METHOD FOR DETERMINING THE SPATIOTEMPORAL DISTRIBUTION OF ACTIVITY OF A PROTEOLYTIC ENZYME IN A HETEROGENEOUS SYSTEM (VARIATIONS), A DEVICE FOR REALIZING SAME AND A METHOD FOR DIAGNOSING THE DEFECTS IN THE HEMOSTATIC SYSTEM ON THE BASIS OF A CHANGE IN THE SPATIOTEMPORAL DISTRIBUTION OF ACTIVITY OF A PROTEOLYTIC ENZYME IN A HETEROGENEOUS SYSTEM

摘要

The invention relates to the field of biotechnology. The method for determining the spatial and temporal distribution of the activity of a proteolytic enzyme in an in vitro heterogeneous system, such as blood or blood plasma, involves the introduction of a luminescent, fluorogenic or chromogenic substrate into a sample with the subsequent release of a detectable tag as the proteolytic enzyme cleaves the substrate, and the recording of the optical characteristics of the sample, which makes it possible to assess the spatial and temporal distribution of the activity of the enzyme. The device for the implementation of the above method comprises an in vitro system, a means for illuminating the sample, a recording means and a control means. A method for diagnosing homeostatic imbalances according to a change in the spatial and temporal distribution of the activity of a proteolytic enzyme in a blood sample is also proposed.

主权项

1. A method for determining the spatiotemporal distribution of activity of a proteolytic enzyme in a heterogeneous system, comprising the steps of:
providing an in-vitro system that includes a sample of a test medium, wherein the test medium is selected from the group consisting of blood plasma, whole blood, water, lymph, colloidal solution, crystalloid solution and a gel, and a proteolytic enzyme or its zymogen, wherein the proteolytic enzyme or its zymogen is distributed in the sample of the test medium; combining a fluorogenic substrate with the in-vitro system, wherein the fluorogenic substrate is cleaved by the proteolytic enzyme, and a fluorescent mark is released upon cleavage of the fluorogenic substrate; illuminating the sample of the test medium to excite a fluorescence signal of the fluorescent mark; recording the spatial distribution of the fluorescence signal of the fluorescent mark in the sample of the test medium, and obtaining a set of distributions of the fluorescence signal; and calculating the spatiotemporal distribution of the proteolytic enzyme activity by using the set of distributions of the fluorescence and solving the inverse “reaction-diffusion-convection” problem, wherein the step of calculating includes correcting the spatiotemporal distribution by using at least one numerical algorithm for reducing noise and distortion of the fluorescence signal.