Hcy detecting method and detecting kit

A homocysteine ​​and determination method technology, applied in the field of homocysteine ​​determination methods and determination kits, can solve time-consuming problems and achieve the effects of long storage time, good stability and low test cost

Active Publication Date: 2014-07-16
浙江夸克生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that most of the operations are still manual, which is time-consuming.

Method used

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  • Hcy detecting method and detecting kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 (double dose)

[0087] Prepare the homocysteine ​​assay kit according to the following ingredients and dosage:

[0088] Reagent 1:

[0089] Lactate dehydrogenase (LD) 35KU / L;

[0090] Serine (SER) 0.76 mmol / L;

[0091] NADH 0.47 mmol / l;

[0092] Reagent 2:

[0093] Cystathionine β-synthase (CBS) 20KU / L;

[0094] Cystathionine β-decomposing enzyme (CBL) 10KU / L.

[0095] The reagents 1 and 2 may also include a buffer composed of 12mmol / L pH8.3 Tris-HCL and KCL60mmol / L; a mixture of glycerol and mannitol, wherein glycerol:mannitol=15:1. The buffer and stabilizer can significantly improve the stability of the kit.

[0096] Detection method:

[0097] 1. Basic parameters

[0098] Method: rate method Sample / reagent: 1 / 17

[0099] Dominant wavelength: 340nm Reaction temperature: 37°C

[0100] Secondary wavelength: 405nm Response time: 10min

[0101] 2. Determination

[0102] Blank Sample Calibration

[0103] Normal s...

Embodiment 2

[0118] Example 2 (single dose)

[0119] Prepare the homocysteine ​​assay kit according to the following ingredients and dosage:

[0120] Lactate dehydrogenase (LD) 30.33KU / L;

[0121] Serine (SER) 0.66 mmol / L;

[0122] NADH 0.41 mmol / l;

[0123] Cystathionine β-synthase (CBS) 2.67KU / L;

[0124] Cystathionine β-decomposing enzyme (CBL) 1.33KU / L.

[0125] The reagent may also include a buffer composed of 12mmol / L pH8.3 Tris-HCL and KCL60mmol / L; a mixture of glycerol and mannitol, wherein glycerol:mannitol=15:1.

[0126] The detection method can be adjusted accordingly with reference to Example 1. for example:

[0127] Detection method:

[0128] 1. Basic parameters

[0129] Method: rate method Sample / reagent: 1 / 17

[0130] Dominant wavelength: 340nm Reaction temperature: 37°C

[0131]Secondary wavelength: 405nm Response time: 10min

[0132] 2. Determination

[0133] Blank Sample Calibration

[0134] Normal saline / H2O (μl) 16 0 0

...

Embodiment 3

[0147] Example 3 (three doses)

[0148] Prepare the homocysteine ​​assay kit according to the following ingredients and dosage:

[0149] Reagent 1: NADH 0.94mmol / l;

[0150] Reagent 2:

[0151] Lactate dehydrogenase (LD) 70KU / L;

[0152] Serine (SER) 1.52mmol / L;

[0153] Reagent 3:

[0154] Cystathionine β-synthase (CBS) 20KU / L;

[0155] Cystathionine β-decomposing enzyme (CBL) 10KU / L.

[0156] The reagents 1, 2 and 3 may also include a buffer composed of 12mmol / L pH8.3 Tris-HCL and KCL60mmol / L; a stabilizer sodium azide.

[0157] The detection method can be adjusted accordingly with reference to Example 1. for example:

[0158] Detection method:

[0159] 1. Basic parameters

[0160] Method: rate method Sample / reagent: 1 / 17

[0161] Dominant wavelength: 340nm Reaction temperature: 37°C

[0162] Secondary wavelength: 405nm Response time: 10min

[0163] 2. Determination

[0164] Blank Sample Calibration

[0165] Normal saline / H2O (μl) ...

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PUM

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Abstract

The invention provides an Hcy concentration detecting method. According to the principle, oxidized form Hcy is changed into free Hcy, the free Hcy reacts with serine under the catalysis of CBS to generate L-cystathionine, and then the L-cystathionine generates Hcy, pyruvic acid and NH3 under the catalysis of CBL. The pyruvic acid generated through circular reaction can be detected through LDH and NADH, the rate of the NADH which is changed into NAD is in direct proportion to the content of the Hcy in a sample, and the content of the Hcy can be detected by detecting the lowering rate of NADH+ at the position of 340 nm. The invention further provides a high-sensitivity Hcy concentration detecting kit.

Description

[0001] technical field [0002] The invention relates to the technical field of medical testing and determination, in particular to a homocysteine ​​determination method and a determination kit. [0003] Background technique [0004] At present, there are many methods for determining the concentration of homocysteine ​​in plasma, including circulating enzyme method, FPIA method, high performance liquid chromatography (HPLC), enzyme-linked immunoassay (EIA), etc. [0005] 1. Cyclic enzymatic method. Determination of serum Hcy can be used in automatic biochemical analyzers, the reaction principle is that oxidized Hcy is converted into free Hcy under the action of phosphine hydrogen chloride (TCEP), and the covalent substrate S-adenosylmethionine SAM catalyzes the reaction to form methionine with S-adenosylhomocysteine ​​SAH. SAH is hydrolyzed into adenosine and Hcy by SAH hydrolase, and the formed Hcy can be recycled into the reaction, thereby amplifying the detection sign...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78G01N21/31G01N31/22
Inventor 邢力策陈青松林耀文
Owner 浙江夸克生物科技有限公司
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