Method of Determining Enzymatic Activity in Biological Media

a biological media and enzymatic activity technology, applied in the field of diagnostics, can solve the problems of affecting the inability to calculate the amount of enzyme present during the time course of the reaction, and the inability to completely consume substrate before the end of the enzyme generation process

Inactive Publication Date: 2012-05-31
THROMBINOSCOPE
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  • Claims
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AI Technical Summary

Benefits of technology

This approach provides a reliable, fast, and convenient method for accurately determining thrombin activity in real-time, correcting for substrate consumption and medium-dependent variations, thereby improving measurement accuracy and efficiency.

Problems solved by technology

Drawbacks of this method are that the activity of the enzyme towards any physiological substrate that may also be present in this medium will compete with the signal substrate that is added and that the substrate may be completely consumed before the enzyme generation has finished.
Another problem is that the measured signal is usually dependent on the turbidity and the color of the medium in which the reaction takes place.
This makes it more difficult to calculate the amount of enzyme present during the time course of the reaction.

Method used

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  • Method of Determining Enzymatic Activity in Biological Media
  • Method of Determining Enzymatic Activity in Biological Media
  • Method of Determining Enzymatic Activity in Biological Media

Examples

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example

[0092]FIG. 8A shows the uncorrected fluorescent signal measured from a reaction mixture containing ⅔rd platelet poor plasma, 416 μM of the fluorogenic thrombin substrate Z-G-G-R-AMC, 4 μM of procoagulant phospholipid vesicles and 5 pM recombinant tissue factor. At time zero the reaction is started by the addition of the fluorogenic substrate and calcium to the citrated plasma. The dashed line shows the corrected signal for this reaction in which correction has been made for inner filter effect and substrate consumption.

[0093]FIG. 8B shows a measurement of 100 nanomolar of thrombin in Hepes buffered saline pH 7.35. This calibrator curve is mathematically fit according to the formula FU(t)=FU(0)+MAX*(1−exp(K*t)). A single point measurement of a concentration of fluorophore that equals the substrate concentration in another sample of the same plasma as in which thrombin generation takes place is used to correct the fluorescent signal in FIG. 8A using the formula:

FU_corrected=−SPM*LN[FU...

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Abstract

A reliable, fast and convenient method is provided to determine the change in concentration of enzyme in time in a biological medium in the presence of a signal substrate that corrects for substrate consumption, color-dependency of the signal and non-linearity between the concentration of the leaving group of the signal substrate and the amount of signal. The method comprises the measurement of a calibrator curve in a suitable medium, such as a buffer, to determine the characteristics of the measured curve. These characteristics will then allow to obtain this whole curve or a sufficient part thereof again through a mathematical procedure based on a single-point measurement in a sample of a medium in which the enzyme generation takes place. This has the great advantage that there is no need to measure the whole calibrator curve in each individual medium, since a single-point measurement is adequate.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of U.S. Ser. No. 11 / 814,882, filed Oct. 15, 2010, which is a national stage application of PCT / EP2007 / 005044, filed Jun. 6, 2007, which is claims priority to EP 06011678.7, filed Jun. 6, 2006. The present application claims the benefit of the filing date of each of the aforementioned applications, and the entire disclosure of each of the aforementioned applications is incorporated by reference.FIELD OF THE INVENTION[0002]The present invention is in the field of diagnostics and relates in particular to a new method of determining biologically active forms of proteolytic enzymes, such as thrombin, in blood and other fluids, and to a test kit for carrying out this method.BACKGROUND[0003]Many laboratories in the fields of biochemistry, chemistry, medicine, diagnostics and the like are interested in the measurement of generation of enzymes in biological media. To this end, signal substrates are generally used t...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12Q1/56
CPCC12Q1/56G01N33/52G01N33/573G01N33/86
InventorGIESEN, PETER L.A.VAN ASTEN, TIMOTHY C.P.
OwnerTHROMBINOSCOPE