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Detection method of alkaline phosphatase

A phosphatase, alkaline technology, applied in the field of photochemistry, can solve the problem of not being able to monitor enzyme activity in real time, and achieve the effects of high sensitivity, high accuracy and low detection limit

Active Publication Date: 2016-11-02
江苏润熵生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some of these methods have high sensitivity, most of them cannot monitor enzyme activity in real time

Method used

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  • Detection method of alkaline phosphatase
  • Detection method of alkaline phosphatase
  • Detection method of alkaline phosphatase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] A method for detecting alkaline phosphatase, comprising the following steps:

[0028] Dissolve the purchased alkaline phosphatase ALP and ascorbic acid AAP with secondary water to prepare 0.1U / mL alkaline phosphatase ALP solution and 1mg / mL ascorbyl phosphate AAP solution respectively, and then prepare 100μg / mL CuSO 4 solution, 10ng / mL HRP and 8.3mM TMB-H 2 o 2 solution and stored at 4°C for later use.

[0029] a. Mix 10 μL 1 mg / mL ascorbyl phosphate AAP solution and 15 μL alkaline phosphatase ALP solution with a series of concentration gradients in 100 mL PBS buffer solution (pH=7.4), the concentration gradients are 0 mU / mL and 10 mU / mL respectively , 20mU / mL, 40mU / mL, 60mU / mL, 80mU / mL, 100mU / mL and 120mU / mL, then add 10μL, 100μg / mL of CuSO 4 Solution, react at room temperature for 40min;

[0030] b. Add 50 μL of 10 ng / mL HRP solution to the mixed solution obtained in the above step a, react at room temperature for 20 minutes, and finally add 100 μL of 8.3 mM TMB-H...

Embodiment 2

[0033] A kind of method that detects alkaline phosphatase, the pH of PBS buffer solution is respectively 5,6,7.4,8 and 9, other operation is identical with embodiment 1, and the result is as follows Figure 3A shown. Example 3

Embodiment 3

[0034] A method for detecting alkaline phosphatase, the reaction time in step a is 5min, 10min, 20min, 40min, 60min and 90min, other operations are the same as in Example 1, and the results are as follows Figure 3B shown.

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Abstract

The invention provides a detection method of alkaline phosphatase. The method uses alkaline phosphatase ALP for reduction of ascorbyl phosphate AAP to generate alkaline phosphatase; and then the ascorbic acid reduces Cu<2+> into Cu<+>. Cu<+> can inhibit the activity of horseradish peroxidase, so that TMB-H2O2 solution does not change color, and the detection of ALP is achieved. Compared with the prior art, the method uses colorimetric method for the quantitative determination of ALP, and has the advantages of high sensitivity, low detection limit and high accuracy.

Description

technical field [0001] The invention belongs to the technical field of photochemistry, in particular to a method for detecting alkaline phosphatase, constructing Cu 2+ and HRP-TMB-H 2 o 2 The colorimetric system realizes the sensitive detection of alkaline phosphatase. Background technique [0002] In recent years, human health has attracted more and more attention. As a typical phosphomonoesterase, alkaline phosphatase (ALP) is one of the important biomarkers of various human diseases. It can catalyze the hydrolysis of various And transphosphorylation, including some biological macromolecules and small molecules. Therefore, alkaline phosphatase plays a key role in cell cycle, growth, apoptosis signal transduction and intracellular regulatory processes. The abnormal level of ALP in the human body is a signal of many diseases, especially the ALP activity in liver and bone serum is often used as a diagnostic indicator of some diseases, including bone diseases (bone tumors,...

Claims

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Application Information

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IPC IPC(8): G01N21/78
CPCG01N21/78
Inventor 王广凤史东敏孙悦
Owner 江苏润熵生物科技有限公司
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