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A sensitive ELISA for human tissue-type plasminogen activator applicable to the study of acute release from cultured human endothelial cells

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Author: Schrauwen, Y. · Emeis, J.J. · Kooistra, T.
Type:article
Date:1994
Institution: TNO Preventie en Gezondheid
Source:Thrombosis and Haemostasis, 2, 71, 225-229
Identifier: 232484
Keywords: Antigen · Cell extract · Plasminogen activator inhibitor 1 · Controlled study · Diagnostic accuracy · Endothelium cell · Human cell · Umbilical vein · Avidin · Biotin · Cells, Cultured · Comparative Study · Culture Media · Endothelium, Vascular · Enzyme-Linked Immunosorbent Assay · Human · Plasminogen Activator Inhibitor 1 · Reference Standards · Reproducibility of Results · Sensitivity and Specificity · Subcellular Fractions · Support, Non-U.S. Gov't · Thrombin · Tissue Plasminogen Activator

Abstract

The development of a highly sensitive, enzyme-linked immunosorbent assay (ELISA) for tissue-type plasminogen activator (tPA) is described. The use of a biotin-avidin system resulted in a detection limit of 10 pg of tPA per ml, which is 50 to 150 times more sensitive than commercially-available ELISAs. Free tPA and tPA complexed to PA inhibitor type-1 were detected with equal efficiency. The ELISA proved suitable for measuring tPA antigen in conditioned media and in cell extracts. The intra-assay coefficient of variation varied in six different experiments from 1.9% to 3.4% in the range 0 to 250 pg of tPA antigen per ml. The inter-assay coefficient of variation was 7% (n = 6). The ELISA was used to study the acute release of tPA from human umbilical vein endothelial cells in vitro. Upon addition of thrombin (1 NIH U/ml) to endothelial cells, tPA was rapidly released into the medium, the highest release occurring during the first minute. Concomitantly, the tPA concentration in the cell extracts decreased. Evidence is presented that tPA is released from an intracellular source. Chemicals/CAS: biotin, 58-85-5; plasminogen activator inhibitor 1, 140208-23-7; tissue plasminogen activator, 105913-11-9; Avidin, 1405-69-2; Biotin, 58-85-5; Culture Media; Plasminogen Activator Inhibitor 1; Thrombin, EC 3.4.21.5; Tissue Plasminogen Activator, EC 3.4.21.68