Homotypic cysteine measuring method and its reagent

A homocysteine ​​and reagent technology, applied in biological testing, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as inability to apply clinical large-flow automated analysis, complex reagents, and inability to achieve assay sensitivity.

Active Publication Date: 2009-10-28
北京安百胜诊断科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The common features of the above methods are that special measuring instruments are required, the operation is complicated, the test takes a long time, it cannot be applied to the automatic analysis of clinical large flow, and it is expensive
[0004] Due to the low content of homocysteine ​​in the sample, conventional clinical enzymology assay methods cannot achieve the required assay sensitivity, which limits the adoption of conventional methods
In terms of technology for the determination of homocysteine ​​by enzymatic methods, Naoto Matsuyama et al. (see US Patent #6686172) invented an acyclic method using homocysteine ​​methyltransferase and adenosylhomocysteinase Enzyme determination technology, a method to improve the sensitivity of the determination, but the method is cumbersome to operate, the reagents are complicated, and the sulfhydryl compound needs to be added before the determination, and multiple blank determinations must be carried out at the same time during the determination, which increases the inaccuracy of the determination method

Method used

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  • Homotypic cysteine measuring method and its reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: The hydrogen peroxide chromogenic reaction (Trinder's reaction) produced by the reagent system is determined by using an enzyme cycle reaction consisting of homocysteine ​​methyltransferase and adenosyl homocysteine.

[0020] Reagent 1: (R1:R2=3:1)

[0021] Reagent components

[0022] AdoHcyase

[0023] Reagent 2: (R1:R2=3:1)

[0024] Reagent components

[0025]Example 1 Reagent 1 formula is used to reduce oxidized homocysteine ​​to eliminate the interference of adenosine in the sample, and the complete enzyme cycle assay reagent of reagent 1 is used to eliminate side reactions. When measuring samples, the fixed time method is adopted, and the ratio of reagents to samples is not fixed, but it should be consistent within the same measurement batch. For example, the ratio of reagent 1: sample: reagent 2 is 300: 20: 100, the temperature is 37°C, and the measurement wavelength is 540 nm. After adding the sample or calibrator to r...

Embodiment 2

[0026] Example 2: Using an enzyme cycle reaction consisting of homocysteine ​​methyltransferase and adenosylhomocysteinase, the rate of oxidation of reduced coenzyme within a fixed period of time was determined by ammonia reagent method.

[0027] Reagent 1: (R1:R2=4:1)

[0028] Reagent components

[0029] DTT

[0030] Reagent 2: (R1:R2=4:1)

[0031] Reagent components

[0032] Example 2 Reagent 1 formula is used to reduce oxidized homocysteine, and Reagent 2 and Reagent 1 are combined to form a complete enzyme cycle assay reagent. When measuring samples, the fixed time method is adopted, and the ratio of reagents to samples is not fixed, but it should be consistent within the same measurement batch. For example, the ratio of reagent 1: sample: reagent 2 is 200: 25: 50, the temperature is 37°C, and the measurement wavelength is 340 nm. After adding sample or calibrator to reagent 1, incubate at the measurement temperature for 300 seconds, then add...

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Abstract

The invention relates to an enzymatic determination method and a reagent thereof using a circulation increment technique, which are used for determining the content of homocysteine ​​in a body fluid sample. Its characteristics are: by adopting a cycle incremental technology, the determination sensitivity is improved, so that it can automatically measure homocysteine ​​in body fluid samples like ordinary enzyme diagnostic reagents. Homocysteine ​​in body fluid samples reacts repeatedly with S-adenosyl-L-methionine under the action of homocysteine ​​methyltransferase and adenosylhomocysteinase to produce adenosine and adenosine The rate of adenosine is directly proportional to the content of homocysteine ​​in the sample. By measuring the rate of generation of adenosine, the purpose of measuring the content of homocysteine ​​in the body fluid sample can be achieved. The quantitative determination reagent prepared by the method has the characteristics of convenience, quickness, automation and high sensitivity.

Description

Technical field: [0001] The invention relates to a method for determining homocysteine ​​and its reagents Background technique: [0002] Homocysteine ​​(Homocysteine) is a sulfur-containing amino acid that is produced in cells by demethylation of methionine. Recent studies have shown that homocysteine ​​produces superoxide and peroxide damages vascular endothelial cells, changes the function of coagulation factors, increases the tendency of thrombosis, promotes atherosclerosis and thrombosis, and increases the incidence of cardiovascular diseases and Mortality increases, so measuring the concentration of homocysteine ​​in blood is of great clinical significance. When homocysteine ​​accumulates in cells and enters the blood circulation, most of it exists in the oxidized form and binds to proteins, while the reduced form of homocysteine ​​accounts for only 1% in the blood. To determine the content of total homocysteine ​​in blood, it is generally necessary to use a reducing ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/78G01N33/68C12Q1/34C12N9/14
Inventor 王学忠
Owner 北京安百胜诊断科技有限公司
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