Quantitation of tissue-type plasminogen activator in human endothelial cell cultures by use of an enzyme immunoassay

Endothelial cells from human umbilical cord were cultured to study plasminogen activator synthesis and secretion. Since simultaneous production of plasminogen activator inhibitor(s) prevented detection of plasminogen activators by use of fibrinolytic assays, an enzyme immunoassay for tissue-type plasminogen activator (t-PA) was developed. In this assay, t-PA in test samples was adsorbed onto microtiter plates coated with rabbit antibody against t-PA and then quantitated by successive incubation with goat antibody against t-PA and enzyme labeled rabbit antibody against goat IgG. The sensitivity of the assay was found to be 1 ng t-PA/ml. In the absence of serum, arterial and venous endothelial cells continuously produce t-PA antigen during a 24 h period, reaching a level of 5.1 +/- 2.5 ng t-PA/ml (n = 8). In serum containing (20%) medium, 9.3 +/- 6.0 ng t-PA/ml (n = 17) was produced during this period (0.1 - 0.2 ml medium per cm2 confluent cells). It is concluded that the enzyme immunoassay is a useful method for quantitating t-PA secretion by endothelial cells in the presence of proteinase inhibitors.