An Enzyme-Linked Immunosorbent Assay (ELISA) Used to Study the Cellular Secretion of Endothelial Plasminogen Activator Inhibitor (PAI-1)

Summary

A two-site sandwich ELISA was developed to measure PAI-1 antigen and utilised a polyclonal antiserum produced against PAI-1 purified from human endothelial cell secretory products. The assay was calibrated against a preparation of pure PAI-1 whose protein concentration had been determined by amino acid analysis and the detection limit was 30 pg PAI-1 ml“³ sample. PAI-1 was detected in primate sera but not in a wide range of nonprimate sera and no cross-reactivity with α₂-antiplasmin or antithrombin III was observed.

The ELISA was used to study cellular secretion of PAI-1 which was confirmed as a major secretory protein in human umbilical vein endothelial cells (HUVEC). PAI-1 antigen accumulated in the medium in a linear fashion with time and accounted for ≃10% of total secreted protein. Specific activity of intracellular PAI1 was typically 20-fold greater than that of PAI-1 in 24 h conditioned medium and a t^½ for inactivation of secreted PAI-1 of 0.53 h was calculated. Purified endotoxin stimulated the secretion of PAI-1 antigen and raised the intracellular levels in HUVEC cultures showing that the anti-fibrinolytic actions of endotoxin are effected by increasing the rate of synthesis and secretion of PAI-1.

Key words

ELISA - Plasminogen activator inhibitor - Endothelial - Cell cultures

• References

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