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Method, reagent and kit for quantitative determination of mitochondrial AST activity in human serum

A technology for the quantitative determination of aspartic acid amino groups, which is applied in the field of quantitative determination of mitochondrial aspartate aminotransferase activity in human serum, reagents and kits, and can solve the problems of being unsuitable for routine testing, expensive, and time-consuming. problems, to achieve the effect of fully automated analysis and easy operation

Inactive Publication Date: 2015-08-26
ZHEJIANG LANSEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have the disadvantages of cumbersome operation, long time-consuming, expensive, excessive waste of resources, and are not suitable for routine testing, especially large-scale epidemiological examination or simultaneous detection of many items of clinical large-scale specimens

Method used

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  • Method, reagent and kit for quantitative determination of mitochondrial AST activity in human serum
  • Method, reagent and kit for quantitative determination of mitochondrial AST activity in human serum
  • Method, reagent and kit for quantitative determination of mitochondrial AST activity in human serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Prepare the following reagent I and reagent II of the present invention according to the following components and ratios:

[0024] Reagent I:

[0025]

[0026] Reagent II:

[0027] α-ketoglutarate 1mmol / L

[0028] Reducing coenzyme 0.05mmol / L

[0029] Mix 200μl reagent I and 20μl serum sample in the sample tube, incubate at 37°C for 5 minutes, use Hitachi 7060 automatic biochemical analyzer, adjust the blank tube to zero, then add 40μL reagent II to the sample, continue incubating 1-2 Minutes later, continuously monitor the absorbance change of the sample for 1-2 minutes at a wavelength of 340nm, and calculate the average absorbance change per minute of the sample △A sample / min; Use the same method to determine the average change in absorbance per minute of the blank tube △A blank / min; The mitochondrial aspartate aminotransferase activity of the serum sample is calculated by the following formula (1):

[0030] m-AST activity (U / L) = (△A sample / min-△A blank / min)×K (1)

[00...

Embodiment 2

[0034] Prepare the following reagent I and reagent II of the present invention according to the following components and ratios:

[0035] Reagent I:

[0036]

[0037] Reagent II:

[0038] α-ketoglutarate 50mmol / L

[0039] Reducing coenzyme 1mmol / L

[0040] Mix 200μl reagent I and 20μl serum sample in the sample tube, incubate at 37°C for 5 minutes, use Olympus 400 automatic biochemical analyzer, adjust the blank tube to zero, then add 40μL reagent II to the sample and continue incubating After 1-2 minutes, continuously monitor the absorbance change of the sample for 1-2 minutes at a wavelength of 340nm, and calculate the average absorbance change per minute of the sample △A sample / min; Use the same method to determine the average change in absorbance per minute of the blank tube △A blank / min; The mitochondrial aspartate aminotransferase activity of the serum sample is calculated by the following formula (1):

[0041] m-AST activity (U / L) = (△A sample / min-△A blank / min)×K (1)

[00...

Embodiment 3

[0045] Prepare the following reagent I and reagent II of the present invention according to the following components and ratios:

[0046] Reagent I:

[0047]

[0048] Reagent II:

[0049] α-ketoglutarate 10mmol / L

[0050] Reducing coenzyme 0.2mmol / L

[0051] Mix 200μl reagent I and 20μl serum sample in the sample tube, incubate at 37°C for 5 minutes, use Beckman LX20 automatic biochemical analyzer, adjust the blank tube to zero, then add 40μL reagent II to the sample, and continue to incubate 1- After 2 minutes, continuously monitor the absorbance change of the sample for 1-2 minutes at a wavelength of 340nm, and calculate the average absorbance change per minute of the sample △A sample / min; Use the same method to determine the average change in absorbance per minute of the blank tube △A blank / min; The mitochondrial aspartate aminotransferase activity of the serum sample is calculated by the following formula (1):

[0052] m-AST activity (U / L) = (△A sample / min-△A blank / min)×K (1...

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Abstract

The invention relates to a reagent for the quantitative determination of mitochondrial AST (Aspartate Aminotransferase) activity in human serum. The reagent comprises a reagent I and a reagent II which are placed separately, wherein the reagent I contains Tris buffer, L-aspartic acid, malate dehydrogenase, lactate dehydrogenase, a c-AST goat anti-human antibody and an enzyme stabilizer; the reagent II contains alpha-ketoglutaric acid and reductive coenzyme. A kit and a detection method adopted in the invention only require tens of microlitres of serum, need no centrifugation or electrophoresis and other separation treatments, are easy to operate, can meet the requirements of automatic analysis, and are applicable to the timely and accurate detection of large-scale samples.

Description

Technical field [0001] The application relates to a quantitative determination method, reagent and kit for the activity of mitochondrial aspartate aminotransferase in human serum. Background technique [0002] Aspartate aminotransferase (AST) is widely present in various tissues and cells of the human body, especially in liver and heart tissue cells, while the activity of AST in normal human serum is very low. There are two isoenzymes of AST in the human body, one is cAST, which is mainly derived from cytoplasm, and the other is mAST, which exists in mitochondria. These two isoenzymes are different in gene composition, in terms of amino acid composition, immunological properties and half-life in vivo. [0003] When the body is mildly diseased, the cell membrane permeability of tissue cells increases. CAST penetrates the cell membrane and enters the blood system. However, mAST is protected by two layers of mitochondrial membranes and is not easily released into the blood. When the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33
Inventor 张益军
Owner ZHEJIANG LANSEN BIOTECH
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