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Glutamate-pyruvate transaminase determination method and glutamate-pyruvate transaminase determination reagent kit

A technology of alanine aminotransferase and kit, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, measuring devices, etc. It can solve the problems of high false results, misdiagnosis and inaccuracy of clinical diseases, and achieve detection accuracy High, accurate reflection, the effect of eliminating interference

Active Publication Date: 2007-07-11
BEIJING BGI GBI BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is a common problem in this detection method: when the sample is not separated in time, the endogenous pyruvate produced by the glycolysis of red and white blood cells in the blood sample can also participate in the reaction, resulting in the failure of the one-step method to determine ALT activity. The result is falsely high, and the test result does not really reflect the active concentration of serum ALT
As a result, the accuracy of the serum ALT test item is low, which has a negative impact on the development of voluntary blood donation in my country, and may also lead to misdiagnosis of clinical diseases due to inaccurate ALT measurement results

Method used

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  • Glutamate-pyruvate transaminase determination method and glutamate-pyruvate transaminase determination reagent kit
  • Glutamate-pyruvate transaminase determination method and glutamate-pyruvate transaminase determination reagent kit
  • Glutamate-pyruvate transaminase determination method and glutamate-pyruvate transaminase determination reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, the composition of reagent 1 and reagent 2 of alanine aminotransferase assay kit are as follows:

[0041] The main components of reagent 1 are as follows:

[0042] Buffer 100mmol / L

[0043] Pyruvate oxidase 2KU / L

[0044] Catalase 2KU / L

[0045] Peroxidase 3KU / L

[0046] Stabilizer 1000mmol / L

[0047] The main components of reagent 2 are as follows:

[0048] Buffer 100mmol / L

[0049] Sodium azide 30mmol / L

[0050] L-alanine 1mol / L

[0051] α-ketoglutarate 30mmol / L

[0052] 4-Aminoantipyrine 20mmol / L

[0053] TBHBA 2mmol / L

Embodiment 2

[0054] Example 2. A two-step method for eliminating interference from endogenous pyruvate The method for measuring ALT activity is as follows:

[0055] 1. On the ELISA plate, there is one reagent blank well, two standard wells, and the rest are assay wells. Add 50ul of Reagent 1 to each well.

[0056] 2. Add 20ul of distilled water to the blank wells, add 20ul of calibration serum to the standard wells, add 20ul of serum to be tested to the measurement wells, mix well and incubate at 37°C for 10 minutes.

[0057] 3. Add 50ul of Reagent 2 to each well, mix well and incubate at 37°C for 10 minutes.

[0058] 4. Add 100ul of stop solution to each well, mix well and perform colorimetry on a microplate reader. The main wavelength is 492nm, the secondary wavelength is 620nm, input the position of the reagent blank well, standard well and measurement well, input the calibration serum ALT activity concentration, adjust to zero with the reagent blank well for detection, and calculate ...

Embodiment 3

[0060] Example 3. Interference of endogenous pyruvate on samples detected by one-step method:

[0061] When serum / plasma is not separated in time after blood sample collection, the glycolysis of red and white blood cells in the sample produces endogenous pyruvate. Concentration determination interferes. The specific test is as follows:

[0062] 1. On the ELISA plate, add samples respectively: 0.125mg / ml sodium pyruvate, 0.0625mg / ml sodium pyruvate, 0.0313mg / ml sodium pyruvate, 44.5U / LALT, 0.125mg / ml sodium pyruvate+ 44.5U / LALT, 0.0625mg / ml sodium pyruvate+44.5U / LALT, 0.0313mg / ml sodium pyruvate+44.5U / LALT.

[0063] 2. Mix reagent 1 and reagent 2 at a ratio of 1:1 to make a working solution.

[0064] 3. Add 100ul working solution to each well, mix with the sample and react at 37°C for 15 minutes.

[0065] 4. Add 100ul stop solution to each well, and compare the color on a microplate reader. The main wavelength is 492nm, the secondary wavelength is 620nm, and the OD value i...

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Abstract

The invention discloses a measuring method of Alanine Aminotransferase (ALT) and agent box in the external diagnostic technical domain, which comprises the following steps: reacting agent 1 and sample; generating hydrogen peroxide through reacting acetonic acid oxidase and endogenous acetonic acid; producing water and oxygen from hydrogen peroxide acted by catalase; reacting with agent 2; utilizing L-Ala and alpha-ketoglutaric acid starting serum ALT to react; detecting the activity of serum ALT of absorbance change of linear reaction.

Description

technical field [0001] The invention relates to a method for detecting the activity concentration of alanine aminotransferase in serum or plasma and a kit for measuring alanine aminotransferase, belonging to the technical field of in vitro diagnostic reagents. Background technique [0002] Elevated serum alanine aminotransferase (ALT) activity is the earliest abnormal indicator of viral hepatitis in the early stage, and is recommended by the World Health Organization as the most sensitive detection indicator of liver function damage. During disease screening and screening of blood donors, the detection of serum alanine aminotransferase activity is a must. [0003] At present, the detection of ALT enzyme activity is mainly divided into Rei's method, rate method and pyruvate oxidase method in terms of methodology. The pyruvate oxidase method appeared in China in the early 1990s. The principle is: ALT catalyzes the reaction of L-alanine and α-ketoglutaric acid to generate pyru...

Claims

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Application Information

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IPC IPC(8): C12Q1/52C12Q1/30G01N33/576
Inventor 余燕文洁尹烨
Owner BEIJING BGI GBI BIOTECH
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