Improved homocysteine detection reagent and method

A homocysteine ​​and detection reagent technology, applied in the field of improved homocysteine ​​detection reagents, can solve problems such as poor stability

Active Publication Date: 2015-05-20
BEIJING AMBITION BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing methyltransferase and hydrolase cycle enzyme methods still have the disadvantage of poor stability, and the sensitivity needs to be further improved

Method used

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  • Improved homocysteine detection reagent and method
  • Improved homocysteine detection reagent and method
  • Improved homocysteine detection reagent and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: The effect of component exchange and concentration changes on the kit

[0049] As a kit with clinical detection and diagnostic significance, the replacement of its reagent components, changes in concentration, and even changes in buffers and corresponding pH will change the performance of the kit, and these changes cannot be simple from the existing formula Derived from the ground, but it takes a lot of trials to get the ideal formula. The following content is several sets of examples involved in the development process of the kit of the present invention, which verify the influence of factors such as component exchange and concentration change on the efficacy and stability of the kit.

[0050] First, the formula of the kit 1 disclosed in the prior art is as follows:

[0051] Reagent 1 (R1: R2=3:1)

[0052] Reagent components

Final concentration

Good's buffer

15mM

NAD(P)H

5mM

GLDH

2KU / L

BSA

1.2g / L

TCEP

0.2mM

ATP

10mM

alpha-ketoglutarate

30...

Embodiment 2

[0095] Example 2 Composition and method of use of the kit 1 of the present invention

[0096] After various attempts in the above-mentioned Example 1, the present invention adjusts part of the active enzyme to reagent 2, adds different stabilizers to reagent 2, adjusts the pH of reagents 1 and 2, and adjusts the content and position of reduced coenzymes. , Adjust the position and concentration of the reducing agent to obtain the kit of the present invention.

[0097] The following is a specific example of the kit of the present invention, called kit 1. The kit also adopts an enzyme cycle reaction consisting of homocysteine ​​methyltransferase and adenosine homocysteine ​​hydrolase, by using The method of detecting ammonia is to measure the rate of oxidation of reduced coenzyme within a fixed period of time to quantify the Hcy content in the sample.

[0098] Reagent 1: (R1:R2=3.75:1)

[0099] Reagent components

[0100] Reagent 2: (R1:R2=3.75:1)

[0101] Reagent components

[010...

Embodiment 3

[0104] Example 3: Performance measurement of kit 1 of the present invention

[0105] Purpose: To evaluate the precision, accuracy, reportable range, sensitivity, and the appropriate range of the reference visible interval of the kit, to test whether it can meet the requirements of clinical testing and scientific research, and not cause obvious bias to clinical introduction.

[0106] Method: A Hitachi 7020 automatic biochemical analyzer was used to verify the following performance indicators of kit 1.

[0107] Conclusion: The test results of all performance indicators meet the requirements of clinical testing and scientific research.

[0108] 1 Test materials

[0109] 1.1 Instruments and reagents

[0110] 1.1.1 Instrument: Hitachi 7020 automatic biochemical analyzer

[0111] 1.2 Reagent: Beijing Aibixin batch number: 20130831

[0112] 1.3 The performance index requirements of the kit:

[0113] 1.3.1 Measuring range: within 3~50μmol / L, r≥0.990.

[0114] 1.3.2 Precision: intra-assay precision (...

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Abstract

The invention relates to an improved homocysteine detection reagent and method. The method comprises the following steps: enabling homocysteine to perform repeated circular reaction with S-adenosylmethionine under the action of homocysteine methyltransferase and S-adenosylhomocysteine hydrolase to produce adenosine, and determining the concentration of homocysteine in a sample by detecting the production rate of adenosine. According to the improved homocysteine detection reagent and method disclosed by the invention, by adopting measures of regulating positions and pH values of components in a kit and adding a stabilizing agent, a method and kit which have high specificity, high sensitivity and strong stability and can be used for automatically detecting homocysteine with extremely low content in body fluid can be obtained.

Description

Technical field [0001] The invention relates to an improved reagent and method for detecting homocysteine, in particular to a method and reagent for measuring homocysteine ​​by using an enzyme-catalyzed reaction, an enzyme cycle amplification method, and an enzyme colorimetric method. Background technique [0002] Homocysteine ​​(Hcy) is a sulfhydryl amino acid, which is a metabolic intermediate product in the methionine cycle in the body. The pathological increase of the total Hcy concentration in the blood can lead to hyperhomocysteinemia. Hyperhomocysteinemia is closely related to the occurrence of various cardiovascular diseases such as atherosclerosis, hypertension, and myocardial infarction, which are the number one killer of humans. It is a new independent and important part of cardiovascular disease. Risk factors. The detection of homocysteine ​​has been regarded as an independent risk factor of clinical coronary heart disease, and its high pathologicalness is the chief...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/34C12Q1/32
Inventor 不公告发明人
Owner BEIJING AMBITION BIOTECH
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