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Enzyme-linked coagulation assay: a clot-based, solid-phase assay for thrombin.

G J Doellgast, H Rothberger

Analytical Biochemistry

|June 1, 1985

Summary

A novel enzyme-linked coagulation assay offers a sensitive method for measuring thrombin. This microtiter plate assay detects low thrombin concentrations, serving as a convenient alternative to traditional clot-based coagulation tests.

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Area of Science:

Biochemistry
Hematology
Assay Development

Background:

Thrombin is a key enzyme in the coagulation cascade.
Conventional clot-based assays for thrombin have limitations in sensitivity and convenience.

Purpose of the Study:

To develop a novel, sensitive, and convenient solid-phase microtiter plate assay for quantifying thrombin.
To characterize the performance of the new assay using specific thrombin inhibitors.

Main Methods:

A solid-phase microtiter plate assay was developed utilizing fibrinogen immobilized on plate wells.
An indicator system with peroxidase-fibrinogen was employed to detect thrombin activity.
The assay measures the binding of peroxidase-fibrin to plate-bound fibrin, which is proportional to thrombin concentration.

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Main Results:

The developed assay can measure thrombin at concentrations as low as 0.25 ng/ml (0.006 nM) in 150 microliters of sample.
Specific inhibitors, benzamidine and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, effectively reduced thrombin activity.
Inhibitor concentrations correlated with their known affinities and mechanisms of action.

Conclusions:

The enzyme-linked coagulation assay is a highly sensitive and convenient method for thrombin measurement.
This assay provides a valuable alternative to conventional clot-based coagulation tests.
The assay's performance is validated by its response to specific thrombin inhibitors.