CN117664885B - Quantitative detection method for content of hyaluronic acid in fabric - Google Patents
Quantitative detection method for content of hyaluronic acid in fabric Download PDFInfo
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 140
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- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- General Physics & Mathematics (AREA)
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- Analytical Chemistry (AREA)
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- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biotechnology (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a quantitative detection method for the content of hyaluronic acid in a fabric, which comprises the steps of firstly combining CD44 protein with the hyaluronic acid on the fabric, quantifying the rest free unbound CD44 protein by adopting a double antibody sandwich principle of an enzyme-linked immunosorbent assay, and finally catalyzing a substrate TMB to develop color under the combined action of hydrogen peroxide by the obtained compound, and obtaining the amount of horseradish peroxidase according to the absorbance value of a developing solution at 450nm, thereby obtaining the amount of the hyaluronic acid on the fabric. The detection method disclosed by the invention is based on the competitive binding principle and the double-antibody sandwich method principle, can be used for quantitatively detecting hyaluronic acid which is difficult to elute in a complex matrix, especially in a fabric, and effectively improves the accuracy of quantitative detection of hyaluronic acid.
Description
Technical Field
The invention relates to the technical field of hyaluronic acid detection in complex matrixes, in particular to a method for detecting the content of hyaluronic acid in fabrics.
Background
Hyaluronic acid (i.e. hyaluronic acid, HA) is a straight-chain high-molecular glycosaminoglycan formed by alternately connecting D-glucuronic acid and N-acetylglucosamine disaccharide units, the molecular weight can reach 5-20000 kDa, and the hyaluronic acid is an important structural substance in cartilage, synovial fluid, gingiva, skin and vitreous humor of vertebrates, and the hyaluronic acid maintains extracellular space of the parts in vivo, HAs the functions of water retention and lubrication, can regulate cell osmotic pressure, and promotes cell repair.
Hyaluronic acid is a biochemical drug with high value in clinic, can be used as a human body structural substance in various operations, such as glaucoma, crystal implantation, cornea transplantation and the like in ophthalmic operations, can be used as an arthritis treatment drug, and can be used as a humectant in cosmetics.
The hyaluronic acid has excellent water retention performance, hydroxyl groups and other polar molecular groups in the hyaluronic acid structure and H 2 O are combined together due to the formation of hydrogen bonds, more than 400 times of water can be combined, the high-concentration hyaluronic acid can be crosslinked into a film-shaped structure and has certain viscoelasticity, the aging of skin is related to the loss of collagen, and the loss of collagen is caused by the reduction of the content of the hyaluronic acid, so the hyaluronic acid is often used as a water supplementing agent for the skin in cosmetics.
Common hyaluronic acid detection ranges include fermentation broth stock solutions for producing hyaluronic acid, human biological samples (such as blood) in which hyaluronic acid is present, cosmetic or pharmaceutical raw materials, and finished products such as textiles containing hyaluronic acid. The samples are various in sources and complex in components, and hyaluronic acid is used as a polysaccharide polymer, the molecular weight is different, and especially the hyaluronic acid in the fabric can be combined with the fabric fiber, elution is difficult, and the factors increase the difficulty in quantifying the hyaluronic acid.
At present, common detection methods of hyaluronic acid include: HPLC, colorimetry, carbazole chromogenic method and CTAB turbidimetry. These methods all require the breakdown of hyaluronic acid into monomers for initial quantification.
The HPLC method is to detect the hyaluronic acid which is pretreated by the sample by utilizing the condition of a specific chromatographic column, and common methods include the steps of acid hydrolysis or specific biological enzymolysis, pre-column derivatization and the like of the hyaluronic acid, so that the hyaluronic acid is quantified by using chromatographic peak area after being decomposed into micromolecular substances with ultraviolet absorption, or the hyaluronic acid is quantified by gradient elution by adopting a gel chromatographic column method. The detection conditions for detecting the hyaluronic acid by using the HPLC are harsh, a chromatographic column is needed, the cost is high, the requirements on operators are high, and the method is only suitable for detecting samples with single components.
The colorimetric method for detecting the hyaluronic acid is a detection method based on aminosugars, the hyaluronic acid is disaccharide unit glycosaminoglycan composed of two minimum structural units of D-glucuronic acid and N-acetylglucosamine, the aminosugars form chromophores under alkaline conditions, the chromophores react with Eulich's reagent (N, N-DIMETHYL P-aminobenzaldehyde), namely acid p-dimethylaminobenzaldehyde to generate a mauve compound, the absorbance value can be detected at the wavelength of 530nm, so that the hyaluronic acid is quantified, the interference of other impurities such as aminosugars and the like is difficult to be removed, and the toxic reagent is used, so that the experimental operation is dangerous.
The carbazole chromogenic method is to take borax as a catalyst, decompose hyaluronic acid into glucuronic acid monomers by concentrated sulfuric acid, then adopt carbazole and glucuronic acid to form a purple complex, and indirectly calculate the content of hyaluronic acid according to the absorbance value of the complex and the concentration scale of glucuronic acid.
The CTAB turbidity method is characterized in that a cationic surfactant such as Cetyl Trimethyl Ammonium Bromide (CTAB) and hyaluronic acid are adopted to carry out a complexation reaction, the complex enables a solution to be turbid and to follow the lambert-beer law in a certain concentration range, and therefore the content of the hyaluronic acid can be quantitatively measured by an ultraviolet spectrophotometer. The method is less influenced by impurities in the sample, but is more influenced by the buffer solution of the reaction, and the pH value of the system has more influence on the quantitative determination result.
At present, the main problems existing in the quantitative detection process of hyaluronic acid are as follows:
(1) Hyaluronic acid is polysaccharide, has large molecular weight, and some detection methods such as an HPLC method and a carbazole method need complex pretreatment process, so that the operation difficulty and cost are increased, and the practicability of the method is reduced;
(2) The specific requirements of impurities in the complex matrix on the detection method are high, and the detection results of methods such as a turbidimetry method, a colorimetry method and the like cannot well exclude the interference of impurities with similar structures;
(3) Some samples such as hyaluronic acid on fabrics are difficult to elute, so that the content of hyaluronic acid in an eluent is low, the detection limit of the detection requirement method is low, and the elutable hyaluronic acid cannot be accurately quantified by a conventional method, so that a detection result has a large error.
Such as: CN106501503a discloses a Hyaluronic Acid (HA) test kit and a preparation method thereof, comprising: magnetic separation reagent, nanometer magnetic microsphere of labeled HABP protein, concentration is 167 mug/mL; reagent R1, containing alkaline phosphatase-labeled aggrecan protein, wherein the concentration of the alkaline phosphatase-labeled aggrecan protein is 1 mug/mL; reagent R2, buffer containing bovine gamma globulin component; standard, quality control and calibrator for HA antigen containing bovine serum albumin component five (BSAV) solution; washing the concentrated solution, and a buffer solution containing Tween-20 and Proclin-300; a diluent comprising a solution of bovine serum albumin component five (BSAV); a luminescent substrate, derivative IUMIPHOS of adamantane 530, the concentration of derivative IUMIPHOS of adamantane 530 being 10 μg/mL.
CN112595845A discloses a hyaluronic acid detection kit and a detection system, the hyaluronic acid detection kit comprises a first reagent, a second reagent, a third reagent and a fourth reagent, the first reagent comprises a hyaluronic acid conjugate marked by a first marker, the hyaluronic acid conjugate comprises gelatin and hyaluronic acid, the average molecular weight of gelatin in the hyaluronic acid conjugate is 50 KD-100 KD, the second reagent comprises hyaluronic acid binding protein, the third reagent comprises an antibody of hyaluronic acid binding protein marked by a second marker, and the fourth reagent comprises magnetic particles marked by a third marker.
The detection method adopted in the above documents is mainly suitable for quantitative detection of hyaluronic acid in-vitro diagnosis (such as serum, blood and the like), and cannot be suitable for quantitative detection of hyaluronic acid on fabrics. Because fabrics as solid fiber materials differ greatly from other matrix samples (e.g., blood, etc.): (1) The hyaluronic acid serving as a long-chain high molecular compound is difficult to completely elute by ultrasonic with an aqueous solution on a fabric, and residues on the fabric cannot be determined, so that the hyaluronic acid in the aqueous solution cannot be directly and accurately quantitatively detected by a conventional method or a kit in the above documents; (2) Fabric fibers are prone to cause nonspecific adsorption, and the conventional methods or the kit in the above documents can cause large errors in the direct coupling of antibodies to hyaluronic acid on the fabric and the re-quantification.
Therefore, based on the advantages and disadvantages of the existing methods for quantifying hyaluronic acid, there is a need in the art to develop a quantitative detection method for the content of hyaluronic acid in complex matrices, especially in textiles.
Disclosure of Invention
The invention aims to provide a quantitative detection method for the content of hyaluronic acid in a fabric.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
A quantitative detection method for the content of hyaluronic acid in a fabric comprises the following steps:
(1) Pretreatment of
Cutting a hyaluronic acid fabric sample, adding an aqueous solution, and performing ultrasonic treatment to obtain a working solution 1; the working solution 1 comprises hyaluronic acid solution 1 eluted from the fabric sample and hyaluronic acid fabric sample 2 which is not completely eluted;
(2) Coating
The working reagent 1 is diluted by a sodium Carbonate Buffer Solution (CBS) special for an enzyme-linked immunosorbent assay, then added into a 96-well plate, and incubated overnight with a film, and the diluted working reagent 1 is coated on the bottom of the plate. Removing the working reagent 1 in the pore plate after overnight, washing 3 times by using a washing liquid, and adding a sealing liquid to seal for 1 hour;
The working reagent 1 is a CD44 protein capture antibody; the blocking solution is a 20mM Tris,150mM NaCl,pH 7.4 buffer solution containing 2% BSA and 0.05% Tween 20; the washing solution is a buffer solution containing 20mM Tris,150mM NaCl,pH 7.4 percent of Tween 20;
(3) Bonding of
Mixing and incubating the working reagent 2 and the working solution 1 for 1 hour to obtain the working solution 2;
Wherein the working reagent 2 is a solution containing CD44 protein; the working solution 2 is a mixed solution of hyaluronic acid and a CD44 protein conjugate and free excessive CD44 protein;
(4) Capturing
Adding the working solution 2 into a 96-well plate coated with the working reagent 1 for incubation for 2 hours;
(5) Marking
Removing the working solution 2 from the pore plate and washing the pore plate with a washing solution for 3 times, adding the working reagent 3 into the 96 pore plate, and mixing and incubating the working solution 2 and the working reagent 3 for 1 hour to obtain the working solution 3;
wherein the working reagent 3 is an anti-CD 44 antibody with horseradish peroxidase label, and the working solution 3 comprises a free horseradish peroxidase labeled CD44 secondary antibody;
(6) Color development
Removing the working solution 3 from a 96-well plate, washing the solution for 3 times, adding the working reagent 4 into the 96-well plate, enabling the HRP contained in the working solution 3 to enable the 3,3', 5' -tetramethylbenzidine to be blue under the action of hydrogen peroxide, and adding a reaction stopping solution to obtain the working solution 4, wherein the solution is yellow;
Wherein the working reagent 4 is a solution containing 3,3', 5' -tetramethyl benzidine substrate and hydrogen peroxide; the working solution 4 is a solution of the working solution 3 catalyzed by the working reagent 4; the reaction stopping solution is 2N sulfuric acid;
(7) Detection of
And (3) placing the 96-well plate filled with the working solution 4 in an enzyme-labeled instrument to detect absorbance, and calculating the relation between the absorbance and the content of hyaluronic acid in the fabric.
Further, the working reagent 1 is a CD44 protein capture antibody solution with the concentration of 0.2mg/mL, and the sodium Carbonate Buffer (CBS) is 0.05M Na 2CO3,0.05M NaHCO3, and a buffer solution filtered by a pH 9.6,0.2 μm filter membrane; the working reagent 1 is diluted by 100 times by CBS buffer solution;
Further, the working reagent 2 is a CD44 protein standard solution provided with a series of concentration gradients, and the concentration range of the working reagent is 0 ng/mL-50 ng/mL; when preparing the CD44 protein standard solution, the CD44 protein mother solution needs to be diluted by a 20mM Tris,150mM NaCl,pH 7.4 buffer solution containing 0.1% BSA and 0.05% Tween 20;
Further, the working reagent 3 is an anti-CD 44 antibody with horseradish peroxidase mark at a concentration of 0.25 mu g/mL, and the working reagent 3 is a high concentration mother solution of 0.25mg/mL diluted 1000 times by a buffer solution containing 20mM Tris,150mM NaCl,pH 7.4% of BSA and 0.05% of Tween 20.
Wherein, the working solution 4 is placed in an enzyme label instrument, and the absorbance value of the solution at 450nm is detected.
And (3) the absorbance value of the working solution 4 and the fabric standard containing different concentrations of hyaluronic acid are subjected to corresponding standard curves, so that the concentration of the hyaluronic acid in the fabric is calculated.
Bringing the absorbance value a into a fitting equation C x＝0.4322C0-3.3117Ax +0.0176 derived from the relationship between absorbance values and fabrics containing different hyaluronic acid concentrations,
Wherein Cx is the content of hyaluronic acid (w/w) on the fabric, in ng/g;
C 0 is the specific concentration of CD44 protein prior to initial mixing with hyaluronic acid;
A x is the absorbance value of the color development liquid.
The coefficients in the formula are calculated according to the concentration C a of the hyaluronic acid on the fabric, the content C m of the CD44 protein and the absorbance value A a, which are obtained by fitting the standard curve, and are calculated according to the stoichiometric ratio relation between the hyaluronic acid and the CD44 protein.
Wherein, the stoichiometric ratio of the mass concentration of the CD44 protein to the hyaluronic acid is 55-66 mol/L:1mol/L.
The detection method of the invention has the working principle that:
Firstly, ultrasonically oscillating the fabric by using an aqueous solution so that part of hyaluronic acid on the fabric is dissolved in water to obtain an extracting solution; then adding a CD44 protein solution capable of specifically binding with hyaluronic acid to respectively bind with the eluted hyaluronic acid and the unextracted hyaluronic acid in the extracting solution; then taking out the fabric and washing the fabric for 3 times, and combining the washing solutions; adding the washing solution into an ELISA plate coated with a CD44 protein capture antibody, so that redundant free CD44 protein which is not combined with hyaluronic acid is captured by the CD44 protein capture antibody, and a CD44 protein compound combined with hyaluronic acid cannot be captured due to the fact that the molecular weight is large and long chains are intertwined, thereby achieving the purpose of detecting only the free CD44 protein; labeling the captured free CD44 protein by using a horseradish peroxidase-coupled CD44 protein secondary antibody, and then washing for 3 times to remove the excessive horseradish peroxidase-coupled CD44 protein secondary antibody in the solution; and finally, obtaining the content of free CD44 protein according to the absorbance value, and calculating the initial concentration of the CD44 protein in the washing liquid after the known ultrasonic vibration of the fabric, thereby indirectly obtaining the content of hyaluronic acid in the fabric.
Compared with the prior art, the invention has the outstanding effects that:
(1) The detection method adopts indirect quantification for quantitative detection of the hyaluronic acid on the special material of the fabric, utilizes a competitive binding method, introduces a proper amount of CD44 protein capable of being specifically bound with the hyaluronic acid into an ultrasonic fabric sample, and detects the content of the CD44 protein which cannot be bound with the hyaluronic acid on the fabric so as to indirectly obtain the content of the hyaluronic acid on the fabric.
(2) The invention can indirectly quantify the hyaluronic acid which is difficult to elute or extract in the sample based on the principle of the enzyme-linked immunosorbent assay, thereby effectively improving the accuracy of quantitative determination of the hyaluronic acid.
The quantitative detection method of the hyaluronic acid content in the fabric according to the invention is further described below with reference to the accompanying drawings and specific examples.
Drawings
FIG. 1 is a standard curve obtained by fitting the concentration of CD44 protein on the x-axis and the absorbance value A on the y-axis.
FIG. 2 is a graph of simulated results of hyaluronic acid at various concentration gradients versus absorbance measurements.
Detailed Description
Example 1 search for the binding ratio of hyaluronic acid to CD44 protein and Standard Curve preparation
Diluting CD44 protein to prepare a series of concentration gradient solutions, wherein the concentration gradient is as follows: 0,0.125,0.25,0.5,1,2,4,8ng/mL, and making a standard curve of absorbance-CD 44 protein concentration according to absorbance values to obtain a standard curve equation: a= 0.30196C 0+0.00355,C0 units are ng/mL; see fig. 1.
Preparing a series of concentration gradient solutions by using a hyaluronic acid calibrator mother solution to prepare a standard curve and obtaining a fitting equation of the relationship between the concentration and the absorbance of the hyaluronic acid, wherein the fitting equation C x＝0.4322C0-3.3117Ax +0.0176,
Wherein Cx is the content of hyaluronic acid (w/w) on the fabric, in ng/g;
C 0 is the specific concentration of CD44 protein prior to initial mixing with hyaluronic acid;
A x is the absorbance value of the color development liquid.
The coefficients in the formula are calculated according to the concentration C a of the hyaluronic acid on the fabric, the content C m of the CD44 protein and the absorbance value A a, which are obtained by fitting the standard curve, and are calculated according to the stoichiometric ratio relation between the hyaluronic acid and the CD44 protein.
The fitting equation is derived by the steps of:
Configuration of the calibrator: preparing a series of concentration gradient solutions of 12.5ng/mL,10ng/mL,7.5ng/mL,5ng/mL,2.5ng/mL and 0ng/mL of hyaluronic acid standard;
Combining: mixing 50 mu L of serial concentration gradient hyaluronic acid with 50 mu L of CD44 protein with concentration of 35ng/mL by shaking vortex for half an hour, adding into a 96-well plate coated with CD44 binding protein, incubating for 2 hours, and removing hyaluronic acid-CD 44 protein conjugate in the 96-well plate;
Capturing: adding the working solution 2 into a 96-well plate coated with the working reagent 1 for incubation for 2 hours; wherein, the working reagent 1 is a CD44 protein capture antibody solution with the concentration of 0.2 mg/mL; the working reagent 1 is diluted by 100 times by CBS buffer solution; the working solution 2 is a mixed solution of hyaluronic acid and a CD44 protein conjugate and free excessive CD44 protein;
marking: removing the working solution 2 from the pore plate and washing with 300 mu L of washing solution for 3 times, adding 100 mu L of working reagent 3 into the 96 pore plate, and mixing and incubating the working solution 2 and the working reagent 3 for 1 hour to obtain a working solution 3; wherein the working reagent 3 is an anti-CD 44 antibody with horseradish peroxidase label, and the working solution 3 comprises a free horseradish peroxidase labeled CD44 secondary antibody;
Color development: removing the working solution 3 from the 96-well plate, washing with 300 mu L of washing solution for 3 times, adding 200 mu L of working reagent 4 into the 96-well plate, enabling the HRP contained in the working solution 3 to enable 3,3', 5' -tetramethylbenzidine to be blue under the action of hydrogen peroxide, and adding 50 mu L of reaction stopping solution after 20 minutes to obtain the working solution 4, wherein the solution is yellow;
Wherein the working reagent 4 is a solution containing 3,3', 5' -tetramethyl benzidine substrate and hydrogen peroxide; the working solution 4 is a solution of the working solution 3 catalyzed by the working reagent 4; the reaction stopping solution is 2N sulfuric acid;
and (3) detection: and (3) placing the 96-well plate filled with the working solution 4 in an enzyme-labeled instrument to detect absorbance, and calculating the relation between the absorbance and the content of hyaluronic acid in the fabric.
The following data were obtained by measuring the addition recovery rate by adding different concentrations of hyaluronic acid calibrator mother liquor to a blank fabric sample containing no hyaluronic acid. Wherein, the recovery rate represents the difference of the detection result compared with the concentration of the sample of hyaluronic acid actually added, and the accuracy of the detection can be reflected.
| Hyaluronic acid calibrator added concentration (ng/mL) | Recovery (%) | RSD |
| 12.5 | 82.3-90.1 | 4.3 |
| 10 | 81.2-89.0 | 5.6 |
| 7.5 | 79.0-86.5 | 5.9 |
| 5 | 73.2-84.7 | 6.7 |
| 2.5 | 65.3-75.2 | 8.5 |
Example 2 quantitative determination of the content of hyaluronic acid in fabrics
The quantitative detection method for the content of the hyaluronic acid in the fabric adopts the detection method of the embodiment 1, and specifically comprises the following steps:
(1) Pretreatment of
Cutting 0.1g of fabric sample (10 mu g of hyaluronic acid is known to be contained in each mg of fabric), adding 50mL of deionized water, and carrying out ultrasonic treatment to obtain a working solution 1; the working solution 1 contains hyaluronic acid solution eluted from the fabric sample and hyaluronic acid fabric sample 2 which is not completely eluted;
(2) Coating
Diluting 0.2mg/mL of working reagent 1 mother solution by using sodium Carbonate Buffer (CBS) special for an enzyme-linked immunosorbent assay for 100 times, adding into a 96-well plate, adding 100 mu L of each well, adding a film, incubating overnight, and coating the diluted working reagent 1 on the bottom of the plate; removing the working reagent 1 in the pore plate after overnight, washing 3 times by using a washing liquid, and adding a sealing liquid to seal for 1 hour;
The working reagent 1 is a CD44 protein capture anti-solution with the concentration of 0.2mg/mL, and the sodium Carbonate Buffer (CBS) is a buffer solution filtered by a filter membrane with the pH of 9.6,0.2 μm and the concentration of 0.05M Na 2CO3,0.05M NaHCO3; the working reagent 1 is diluted by 100 times by CBS buffer solution; the blocking solution is a 20mM Tris,150mMNaCl,pH 7.4 buffer solution containing 2% BSA and 0.05% Tween 20; the washing solution is a buffer solution containing 20mM Tris,150mM NaCl,pH 7.4 percent of Tween 20;
(3) Bonding of
Mixing and incubating 50 mu L of working reagent 2 with 50 mu L of working solution 1 with the concentration of 50ng/mL for 1 hour to obtain working solution 2;
wherein the working reagent 2 is a CD44 protein standard solution with the concentration of 50 ng/mL; when preparing the CD44 protein standard solution, the CD44 protein mother solution needs to be diluted by a 20mM Tris,150mM NaCl,pH7.4 buffer solution containing 0.1% BSA and 0.05% Tween 20; the working solution 2 is a mixed solution of hyaluronic acid and a CD44 protein conjugate and free excessive CD44 protein;
(4) Capturing
Adding the working solution 2 into a 96-well plate coated with the working reagent 1 for incubation for 2 hours;
(5) Marking
Removing the working solution 2 from the pore plate and washing with 300 mu L of washing solution for 3 times, adding 100 mu L of working reagent 3 into the 96 pore plate, and mixing and incubating the working solution 2 and the working reagent 3 for 1 hour to obtain a working solution 3;
Wherein the working reagent 3 is an anti-CD 44 antibody with horseradish peroxidase mark and the concentration of the working reagent 3 is 0.25 mu g/mL, and the working reagent 3 is formed by diluting a high-concentration mother solution of 0.25mg/mL by 1000 times of a buffer solution containing 20mM Tris,150mM NaCl,pH 7.4% of BSA and 0.05% of Tween 20; the working solution 3 comprises a free horseradish peroxidase-labeled CD44 secondary antibody;
(6) Color development
Removing the working solution 3 from the 96-well plate, washing with 300 mu L of washing solution for 3 times, adding 200 mu L of working reagent 4 into the 96-well plate, enabling the HRP contained in the working solution 3 to enable 3,3', 5' -tetramethylbenzidine to be blue under the action of hydrogen peroxide, and adding 50 mu L of reaction stopping solution after 20 minutes to obtain the working solution 4, wherein the solution is yellow;
Wherein the working reagent 4 is a solution containing 3,3', 5' -tetramethyl benzidine substrate and hydrogen peroxide; the working solution 4 is a solution of the working solution 3 catalyzed by the working reagent 4; the reaction stopping solution is 2N sulfuric acid;
(7) Detection of
And (3) placing the 96-well plate filled with the working solution 4 in an enzyme-labeled instrument to detect absorbance, and calculating the relation between the absorbance and the content of hyaluronic acid in the fabric.
Measuring the absorbance value of the heavy suspension at 450nm by using an enzyme-labeled instrument; substituting the absorbance value into a fitting equation C x＝0.4322C0-3.3117Ax +0.0176 obtained by fitting according to the relation between the absorbance value and the concentration of the hyaluronic acid calibrator,
Wherein Cx is the content of hyaluronic acid (w/w) on the fabric, in ng/g;
C 0 is the specific concentration of CD44 protein prior to initial mixing with hyaluronic acid;
A x is the absorbance value of the color development liquid.
The calculated amount of hyaluronic acid in the fabric was 8 μg hyaluronic acid per mg of fabric, and the recovery rate was 80% compared to the result of the sample having a known amount of 10 μg.
In conclusion, the detection method can indirectly quantify the hyaluronic acid which is difficult to elute or extract in the sample, and effectively improves the accuracy of quantitative determination of the hyaluronic acid.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (8)
1. The quantitative detection method for the content of the hyaluronic acid in the fabric is characterized by comprising the following steps of:
(1) Pretreatment of
Cutting a hyaluronic acid fabric sample, adding an aqueous solution, and performing ultrasonic treatment to obtain a working solution 1; the working solution 1 contains hyaluronic acid solution 1 eluted from the fabric sample and hyaluronic acid fabric sample 2 which is not completely eluted;
(2) Coating
Diluting the working reagent 1 with a sodium Carbonate Buffer Solution (CBS) special for an enzyme-linked immunosorbent assay, adding into a 96-well plate, adding a film, incubating overnight, and coating the diluted working reagent 1 on the bottom of the plate; removing the working reagent 1 in the pore plate after overnight, washing 3 times by using a washing liquid, and adding a sealing liquid to seal for 1 hour;
The working reagent 1 is a CD44 protein capture antibody; the blocking solution is a buffer solution of 20mM Tris, 150mM NaCl,pH 7.4 containing 2% BSA and 0.05% Tween 20; the washing solution is a buffer solution of 20mM Tris, 150mM NaCl,pH 7.4 containing 0.05% Tween 20;
(3) Bonding of
Mixing and incubating the working reagent 2 and the working solution 1 for 1 hour to obtain the working solution 2;
Wherein the working reagent 2 is a solution containing CD44 protein; the working solution 2 is a mixed solution of hyaluronic acid and a CD44 protein conjugate and free excessive CD44 protein;
(4) Capturing
Adding the working solution 2 into a 96-well plate coated with the working reagent 1 for incubation for 2 hours;
(5) Marking
Removing the working solution 2 from the pore plate and washing the pore plate with a washing solution for 3 times, adding the working reagent 3 into the 96 pore plate, and mixing and incubating the working solution 2 and the working reagent 3 for 1 hour to obtain the working solution 3;
wherein the working reagent 3 is an anti-CD 44 antibody with horseradish peroxidase label, and the working solution 3 comprises a free horseradish peroxidase labeled CD44 secondary antibody;
(6) Color development
Removing the working solution 3 from a 96-well plate, washing the solution for 3 times, adding the working reagent 4 into the 96-well plate, enabling the HRP contained in the working solution 3 to enable the 3,3', 5' -tetramethylbenzidine to be blue under the action of hydrogen peroxide, and adding a reaction stopping solution to obtain the working solution 4, wherein the solution is yellow;
Wherein the working reagent 4 is a solution containing 3,3', 5' -tetramethyl benzidine substrate and hydrogen peroxide; the working solution 4 is a solution of the working solution 3 catalyzed by the working reagent 4; the reaction stopping solution is 2N sulfuric acid;
(7) Detection of
And (3) placing the 96-well plate filled with the working solution 4 in an enzyme-labeled instrument to detect absorbance, and calculating the relation between the absorbance and the content of hyaluronic acid in the fabric.
2. The method for quantitatively detecting the content of hyaluronic acid in a fabric according to claim 1, characterized in that: the working reagent 1 is a CD44 protein capture antibody solution with the concentration of 0.2mg/mL, and the sodium Carbonate Buffer (CBS) is a buffer solution filtered by a filter membrane with the pH of 9.6,0.2 μm and the MNA 2CO3,0.05M NaHCO3 of which is 0.05; the working reagent 1 was diluted 100-fold with CBS buffer.
3. The method for quantitatively detecting the content of hyaluronic acid in a fabric according to claim 1, characterized in that: the working reagent 2 is a CD44 protein standard solution with a series of concentration gradients, and the concentration range is 0 ng/mL-50 ng/mL; in preparing the CD44 protein standard solution, the CD44 protein stock solution was diluted with a buffer solution containing 20mMTris,150mM NaCl,pH 7.4 of 0.1% bsa and 0.05% tween 20.
4. The method for quantitatively detecting the content of hyaluronic acid in a fabric according to claim 1, characterized in that: the working reagent 3 is an anti-CD 44 antibody with horseradish peroxidase mark and the concentration of the working reagent 3 is 0.25 mu g/mL, and the working reagent 3 is a high concentration mother solution of 0.25mg/mL which is diluted 1000 times by a buffer solution containing 20mM Tris,150mM NaCl,pH 7.4 of 0.5% BSA and 0.05% Tween 20.
5. The method for quantitatively detecting the content of hyaluronic acid in a fabric according to claim 1, characterized in that: the working solution 4 is placed in an enzyme-labeled instrument, and the absorbance value of the solution at 450nm is detected.
6. The method for quantitatively detecting the content of hyaluronic acid in a fabric according to claim 5, wherein: and (3) the absorbance value of the working solution 4 and the fabric standard containing different concentrations of hyaluronic acid are subjected to corresponding standard curves, so that the concentration of the hyaluronic acid in the fabric is calculated.
7. The method for quantitatively detecting the content of hyaluronic acid in a fabric according to claim 6, wherein: bringing the absorbance value a into a fitting equation C x＝0.4322C0-3.3117Ax +0.0176 derived from the relationship between absorbance values and fabrics containing different hyaluronic acid concentrations,
Wherein Cx is the content of hyaluronic acid (w/w) on the fabric, in ng/g;
C 0 is the specific concentration of CD44 protein prior to initial mixing with hyaluronic acid;
A x is the absorbance value of the color development liquid.
8. The method for quantitatively detecting the content of hyaluronic acid in a fabric according to claim 7, wherein: the stoichiometric ratio of the mass concentration of the CD44 protein to the hyaluronic acid is 55-66 mol/L:1mol/L.
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| JPS64469A (en) * | 1987-03-03 | 1989-01-05 | Chugai Pharmaceut Co Ltd | Method and kit for measuring high-molecular hyaluronic acid |
| CN102692408A (en) * | 2012-04-26 | 2012-09-26 | 北京北方生物技术研究所 | One-step chemiluminiscence quantitative detection kit for hyaluronic acid |
| CN104093415A (en) * | 2011-10-24 | 2014-10-08 | 哈洛齐梅公司 | Companion diagnostic for anti-hyaluronan agent therapy and methods of use thereof |
| CN106501503A (en) * | 2016-09-26 | 2017-03-15 | 王键 | Hyaluronic acid（HA）Test kit and preparation method thereof |
| CN112595845A (en) * | 2020-12-09 | 2021-04-02 | 深圳普门科技股份有限公司 | Hyaluronic acid detection kit and detection system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CA2660455A1 (en) * | 2006-08-08 | 2008-02-14 | Seikagaku Corporation | Method for determination of molecular weight of hyaluronic acid |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS64469A (en) * | 1987-03-03 | 1989-01-05 | Chugai Pharmaceut Co Ltd | Method and kit for measuring high-molecular hyaluronic acid |
| CN104093415A (en) * | 2011-10-24 | 2014-10-08 | 哈洛齐梅公司 | Companion diagnostic for anti-hyaluronan agent therapy and methods of use thereof |
| CN102692408A (en) * | 2012-04-26 | 2012-09-26 | 北京北方生物技术研究所 | One-step chemiluminiscence quantitative detection kit for hyaluronic acid |
| CN106501503A (en) * | 2016-09-26 | 2017-03-15 | 王键 | Hyaluronic acid（HA）Test kit and preparation method thereof |
| CN112595845A (en) * | 2020-12-09 | 2021-04-02 | 深圳普门科技股份有限公司 | Hyaluronic acid detection kit and detection system |
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