A Sensitive Solid-Phase Immunosorbent Assay for Tissue-Type Plasminogen Activator Activity in Plasma Using Trinitrobenzoylated Poly-D-Lysine as a Stimulator for Plasminogen Activation

Lars Christian Petersen; Peter Handest; Jytte Brender; Johan Selmer; Maja Jørgensen; Sixtus Thorsen

doi:10.1055/s-0038-1651095

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1987; 57(02): 205-211
DOI: 10.1055/s-0038-1651095

Original Article

Schattauer GmbH Stuttgart

A Sensitive Solid-Phase Immunosorbent Assay for Tissue-Type Plasminogen Activator Activity in Plasma Using Trinitrobenzoylated Poly-D-Lysine as a Stimulator for Plasminogen Activation

Lars Christian Petersen

The Departments of Clinical Chemistry and Microbiology, University of Copenhagen, Hvidovre Hospital, Hvidovre, Denmark

,

Peter Handest

The Departments of Clinical Chemistry and Microbiology, University of Copenhagen, Hvidovre Hospital, Hvidovre, Denmark

,

Jytte Brender

The Departments of Clinical Chemistry and Microbiology, University of Copenhagen, Hvidovre Hospital, Hvidovre, Denmark

,

Johan Selmer

The Departments of Clinical Chemistry and Microbiology, University of Copenhagen, Hvidovre Hospital, Hvidovre, Denmark

,

Maja Jørgensen

The Departments of Clinical Chemistry and Microbiology, University of Copenhagen, Hvidovre Hospital, Hvidovre, Denmark

,

Sixtus Thorsen

The Departments of Clinical Chemistry and Microbiology, University of Copenhagen, Hvidovre Hospital, Hvidovre, Denmark

› Author Affiliations

Abstract

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Summary

A sensitive, specific and precise immunosorbent assay for tissue-type plasminogen activator (t-PA) activity in plasma was developed. It measured the single-chain and the two-chain forms of t-PA with equal sensitivity. The assay involved (I) coating of wells in microtiter plates with a monoclonal antibody directed towards an epitope on t-PA apart from the catalytic active site, (II) binding of t-PA to the solid-phase antibody, (III) activation of plasminogen by antibody-bound t-PA in the presence of a new potent stimulator, trinitrobenzyl alkylated poly-D-lysine (TNB-poly-D-Lysine) and measurement of plasmin activity with D-Val-Leu-Lys-pNA. Plasma samples were acid-treated and diluted 80 times in order to minimize the inhibitory effect of plasma on the assays. The assay could be performed within one working day with precoated microtiter plates.

The sensitivity of the assay for t-PA in plasma was 1 pM (~70 ng/1). The recoveries of single-chain and two-chain t-PA added to plasma was 97-104%. The intraassay coefficient of variation was 3.4-5.1% and the interassay coefficient of variation was 7.8-18%. Resting values of t-PA in plasma for 42 healthy subjects ranged between 0 and 30 pM (median: 4.1 pM). The values after 10 min venous occlusion ranged between 1.2 and 520 pM (median: 100 pM). The t-PA concentrations determined by the immunosorbent assay correlated well with euglobulin clot lysis time measurements (r = 0.940).

Keywords

Assay - Monoclonal antibody - Plasma - Tissue-type plasminogen activator - Trinitrobenzoylated poly-D-lysine

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References

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