinolysis and/or a tendency to thrombosis and investigations of the role of PAI-1 in platelets.

Impaired release of tissue plasminogen activator from the endothelium in Graves' disease — indicator of endothelial dysfunction and reduced fibrinolytic capacity

“…The plasma vWF level is expressed as a percentage of normal pooled plasma, the antigen level of which is de®ned as 100%. The total plasma concentrations of t-PA and PAI-1 were determined using enzyme-linked immunosorbent assay (ELISA) methods [13,14] (Shanghai Bio-products). Brie¯y, acidi®ed plasma was diluted and added to microtitre plates coated separately with a monoclonal anti-human t-PA antibody and a monoclonal anti-human PAI-1 antibody.…”

Structural and Functional Characterization of Vitronectin‐Derived RGD‐Containing Peptides from Human Hemofiltrate

“…In additional experiments, prior to size-exclusion chromatography the radiolabeled peptide was incubated for 1 h at 37°C in NaCl/P, with human albumin (50 mg/ml) or with active PAI-1 (2 pg) and albumin (10 mg/ml). The concentration of PAI-1 in column fractions was analyzed by means of a sandwich ELISA (Biopool) [16].…”

Hyperfibrinolysis Resulting from Clotting Activation in Patients with Different Degrees of Cirrhosis

“…Plasminogen activator inhibitor (PAI-1) antigen was evaluated in frozen specimens on ELISA (Imubind plasma PAI-1 ELISA kit; American Diagnostica) utilizing the double-antibody principle. It detects active and latent forms of PAI (24). Intraassay and interassay coefficients of variation were 8% and 9%, respectively.…”