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Method for measuring alkaline phosphatase activity

A technology of phosphatase activity and measurement method, which is applied in the field of determination of alkaline phosphatase activity based on colorimetric analysis and alkaline phosphatase activity, achieving the effects of short detection time, high degree of popularization of instruments and equipment, and good repeatability

Inactive Publication Date: 2016-01-20
NANJING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, to date, there has been no report of its application to the quantitative determination of biomolecules based on colorimetric assays

Method used

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  • Method for measuring alkaline phosphatase activity
  • Method for measuring alkaline phosphatase activity
  • Method for measuring alkaline phosphatase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Semi-quantitative colorimetric analysis of alkaline phosphatase activity based on the color change of the detection solution.

[0026] 20μL 140mU·mL -1 Alkaline phosphatase solution (replaced with an equal amount of ultrapure water in the control group) was added to 600 μL of 1 M solution containing 10 mM L-ascorbic acid-2-phosphate, 0.2 mM copper sulfate, 0.4 mM sodium saphenanthroline disulfonate and 1 mM magnesium chloride In the 2-(ethylamino)ethanol detection solution (pH9.8), the resulting mixture was incubated at 25°C for 60min, and then the color change of the solution was recorded by a digital camera, as shown in figure 2 shown. from figure 2 It can be seen that after 60 min, the solution in the control group was still bright yellow, while the solution added with alkaline phosphatase turned dark green. It can be seen that the semi-quantitative colorimetric analysis of the alkaline phosphatase activity in the sample can be carried out by observin...

Embodiment 2

[0027] Embodiment 2: Feasibility analysis of an assay method for alkaline phosphatase activity of the present invention.

[0028] 20μL 140mU·mL -1 Alkaline phosphatase solution was added to 600 μL of 1M 2-(ethylamino)ethanol detection solution (pH9. 8), use a UV-visible spectrophotometer to record the kinetic curve of the resulting mixed solution at 25°C at 426nm as a function of time, as shown in image 3 shown. from image 3 It can be seen that under the condition that all components exist in the detection solution (a), the absorption value of the detection solution at 426nm increases gradually with time; and when the detection solution does not add L-ascorbic acid-2-phosphate (b ), sodium bathophenanthroline disulfonate (c) or copper sulfate (d), its absorption value at 426nm does not change with time. It can be seen that the assay method for alkaline phosphatase activity of the present invention has quite good feasibility.

Embodiment 3

[0029] Example 3: The relationship between the ultraviolet-visible absorption spectrum of the detection solution and the activity of alkaline phosphatase.

[0030] 20 μL of 0, 20, 40, 60, 80, 100, 120 and 140 mU·mL -1 The alkaline phosphatase solution was added to 600 μL of 1M 2-(ethylamino)ethanol detection solution ( pH 9.8), the resulting mixture was incubated at 25°C for 60 min and then recorded with a UV-Vis spectrophotometer in its UV-Vis absorption spectrum in the range of 300-800 nm, as shown in Figure 4 shown. from Figure 4 (A) It can be seen that with the increase of alkaline phosphatase activity in the sample, the absorption value of the detection solution at 426nm increases correspondingly. from Figure 4 (B) It can be seen that by plotting the alkaline phosphatase activity in the sample with the absorption value of the detection solution at 426nm, a good quantitative relationship can be obtained, and its linear range is 0-140mU·mL -1 (R 2 =0.999), the detect...

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Abstract

The invention discloses a method for measuring alkaline phosphatase activity. According to the method, L-ascorbic acid-2-phosphate is taken as a catalytic substrate of alkaline phosphatase, the substrate is hydrolyzed to form L-ascorbic acid under the catalysis of alkaline phosphatase, L-ascorbic acid can reduce bivalent copper into monovalent copper, a bright yellow bivalent copper-bathophenanthroline compound is converted into deep green monovalent copper-bathophenanthroline compound, the higher the alkaline phosphatase activity is, the more obviously the color of a detection liquid changes, and accordingly, semiquantitative colorimetric detection of the alkaline phosphatase activity is realized or quantitative detection is realized through an ultraviolet-visible spectrophotometer. By means of the method, alkaline phosphatase with limit of detection being 3.05 mU*mL<-1> can be detected. Compared with conventional methods, the method has high sensitivity and anti-interference capability and good repeatability, and is simple to operate, short in detection time and low in cost, and a simple, convenient, low-cost, high-resolution and sensitive colorimetric analysis method with stable performance is provided for detection of the alkaline phosphatase activity in a clinic sample.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, in particular to a method for measuring alkaline phosphatase activity, in particular to a method for measuring alkaline phosphatase activity based on colorimetric analysis. Background technique [0002] Alkaline Phosphatase (Alkaline Phosphatase, ALP) is a non-specific phosphomonoesterase encoded by the phoA gene. Group transfer reactions. ALP widely exists in microorganisms and animals, can directly participate in phosphorus metabolism, and plays an important role in the digestion, absorption, secretion and ossification of calcium and phosphorus. Important indicators of diseases such as secondary liver cancer, seminoma, ovarian cancer, certain digestive system diseases, autoimmune diseases, malignant tumors, cholestatic hepatitis, osteomalacia and rickets. In the field of immunology, ALP-labeled antibodies can be used in ELISA and Western blot analysis, etc. Compared with horseradi...

Claims

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Application Information

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IPC IPC(8): G01N21/78
Inventor 孔金明胡琼何玟辉梅亚琦张学记
Owner NANJING UNIV OF SCI & TECH
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