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A bio-immuno assay to determine free tissue-type plasminogen activator (t-PA) in Stabilyte® plasma

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Author: Bos, R. · Revet, M. · Nieuwenhuizen, W.
Type:article
Date:1994
Institution: Gaubius Laboratory TNO Preventie en Gezondheid
Source:Fibrinolysis, SUPPL. 2, 8, 163-165
Identifier: 232682
doi: doi:/10.1016/0268-9499(94)90285-2
Keywords: Biology · Assay · Bio-immunoassay (BIA) · Monoclonal antibody · Plasma · Tissue-type plasminogen activator · Anticoagulant agent · Monoclonal antibody · Stabilyte · Tissue plasminogen activator · Unclassified drug · Bioassay · Conference paper · Controlled study · Fibrinolysis · Human · Immunoassay

Abstract

This article describes a two-step bio-immuno assay (BIA), which determines active (free) tissue-type plasminogen activator (t-PA) in (acidified) plasma. Plasma samples, diluted in buffer of pH 6.0, are added to the wells of a microtiter plate containing an immobilised monoclonal antibody which binds t-PA without affecting t-PA activity. After an overnight incubation at 4°C, bound t-PA activity is determined by incubating the wells at 37°C with plasminogen, CNBr-digested fibrinogen and D-Val-Leu-Lys-pNA, followed by measurement(s) of the absorbance (A) at 405 nm at timed intervals. The ΔA/t2 or, alternatively, the AA after 4 hours (endpoint determination) give an accurate value for the active t-PA concentration. The assay has a lower detection limit of 3 pg t-PA/ml and does not discriminate between various forms of active t-PA. The intra-assay coefficients at 0.2 and 0.05 ng t-PA/ml were 5.5% and 4.3%, respectively. Active t-PA values in samples (N = 66) determined with this assay and with the t-PA BIA from Chromogenix correlated relatively well (r = 0.78). Within a subset of these samples (N = 18), a negative correlation with both t-PA/PAI-1 (r = 0.79) and PAI-1 antigen (r = 0.78) values was observed. Surprisingly, no correlation was found with values for t-PA antigen (r = 0.21).