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Method for rapid determination of hyaluronidase activity

A hyaluronidase and rapid assay technology, applied in the direction of biochemical equipment and methods, microbial assay/inspection, etc., can solve the problems of insufficient response to hyaluronidase activity, high requirements for experimental conditions, and high requirements for conditions, and achieve Determination of good stability, high application value, and high sensitivity

Inactive Publication Date: 2016-04-27
GUANGZHOU MEDICAL UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] DorfmanA et al. used bovine testis to enzymatically hydrolyze hyaluronic acid, and reacted acidic protein with small molecule hyaluronic acid to generate turbidity. The absorbance was measured at 600nm. The acidic protein needs to be operated at 4°C. The experimental conditions are high and the repeatability is poor.
SternM et al. used ELISA to cover the hyaluronic acid in the wells of the plate for 10 hours, and then used hyaluronidase for enzymolysis to wash away the degraded hyaluronic acid, reacted with bovine serum albumin, and measured the absorbance at 492nm. This method takes a long time to react, requires high conditions, and is not enough to reflect the activity of hyaluronidase

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  • Method for rapid determination of hyaluronidase activity
  • Method for rapid determination of hyaluronidase activity
  • Method for rapid determination of hyaluronidase activity

Examples

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Embodiment 1

[0041] Embodiment 1: Microplate reader replaces ultraviolet spectrophotometer to make the standard curve of hyaluronic acid content

[0042] Food and Fermentation Industry. 2009,35(1) "Research on Rapid Detection Method of Hyaluronic Acid Content in Fermentation Broth [J]" disclosed CTAB method to measure hyaluronic acid content. Most of the detection instruments are ultraviolet spectrophotometers, but ultraviolet spectrophotometers The photometer cannot measure multiple samples at the same time, and requires a large sample volume. The microplate reader can measure multiple samples at the same time. It uses a disposable 96-well plate, which is very convenient to operate and can reduce the error of the cuvette.

[0043] Add 5-50 μl of hyaluronic acid (concentration 1 μg / μl) into 1.5ml EP tube with 200 μl of sodium acetate buffer solution, supplement 250ul with sodium acetate buffer solution, then add 500ul of CTAB, put it into a 37℃ water bath, and start timing from the additio...

Embodiment 2

[0044] Embodiment 2: The impact of ionic strength on CTAB nephelometry

[0045] Based on the 0.2M sodium acetate buffer solution, adjust the ionic strength with sodium chloride to 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9M under the condition of constant pH=6 The above-mentioned ionic strength is the ionic strength of sodium chloride), 10 sodium acetate buffer solutions with different ionic strengths and 50 μg of hyaluronic acid are mixed with it to form a 1 μg / μl hyaluronic acid solution, then add 500ulCTAB, put into 37 ℃ water bath, start timing from the addition, and the reaction time is 10 minutes; then accurately take 200 μl of the solution to a 96-well plate, and use a microplate reader to measure the OD value of the solution at a wavelength of 400 nm.

[0046] From figure 2 It can be seen that as the ionic strength increases, the OD value decreases; Table 1 shows that the ionic strength above 0.6M hardly produces turbidity compared with the control. From Figur...

Embodiment 3

[0053] Embodiment 3: pH value influences on CTAB turbidity method

[0054] Hyaluronidase activity pH is generally between 3-7, so based on 0.2M sodium acetate buffer, adjust the pH value with glacial acetic acid, prepare hyaluronic acid solutions with different pH values, the concentration is 1 μg / μl, the solution pH The values ​​are 3, 4, 5, 6, 7 respectively, and it is found that no turbidity is generated when pH=3 during the CTAB turbidimetry experiment. Take 50ul of hyaluronic acid solutions with different pH values ​​and add them into different 1.5ml EP tubes that have been marked. There are 200ul of sodium acetate buffer solutions with different pH values ​​in the tubes. Add 500 μl of CTAB solution, react in a 37°C water bath for 10 minutes, accurately take 200ul of the reaction solution and add it to a 96-well plate, and use a microplate reader to measure the OD value at a wavelength of 400nm

[0055] Figure 4 It can be seen that as the pH increases, the OD value als...

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Abstract

The present invention discloses a method for rapid determination of hyaluronidase activity, and the method comprises the following steps: (1) using a sodium acetate-acetic acid buffer solution for preparation of a hyaluronic acid solution with the pH value greater than 3 and less than or equal to 7; (2) preparing a cetyl trimethyl ammonium bromide solution; (3) taking the hyaluronic acid solution of the step (1), adding a hyaluronidase solution for reacting for 0.5 to 1 hour at 37 DEG C, and adding the cetyl trimethyl ammonium bromide solution for reacting at 37 DEG C for 8 to 15 minutes to obtain a sample solution; (4) preparing a control solution, to be more specific, taking the hyaluronic acid solution of the step (1), and adding the cetyl trimethyl ammonium bromide solution for reacting at 37 DEG C for 8 to 15 minutes to obtain a control solution; (5) using a microplate reader to determine optical density values of the sample solution and the control solution at a wavelength of 400nm; and (6) according to the obtained optical density values of the sample solution and the control solution, calculating hyaluronidase activity and specific activity. The method is simple to operate, good in stability and high in sensitivity and reproducibility, and lays a foundation for scale extraction, preparation and application of hyaluronidase.

Description

technical field [0001] The invention relates to a method for hyaluronidase activity, especially a method for quickly measuring hyaluronidase activity. Background technique [0002] There are three types of isozymes for hyaluronidase. The first type is the classic hyaluronidase (hyaluronidase EC3.2.1.35), which mostly exists in the testes of mammals, and the hyaluronidase in snake venom also belongs to this category. The second category is hyaluronidase EC3.2.1.36, which mostly exists in the salivary glands of leeches; the third category is hyaluronidase EC4.2.2.1, which mostly comes from microorganisms such as bacteria, fungi, and phages. The optimal pH value, isoelectric point and molecular weight of hyaluronidase from different species are different. The activity of hyaluronidase is divided into acid active (acidactive) and neutral active (neutralactive). Between 3 and 4, the latter has an optimum pH between 5 and 6. [0003] Hyaluronic acid, also known as hyaluronic aci...

Claims

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Application Information

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IPC IPC(8): C12Q1/34
CPCC12Q1/34
Inventor 彭享董伟华香咏欣王菡孔天翰
Owner GUANGZHOU MEDICAL UNIV
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