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Kit for simple and rapid detection of glycosaminoglycans and its detection method

A technology of glycosaminoglycans and kits, which is applied in the field of detection kits, can solve the problems of cumbersome detection steps, increase the cost of the experiment, increase the test time, etc., and achieve the effects of simplified operation steps, low cost, and simple preparation

Active Publication Date: 2018-06-19
SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Most of the glycosaminoglycans in the market currently use the Elisa method. This method greatly increases the cost of the experiment and increases the test time. At the same time, the Elisa kit has species selectivity, and the detection steps are relatively cumbersome.

Method used

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  • Kit for simple and rapid detection of glycosaminoglycans and its detection method
  • Kit for simple and rapid detection of glycosaminoglycans and its detection method
  • Kit for simple and rapid detection of glycosaminoglycans and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 preparation kit

[0038] Reagents required:

[0039] Formic acid buffer solution: 4g sodium formate, 4ml formic acid, make up to 1L with deionized water, store in closed storage, room temperature can be stored for 1 year. 10×DMB reagent (1,9-Dimethylmethylene blue, 1,9-dimethylmethylene blue): Weigh 80mg of DMB powder (commercially available, purchased from sigma aldrich), add 12ml of ethanol, after completely dissolved, add 488ml of formic acid Buffer, protected from light and stored at room temperature, at least 1 year.

[0040] Chondroitin sulfate standard product chondroitin sulfate (CS): Weigh 1 g of chondroitin sulfate, dissolve it in 100 ml PBS, and add 0.005% sodium azide at the same time to obtain a 10 mg / ml stock solution. When making a standard curve, dilute to 1mg / ml.

[0041] Lysis buffer: After preparing PBS and adjusting the pH value to 6.4, add 400 mg sodium acetate (S2889, Sigma Aldrich), 200 mg EDTA disodium salt (E5134, Sigma Aldrich) ...

Embodiment 2

[0043] Embodiment 2 detection method

[0044] 1. Detection steps

[0045] 1. Quantify the tissue samples first: count cells (take 100,000 cells as an example) or weigh the tissue (10 mg), take the same amount for each group, add 100 μl of lysate to the cells (500 μl to the tissue) for lysis, and try to keep it on ice Perform sonication. After ultrasonication, perform high-speed centrifugation at 12000g for 10-15min to fully precipitate impurities such as cell debris, and the supernatant contains the GAG ​​to be tested.

[0046] 2. Aspirate 100μl of the supernatant, add 1ml of DMB dye, and shake at room temperature to fully combine GAG ​​and DMB. Centrifuge at a high speed of 12000g for 30min to precipitate the combination of GAG and DMB, add dissociation solution, and place at room temperature for about 10min to dissociate DMB from GAG.

[0047] 3. Preparation of standard curve: Take 5ug, 4ug, 3ug, 2ug and 1ug of chondroitin sulfate standards respectively, add 1ml of DMB reag...

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Abstract

The invention discloses a kit for simple and rapid detection of glycosaminoglycans, which comprises the following components: DMB reagent; chondroitin sulfate standard product; lysate; dissociation solution. In addition, the invention also discloses a detection method of the kit. The kit of the present invention directly combines the DMB dye (reagent) with glycosaminoglycan (GAG). After the dye is dissociated, it detects the absorbance value at a specific wavelength of the dissociated dye of the sample to be tested and the standard product, and directly quantitatively detects the glycosaminoglycan. polysaccharide content. The kit of the invention greatly reduces the experimental cost and shortens the detection time, and at the same time has no selectivity for the species of samples such as tissue cells, and is easy to operate. It can detect the level of glycosaminoglycans in various human or animal tissues (skin, muscle, etc.), cartilage, connective tissue, tumor tissue, body fluid, cell culture supernatant and other samples.

Description

technical field [0001] The invention relates to a detection kit, in particular to a kit for detecting glycosaminoglycans, in particular to a kit for simple and rapid detection of glycosaminoglycans in tissue cells and body fluids. In addition, the present invention also relates to a detection method of the kit. Background technique [0002] Glycosaminoglycans (GAGs) are mainly divided into two types: sulfated and non-sulfated. The former category includes chondroitin sulfate, keratan sulfate, and heparan sulfate, etc., with small molecules; the latter category is hyaluronic acid, which is a long chain of tortuous coils. Large molecules, up to 2.5 μm in length. Small molecule glycosaminoglycans are like bristles on a test tube brush, binding to the core protein, and radially arranged around the core protein to form proteoglycan subunits. The latter binds to the backbone of hyaluronic acid through binding proteins to form proteoglycan polymers. [0003] A large number of p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31G01N1/28
Inventor 丁道芳杜国庆宋奕欧阳霄雯杨晓玲李玲慧曹月龙詹红生
Owner SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M
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