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Cholesterin dehydrogenase stabilization method, composition containing cholesterin dehydrogenase and cholesterin detection reagent

A technology for cholesterol dehydrogenase and detection reagents, which can be used in enzyme stabilization, biological testing, microbial measurement/inspection, etc., and can solve problems such as instability of cholesterol dehydrogenase

Inactive Publication Date: 2013-07-24
SYSMEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, cholesterol dehydrogenase is an unstable enzyme and various efforts have been made to formulate assay reagents

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Prepare a loading solution (pH8.8) containing 0.7U / mL cholesterol dehydrogenase, 0.5M of the compounds shown in Table 1, and 2mg / mL sodium cholate, and leave it at 37°C for 3 days, then obtain it by the method shown below Residual activity of cholesterol dehydrogenase. Table 1 shows the results of the residual activity after storage as a relative value based on the enzyme activity immediately after the preparation of the sample as 100%. As shown in Table 1, by adding glycine-based compounds such as glycine, glycylglycine or tris(hydroxymethyl)methylglycine, enzyme stabilization was clearly achieved.

[0044] 1. A method for measuring cholesterol dehydrogenase activity.

[0045] Prepare a reagent of the following composition.

[0046] Reagent A:

[0047] Cholesterol 1mg / mL

[0048] Triton X-100 20mg / mL

[0049] Reagent B:

[0050] β-NAD 3mg / mL

[0051] Tris buffer (pH8.5) 0.3M

[0052] Diluent:

[0053] Sodium cholate 3mg / mL

[0054] Phosphate buffer (pH7.0) 20...

Embodiment 2

[0059] Concentrations of the compounds shown in Table 2 were examined. Except that the concentration of each compound was varied within the range of 0.1-0.5M, the others were the same as in Example 1. The results are shown in Table 2. As shown in Table 2, a sufficient stabilizing effect in the range of 0.1-0.5M was confirmed.

[0060] Table 2 Unit: residual rate (%)

Embodiment 3

[0062] The stabilization of cholesterol dehydrogenase by combining glycine and glycylglycine was discussed.

[0063] Prepare a loading solution (pH8.8) supplemented with 2U / mL cholesterol dehydrogenase, 14mM dodecyl maltose, 0.5M glycylglycine and various concentrations of glycine, and investigate the cholesterol dehydrogenase after storage at 37°C for a certain period of time residual rate. The residual activity of cholesterol dehydrogenase was determined by the method shown in Example 1.

[0064] The enzyme activity of the freshly prepared loading solution was 100%, and the residual activity after storage was expressed as a relative value. The results are shown in Table 3. As shown in Table 3, it was confirmed that the addition of glycine further enhanced the stabilizing effect.

[0065] table 3

[0066] Glycylglycine (M) Glycine (M)

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PUM

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Abstract

The present invention aims at a new method for stabilizing cholesterol dehydrogenase which is different from conventional methods for stabilizing cholesterol dehydrogenase, and also aims at providing a novel cholesterol-containing composition and a cholesterol detection reagent. This object is achieved by adding a glycine-based compound represented by the following formula (I). R-(NH-CH2-CO)n-NH-CH2-COOH (I) In the formula, R represents hydrogen, an alkyl group that may have a substituent, a phenyl group that may have a substituent, or a carbonyl group that may have a substituent, and n represents 0~2.

Description

technical field [0001] The invention relates to a method for stabilizing cholesterol dehydrogenase, a composition containing cholesterol dehydrogenase and a reagent for detecting cholesterol. Background technique [0002] Conventionally, assay methods using enzymes have been extensively developed due to their specificity, reproducibility, and ease of operation. In particular, many such methods are known in the field of clinical examinations for the determination of components of blood samples. [0003] Recently, however, in the field of clinical examinations, the examination of lipids has increased, especially cholesterol is a risk factor for arteriosclerosis, which is a disease of the elderly, and it is very important, and the examination of cholesterol has gradually increased. Cholesterol detection methods generally use enzyme methods at present, and there are known methods using cholesterol oxidase in these methods, and there are also methods using cholesterol dehydrogen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/96C12N9/04G01N33/92C12N9/02C12Q1/32C12Q1/60
CPCC12N9/96C12Q1/60C12Q1/32
Inventor 酒井康裕一色健二岸浩司
Owner SYSMEX CORP
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