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Method of analyzing enzyme

A technology of enzyme analysis and enzyme substrate, applied in the field of enzyme analysis, can solve the problems of the influence of coloring, turbidity of sample liquid, and difficulty in synthesizing substrates, etc.

Inactive Publication Date: 2008-05-14
MITSUBISHI KAGAKA IATRON INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these synthetic substrates basically develop color and generate fluorescence after the dyes and fluorochromes bound to them are free, instead of expressing signals under any conditions. sites, so it is not easy to develop new synthetic substrates
[0010] In addition, for the enzyme reaction carried out in the liquid phase, when the color development is directly detected, it is easily affected by the turbidity and coloring of the sample liquid
When detecting fluorescence, if the sample is mixed with fluorescent substances, the detection results will be affected by it and become inaccurate

Method used

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  • Method of analyzing enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] "Example 1: Determination of Thrombin Activity"

[0084] (1) Labeling of substrates

[0085] The synthesis of the C-terminal biotin-labeled peptide substrate (N-Gly-Pro-Arg-biotin) was carried out according to the procedure of using 1,2-diaminoethane trityl resin and using N-α-tert-butoxy Carbonylglycine was synthesized by Fmoc chemistry as the N-terminal amino acid residue of the peptide using an automatic peptide synthesizer. After removing the C-terminal amino acid of the peptide from the resin, it was biotin-labeled with 5-(N-succinimidyloxycarbonyl)pentyl D-biotinamide via 1,2-diaminoethane. All protective groups on the side chains of the biotin-labeled peptide were removed by reacting with trifluoroacetic acid. The obtained N-Gly-Pro-Arg-biotin was labeled with alkaline phosphatase at the N-terminal amino group using a commercially available alkaline phosphatase labeling kit (colleague product code: LK12).

[0086] (2) Binding of substrates on magnetic particle...

Embodiment 2

[0091] "Example 2: Determination of ADAMTS13 Activity"

[0092] (1) Preparation of substrate

[0093] A partial fragment of vWF (a fragment composed of amino acids 1596 to 1668. Hereinafter referred to as vWF73) is used as ADAMTS 13 [another name: von Willebrand Factor cleaving protease (von Willebrand Factor cleaving protease)]. Substrate, according to the method described in KoichiKokame, Masanori Matsumoto, Yoshihiro Fujimura, and ToshiyukiMiyata, vWF73, a region from D1596 to R1668 of von Willebrand Factor, provides a minimal substrate for ADAMTS 13, Blood, Jan 2004; 103:607-612 preparation.

[0094] (2) Binding of substrates on magnetic particles

[0095] A 1% solution of magnetic latex (manufactured by JSR) with a particle diameter of 2.4 μm and the vWF73 (500 μg) prepared in (1) were reacted at room temperature for 1 hour in 50 mmol / L MES buffer solution (pH 6). After washing, blocking with 0.3% bovine albumin / 0.1 mol / L Tris buffer (pH 8) was performed to prepare sub...

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Abstract

The invention discloses a method of analyzing an enzyme, which comprises: (1) the step of contacting a test sample likely containing the target enzyme to be analyzed with an immobilized substrate, in which an enzyme substrate cleavable with the enzyme to be analyzed has been bound to an insoluble support, in a liquid; (2) the step of separating the insoluble support from the liquid; and (3) the step of analyzing the substrate or a fragment thereof remaining in the insoluble support and / or a fragment of the substrate released from the insoluble support into the liquid; and a kit for analyzing an enzyme containing at least an immobilized substrate in which an enzyme substrate cleavable with the enzyme to be analyzed has been bound to an insoluble support.

Description

technical field [0001] The present invention relates to a method for analyzing enzymes contained in a sample. More specifically, for example, in a method for measuring enzyme activity, an enzyme analysis method in which a solid-phase substrate formed by binding an enzyme substrate to be measured to an insoluble carrier is reacted with a sample, and the amount of substrate fragments cleaved by the enzyme is detected . Background technique [0002] At present, in the practice of clinical diagnosis and examination, it is required to measure the living body components as the index of disease diagnosis in a short time and with high precision, and to quickly and accurately feed back the results to the treatment site. Appropriate medicaments and methods of treatment; observation of the course of treatment; anticipation of disease recurrence. [0003] Biomaterials (so-called diagnostic markers) used as indicators for diagnosing diseases involve many aspects such as proteins, nucle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/37G01N33/543G01N33/573
CPCG01N33/573G01N33/585C12Q1/34C12Q1/00
Inventor 小野智子小仓实
Owner MITSUBISHI KAGAKA IATRON INC