A sensitive ELISA for human tissue-type plasminogen activator applicable to the study of acute release from cultured human endothelial cells
article
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
Topics
AntigenCell extractPlasminogen activator inhibitor 1Controlled studyDiagnostic accuracyEndothelium cellHuman cellUmbilical veinAvidinBiotinCells, CulturedComparative StudyCulture MediaEndothelium, VascularEnzyme-Linked Immunosorbent AssayHumanPlasminogen Activator Inhibitor 1Reference StandardsReproducibility of ResultsSensitivity and SpecificitySubcellular FractionsSupport, Non-U.S. Gov'tThrombinTissue Plasminogen Activator
TNO Identifier
232484
ISSN
03406245
Source
Thrombosis and Haemostasis, 71(2), pp. 225-229.
Pages
225-229
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