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Evaluation of a bioimmuno assay for t-PA activity and its relation to PAI-1 activity and antigen levels

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Author: Rosén, S. · Wejkum, L. · Billing-Claeson, S. · Ghosh, R. · Grdic, K. · Chmielewska, J. · Meijer, P. · Kluft, C. · Tengborn, L. · Conkie, J. · Walker, I.
Type:article
Date:1998
Institution: Gaubius Instituut TNO
Source:Fibrinolysis and Proteolysis, 6, 12, 340-346
Identifier: 234791
doi: DOI:10.1016/S0268-9499(98)80391-3
Keywords: Health · Alteplase · Monoclonal antibody · Plasminogen activator inhibitor 1 · Polyclonal antibody · Tissue plasminogen activator · Adult · Blood clot lysis · Clinical article · Controlled study · Female · Fibrinolysis · Human · Immunoassay · Male · Reproducibility · Thromboembolism · Thrombogenesis · Thrombosis

Abstract

A chromogenic bioimmunoassay method for determination of t-PA activities has been evaluated on plasmas from healthy individuals and from thromboembolic patients. The assay consists of an initial binding and washing step, whereby plasma t-PA is bound to a specific monoclonal t-PA antibody, which is coated on to microplate wells, followed by a chromogenic plasmin generation step, using the chromogenic plasmin substrate S-2403. Through collection of blood at pH 6 and through maintaining this pH during the binding step, in vitro inhibition of t-PA by PAI-1 is prevented and the resulting t-PA activity reflects the condition in plasma at the time of blood sampling. Through parallel incubation of plasma at pH 8, an estimation of the PAI-1 activity is obtained from the ratio of t-PA activities at pH 6 and 8. A median t-PA activity of 0.46 IU/mL (range 0.05-1.3, n = 69) was obtained for healthy individuals, whereas analysis of plasma from thromboembolic patients resulted in a median value of 0.31 IU/mL (range 0.04-1.5, n = 107). These results were in striking contrast to those obtained from 40 individuals from each group with parallel blood sampling in neutral citrate and incubation at pH 8, where the corresponding activities were 0.03 IU/mL (range 0-0.5) and 0.014 IU/mL (range 0-0.13) respectively, illustrating the severe losses of t- PA activity obtained in vitro unless precautions to avoid inhibition by PAI- 1 are undertaken. Furthermore, the results Showed an inverse correlation of the t-PA activity with PAI-1 activity and antigen levels (r = 0.74-0.91). Elevated concentrations of t-PA antigen in combination with elevated levels of PAI-1 consistently resulted in low t-PA activities. PAI-1 antigen concentrations above 70 ng/mL were always connected with t-PA activities below 0.2 IU/mL and with BIA t-PA ratios below 0.4. Prevention of t-PA inhibition by PAI-1 at pH 8 through addition of the monoclonal PAI-1 antibody MAI-12 resulted in equal t-PA activities as obtained from incubation at pH 6. Altogether, the results suggest that the BIA t-PA assay performed on plasma samples collected in acid medium provides a convenient tool for determination of basal steady state t-PA activities.