Measurement of Plasminogen Activator Inhibitor 1 (Pai-1) in Plasma With an Elisa Based on Two Murine Monoclonal Antibodies

Abstract

An enzyme-linked immunosorbent assay (ELISA) for quantitation of plasminogen activator inhibitor 1 (PAI-1) was developed, based on two murine monoclonal antibodies (MA-7D4B7 and MA-7F5), raised against purified PAI-1 from HT-1080 fibrosarcoma cells, which react with non-overlapping epitopes. MA-7D4B7 was coated on microtiter plates and bound PAI-1 antigen was quantitated with MA-7F5 conjugated with horseradish peroxidase.In normal plasma, collected on citrate at pH 7.4, the PAI-1 level is 27 ± 16 ng/ml (mean ± SD, n=ll), with a corresponding value of 19 ± 11 ng/ml (n=12) in plasma collected on acid citrate pH 4.5, which inhibits the release of PAI-1 from platelets. The lower limit of the assay in plasma is 2 ng/ml; the intra-assay, inter-assay and inter-dilution coefficients of variation are 5.2%, 8.0% and 7.1% respectively.This ELISA was used to measure PAI-1 levels in plasma (collected on citrate, pH 7.4) of non-pregnant women and of women at different stages of pregnancy. A progressive increase is observed : before: 20±9 ng/ml, n=7; first trimester: 25 ± 12 ng/ml, n=5; second trimester: 40 ± 25 ng/ml, n=ll; third trimester: 98 ± 46 ng/ml, n=13. A correlation coefficient of 0.70 is found between the duration of pregnancy and the PAI-1 level.Preliminary data indicate that the PAI-1 antigen level is increased in several disease states, including myocardial infarction and deep vein thrombosis.Thus, this newly developed ELISA allows a direct measurement of the fast-acting inhibitor of plasminogen activator in plasma. Application of this assay to plasma of non-pregnant and pregnant women substantiates previous results obtained with the use of functional assays. In order to quantitate PAI-1 antigen circulating in plasma, blood should be collected under conditions that prevent platelet stimulation.

Characterization of common neoantigenic epitopes generated in plasminogen activator inhibitor‐1 after cleavage of the reactive center loop or after complex formation with various serine proteinases

“…antigen was determined by ELISA [17] or spectrophotometrically at 280 nm using an absorbance coefficient A~) of 10.…”

“…Microtiterplates were coated with the monoclonal antibodies and subsequently blocked with bovine serum albumin [17]. Samples (containing PAI-1/t-PA) were diluted in PBS containing Tween 80 (0.002%), EDTA (5 mM) and bovine serum albumin (1 g/l) (dilution buffer), and added to the wells.…”

Acute Phase Protein α1-Acid Glycoprotein Interacts with Plasminogen Activator Inhibitor Type 1 and Stabilizes Its Inhibitory Activity

“…Under normal physiological conditions, PAI-1 is present in blood plasma at nanomolar concentrations that increase rapidly in response to different stimuli, including cytokines associated with defensive processes such as inflammation (2,3). Among serine proteinase inhibitors (serpins), PAI-1 is the most efficient in vivo inhibitor of both tissue-type and urokinase-type plasminogen activators (4).…”

Purification and characterization of natural and recombinant human plasminogen activator inhibitor‐1 (PAI‐1)

“…The concentration of PAI-1 in normal plasma is very low (20 ng/ml) whereas in conditioned human endothelial cell culture fluid it rarely exceeds 1 pg/ml [19]. Therefore, PAI-1 has been purified from alternative sources including bovine aorta endothelial cell cultures [20] and from malignant human cell line cultures, such as the fibrosarcoma cell line HT 1080 [21,22], the hepatoma cell line HepG2 [22] and the melanoma cell line MJZJ [23].…”