CN113337574B - Hyaluronidase activity detection method - Google Patents
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Abstract
The invention discloses a hyaluronidase activity detection method, which is used for detecting the activity of hyaluronidase by using hyaluronate with the molecular weight of 200-800 kDa as a substrate. According to the detection method, hyaluronate with a specific molecular weight is used as a substrate solution, the solution before and after enzymolysis reacts with acidic bovine serum albumin, and turbidity changes before and after enzymolysis are compared, so that the enzyme activity of hyaluronidase is accurately determined.
Description
Technical Field
The invention relates to the technical field of enzyme activity detection, in particular to a hyaluronidase activity detection method.
Background
Hyaluronidase (HAase), also known as hyaluronidase, is a generic term for an enzyme class that specifically hydrolyzes hyaluronic acid.
Hyaluronidase was discovered in 1928, and was named after the 1940 study, which was approved by the FDA in 1948 for marketing in the united states as a spreading agent for use with other drugs. In 2004, two hyaluronidase preparations were approved by the FDA-sheep hyaluronidase (Vitrase) by ista pharmaceuticals and bovine hyaluronidase (Amphadase) by amphastar pharmaceuticals. In 2005, the FDA has approved 2 new applications for hyaluronidase formulations on the market, namely, purified Niu Gaowan hyaluronidase (Hydase) by PrimaPharm corporation and recombinant human hyaluronidase (Hylenex) by Halozyme pharmaceuticals, inc.
The recombinant human hyaluronidase Hylenex of the American Halozyme company is a product which is obtained by adopting the recombinant DNA technology to express efficiently in CHO cells and highly purifying, and has the advantages of high purity, low immunogenicity and the like, so the recombinant human hyaluronidase Hylenex can be widely used for quickly replacing animal-extracted hyaluronidase. The hyaluronidase is used with other injection drugs clinically to increase the absorption and dispersion of the latter, and can be used for subcutaneous infusion, promoting the dissipation of local edema or hematoma after operation and trauma and in contrast operation of subcutaneous urethra (improving the reabsorption of contrast agent barium sulfate). Hyaluronidase is also often used in combination with anesthetic drugs during ophthalmic surgery to accelerate the onset of action of anesthetic drugs. In addition, the company of the global biopharmaceutical huge headings such as Roche, qiangsheng and the like combines the recombinant human hyaluronidase Hylenex with a plurality of antibody products with the global sales of more than 50 billion dollars under the flag thereof for tumor treatment, and replaces the expensive antibody administration mode from the traditional intravenous infusion to subcutaneous injection, thereby greatly simplifying the treatment scheme of tumor patients and greatly reducing the cost of the patients and medical systems.
The hyaluronidase (hyaluronidase) injection on the market at home is biochemical bovine testicular enzyme in Shanghai, the detection method is executed according to Chinese pharmacopoeia, and the loading amount is 1500 units (IU) per bottle.
Hyaluronidase is used as a medicine, and the most key index of hyaluronidase is the enzyme activity of hyaluronidase. In the literature, it is reported that hyaluronate can complex with serum protein under acidic condition to form suspension, and when hyaluronate is degraded by hyaluronidase, its complexing ability with serum protein under acidic condition is reduced along with the reduction of molecular weight of hyaluronate, and the turbidity of suspension is reduced. According to this principle, in 1957, an international standard was established for the enzyme activity assay of hyaluronidase: niu Gaowan Hyaluronidase lyophilized powder and lactose were mixed to make into tablet, and enzyme activity was measured by nephelometry. This method stipulates that 0.1mg of the hyaluronidase International standard preparation has an enzyme activity of 1IU (International Unit). The United States Pharmacopeia (USP) (2002) uses horse serum as a precipitating agent for reaction with HA. Fresh bovine serum or freeze-dried bovine serum is adopted in the fourth part 1207 of the 2020 edition of Chinese pharmacopoeia, and the serum stock solution is evaporated to dryness and then dried to constant weight, so that the content of the serum solid is 1.67-2%.
Compared with the method that bovine serum or horse serum is dried to constant weight and then diluted to a certain concentration, the bovine serum albumin of the reagent type is directly prepared into reaction liquid with a certain concentration, so that the reagent is simpler and more easily obtained, and the use is convenient.
Correspondingly, the pharmacopoeia does not clearly define the molecular weight range of the hyaluronate serving as a substrate, and in practice, the fact that the hyaluronate with too high or too low molecular weight serving as a substrate solution cannot form a good enzyme activity unit-turbidity standard curve is found. When the molecular weight of the hyaluronic acid salt is too high, the high molecular hyaluronic acid and the bovine serum albumin are complexed into a cluster under an acidic condition, so that a stable turbid liquid cannot be formed, and when the molecular weight of the hyaluronic acid salt is too low, the turbidity formed by the low molecular hyaluronic acid and the bovine serum albumin is too low under the acidic condition, so that the enzyme activity determination is interfered.
Patent application CN202020855194.4 "a kit for detecting the activity of a bacterial hyaluronidase" provides a kit for measuring the activity of a bacterial hyaluronidase-producing enzyme by the DNS method, but the DNS method is based on the prior art Oettl M (2000) Biochemische chararktericerisement boviner, which is referred to the turbidity method
Hyaluronidase und Untersuchungen zum Einfluβvon Hyaluronidase und
The auf das Wachstum von Tumoren reported that 1U specified by the DNS method is equivalent to 1000IU in the nephelometric method, and therefore, the DNS method sensitivity is much lower than that in the nephelometric method.
Disclosure of Invention
In order to solve the problems, the invention provides a hyaluronidase activity detection method, which adopts hyaluronate as a substrate solution, reacts the solution before and after the hyaluronidase enzymolysis with acid bovine serum albumin, and compares the change of turbidity before and after the enzymolysis, thereby accurately determining the enzyme activity of the hyaluronidase.
The specific technical scheme of the invention is as follows:
1. a hyaluronidase activity detection method uses hyaluronate with a molecular weight of 200-800 kDa as a substrate to detect hyaluronidase activity.
2. The detection method according to item 1, comprising the steps of:
preparing a substrate solution: preparing a hyaluronic acid salt solution with the molecular weight of 200kDa-800kDa by using a first buffer solution;
preparing an acidic bovine serum albumin solution: preparing a bovine serum albumin solution by using a second buffer solution, and adjusting the pH range to 3.0-4.0;
preparing a standard solution: adding the first buffer solution into a hyaluronidase standard to obtain a standard solution;
preparing a test solution: adding the first buffer solution into a hyaluronidase test sample to obtain a test sample solution;
and (3) determination: and respectively carrying out enzymolysis on the substrate solution by using a hyaluronidase standard solution and a hyaluronidase test sample solution, reacting the solution before and after the enzymolysis with acid bovine serum albumin, and measuring the absorbance at 550-640 nm to obtain the enzyme activity of the test sample.
3. The detection method according to item 2, wherein in the steps of preparing the standard solution and preparing the test solution, the hydrolyzed gelatin is added to the first buffer solution.
4. The detection method according to any one of items 2 to 3, wherein the pH of the acidic bovine serum albumin solution is 3.1.
5. The detection method according to any one of items 1 to 4, wherein the absorbance at the time of enzyme activity measurement is 640nm.
6. The detection method according to any one of claims 2 to 5, wherein the first buffer is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer.
7. The method of any one of claims 2-6, wherein the hyaluronic acid salt is sodium hyaluronate or potassium hyaluronate.
8. The detection method according to any one of items 2 to 7, wherein the concentration of the acidic bovine serum albumin is 0.2 to 0.4wt%, and preferably, the concentration of the substrate solution is 0.4 to 0.6mg/ml.
9. The test method according to any one of claims 2 to 8, wherein the hyaluronidase standard solution is a solution containing 8 to 12 enzyme activity units per 1 ml.
10. The detection method according to any one of claims 2 to 9, wherein the range of the enzyme activity of the test sample detected by the detection method is 8 to 12 enzyme activity units per 1ml of the solution.
ADVANTAGEOUS EFFECTS OF INVENTION
Compared with a pharmacopeia method, the detection method provided by the invention adopts hyaluronate with a specific molecular weight as a substrate solution, utilizes the reaction between the solution before and after enzymolysis and acidic bovine serum albumin, and compares turbidity changes before and after enzymolysis, thereby accurately determining the enzyme activity of hyaluronidase.
Drawings
FIG. 1 is a standard graph in example 1.
Fig. 2 is a standard graph in example 2.
FIG. 3 is a standard graph in example 3.
Fig. 4 is a standard graph in example 4.
Fig. 5 is a standard graph in example 5.
Fig. 6 is a standard graph in example 6.
FIG. 7 is a standard graph in example 7.
Fig. 8 is a standard graph in comparative example 1.
Fig. 9 is a standard graph in comparative example 2.
Fig. 10 is a standard graph in comparative example 3.
Detailed Description
The present invention is described in detail in the following description in conjunction with the embodiments illustrated in the figures, wherein like numerals indicate like features throughout. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, however, the description is given for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
The invention provides a method for detecting the activity of hyaluronidase, which uses hyaluronate with the molecular weight of 200kDa-800kDa as a substrate to detect the activity of the hyaluronidase.
In one embodiment, the detection method comprises the steps of:
preparing a substrate solution: preparing a hyaluronic acid salt solution with the molecular weight of 200kDa-800kDa by using a first buffer solution;
preparing an acidic bovine serum albumin solution: preparing a bovine serum albumin solution by using a second buffer solution, and adjusting the pH range to 3.0-4.0;
preparing a standard solution: adding the first buffer solution into a hyaluronidase standard to obtain a standard solution;
preparing a test solution: adding the first buffer solution into a hyaluronidase test sample to obtain a test sample solution;
and (3) determination: and respectively carrying out enzymolysis on the substrate solution by using a standard solution and a test sample solution, reacting the solution before and after the enzymolysis with acid bovine serum albumin, and measuring the absorbance at 550-640 nm to obtain the enzyme activity of the test sample. The bovine serum albumin is globulin in bovine serum, comprises 607 amino acid residues, has the molecular weight of 66.446KDa and the isoelectric point of 4.7. Bovine serum albumin has wide application in biochemical experiments.
For example, the molecular weight of the hyaluronate is 200kDa, 300kDa, 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, etc.
According to the invention, hyaluronate is used as a substrate solution, hyaluronidase is adopted to carry out enzymolysis on the substrate solution, the solution before and after enzymolysis is reacted with acidic bovine serum albumin, and the change of turbidity before and after enzymolysis is compared, so that the enzyme activity of hyaluronidase is accurately determined.
The invention uses the hyaluronate with the specific molecular weight as a substrate solution to prevent measurement failure caused by overhigh or overlow molecular weight of the hyaluronate.
In one embodiment, in the step of preparing the standard solution and the test solution, hydrolyzed gelatin is added to the first buffer solution.
The hydrolyzed gelatin is prepared from pure gelatin 50g, water 1000ml, heating to 121 deg.C for 90min, and freeze drying.
In one embodiment, the concentration of the substrate solution is 0.4 to 0.6mg/ml.
In one embodiment, the acidic bovine serum albumin solution has a pH of 3.1.
In one embodiment, the absorbance of the enzyme activity assay is 640nm.
In one embodiment, the first buffer is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer.
In one embodiment, the hyaluronic acid salt is sodium hyaluronate or potassium hyaluronate.
When the substrate solution is prepared using the first buffer, when the hyaluronic acid salt is sodium hyaluronate, the substrate solution is prepared using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, and when the hyaluronic acid salt is potassium hyaluronate, the substrate solution is prepared using a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer.
In one embodiment, the concentration of the acidic bovine serum albumin is 0.2-0.4wt%.
For example, the concentration of the acidic bovine serum albumin may be 0.2wt%, 0.3wt%, 0.4wt%, or the like.
According to the method, hyaluronate with a specific molecular weight is used as a substrate solution, an acidic bovine serum albumin solution with a specific concentration is used for reacting with solutions before and after enzymolysis, and turbidity changes before and after enzymolysis are compared, so that the enzyme activity of hyaluronidase is accurately measured.
In one embodiment, the hyaluronidase standard solution is a solution containing 8-12 enzyme activity units per 1 ml.
Preferably, the hyaluronidase standard is hyaluronidase with calibrated nephelometric activity, preferably standard hyaluronidase provided by Shanghai biochemistry.
In one embodiment, a series of substrate solutions are prepared, the substrate solutions are hydrolyzed by using hyaluronidase standard solutions respectively, after enzymolysis is finished, the solutions before and after enzymolysis are reacted with acidic bovine serum albumin respectively, and absorbance is measured at 640nm, so that a standard curve is obtained.
In one embodiment, the assay detects the enzyme activity of the test agent in a range of about 8 to about 12 enzyme activity units per 1ml of solution.
The hyaluronidase test sample can be any kind of hyaluronidase or enzyme capable of degrading hyaluronic acid, for example, the hyaluronidase test sample can be extracted or heterologously expressed human hyaluronidase, leech hyaluronidase, snake venom hyaluronidase, bacterial hyaluronidase, etc.
According to the invention, the hyaluronate with a specific molecular weight and the acidic bovine serum albumin with a specific concentration are used, so that the problem that the hyaluronate is precipitated with the bovine serum albumin due to overhigh molecular weight can be prevented, the enzyme activity of hyaluronidase is accurately determined, and compared with fresh serum or freeze-dried serum used in pharmacopoeia, the bovine serum albumin is more convenient to purchase, and the solution is simple and convenient to prepare.
Examples
The invention is described generally and/or specifically for the materials used in the tests and the test methods, in the following examples,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The standard enzyme of the method is purchased from Hyaluronidase (hyaluronidase) injection prepared from Shanghai, and the loading amount is 1500 units (IU) per bottle.
Example 1
(1) Preparation of a substrate solution:
A. taking sodium dihydrogen phosphate (NaH) 2 PO 4 ·H 2 O) 2.5g, anhydrous disodium hydrogen phosphate (Na) 2 HPO 4 ) 1.0g and 8.2g of sodium chloride (NaCl) (both accurate to 0.01 g) are added with water to dissolve the mixture into 1000ml, so as to obtain a first buffer solution;
B. taking sodium hyaluronate with molecular weight of 530kDa, which is subjected to decompression drying for 48 hours by phosphorus pentoxide in advance, adding water to prepare solution containing 1.0mg in each 1ml, storing at the temperature below 0 ℃, and stabilizing for 3-4 weeks to obtain sodium hyaluronate stock solution;
C. taking 50ml of sodium hyaluronate stock solution, diluting with 50ml of first buffer solution to prepare a solution with the concentration of 0.5mg in each 1ml, and preparing the solution for use to obtain a substrate solution.
(2) Preparation of acidic bovine serum albumin: dissolving 11.73g of sodium acetate in 200ml of water, adding 20.5ml of glacial acetic acid, and adding 1000ml of water to obtain a second buffer solution; weighing 2.0g of bovine serum albumin, adding 100ml of water for dissolving, adding 400ml of second buffer solution for uniformly mixing to prepare 0.4% bovine serum albumin solution, adjusting the pH value of the solution to 3.1 by using 4mol/L hydrochloric acid solution, and placing the solution in a refrigerator for 18-24 hours to obtain acidic bovine serum albumin solution for later use. The solution can be stored at 0-4 deg.C and can be stabilized for 3-4 weeks.
(3) Preparing a standard solution: weighing 330mg (accurate to 1 mg) of hydrolyzed gelatin, adding 250ml of each of the first buffer solution and water, shaking up to dissolve, and storing at 0-4 ℃ to obtain a hydrolyzed gelatin solution. If the solution is not turbid, the solution can be used continuously. Taking 1 sample of hyaluronidase standard, adding cold hydrolyzed gelatin diluent according to the unit as indicated to prepare solution containing 10 enzyme activity units per 1ml for preparation at the time of use.
(4) Preparing a test solution: diluting with cold hydrolyzed gelatin diluent to obtain a solution containing about 10 enzyme activity units per 1ml, and preparing the solution for use.
(5) And (3) determination:
A. preparation of a standard curve: taking 12 test tubes with the same size, sequentially adding 0.00, 0.10, 0.20, 0.30, 0.40, 0.50ml of standard solution with graduated pipette, 2 test tubes per part, sequentially adding 0.50, 0.40, 0.30, 0.20, 0.10, 0.00ml of hydrolyzed gelatin diluent, and each time at 30 s intervalsZhong Shunxu adding sodium hyaluronate solution 0.50ml, shaking, immediately placing in 37 + -0.5 deg.C water bath, accurately keeping temperature of each tube for 30 min, sequentially taking out every 30 seconds, immediately and sequentially adding acidic bovine serum albumin solution 4ml, shaking, standing at room temperature for 30 min, shaking, and measuring absorbance at 640nm by ultraviolet-visible spectrophotometry. Taking another 1 test tube, adding 0.50ml of phosphate buffer solution and 0.50ml of hydrolyzed gelatin diluent by using a graduated pipette, shaking up, and carrying out the same operation from 'placing in a water bath at 37 +/-0.5 ℃' according to the method and taking a blank. Plotting the absorbance as ordinate and the unit number of the standard solution as abscissa to obtain a standard curve, and calculating a regression equation, wherein the standard curve is shown in FIG. 1 and is y = -0.1271x +0.7666 2 ＝0.9999。
B. Taking 6 test tubes with the same size, respectively adding 0.20ml, 0.30ml and 0.40ml of test solution diluent to each test tube by a pipette in sequence, and adding 0.30ml, 0.20ml and 0.10ml of hydrolyzed gelatin diluent to each test tube in sequence according to the sequence of 2 test tubes, so that the total volume of each test tube is 0.50ml, adding 0.50ml of sodium hyaluronate solution in sequence every 30 seconds, shaking up, immediately placing in a water bath at 37 +/-0.5 ℃, accurately preserving the heat for 30 minutes, taking out in sequence every 30 seconds, immediately adding 4ml of acidic bovine serum albumin solution in sequence, shaking up, placing for 30 minutes at room temperature, shaking up, and measuring the absorbance at the wavelength of 640nm. Taking another 1 test tube, adding 0.50ml of phosphate buffer solution and 0.50ml of hydrolyzed gelatin diluent by using a graduated pipette, shaking up, and carrying out the same operation from 'placing in a water bath at 37 +/-0.5 ℃' according to the method and taking a blank.
C. From the absorbance measured for the test solution, the regression equation is used to calculate the unit number of each test tube (or from the standard curve), and then the following formula is used:
and calculating the average number of 6 test samples to obtain the titer unit of the hyaluronidase, wherein the enzyme activity of the Shanghai biochemical hyaluronidase (1500 IU/branch) of the test samples is 1514IU.
Example 2
Example 2 differs from example 1 in that the molecular weight of sodium hyaluronate is 770kDa, the standard solution is a solution containing 12 enzyme activity units per 1ml, the standard curve is shown in FIG. 2, and the enzyme activity of the sample Shanghai biochemical hyaluronidase (1500 IU/min) is 1524IU.
Example 3
Example 3 differs from example 1 in that 0.2% bovine serum albumin is used, the molecular weight of potassium hyaluronate is 220kDa, the corresponding buffers are potassium dihydrogen phosphate and dipotassium hydrogen phosphate solutions, the standard solution is a solution containing 8 enzyme activity units per 1ml, the standard curve is shown in fig. 3, and the enzyme activity of the sample Shanghai biochemical hyaluronidase (1500 IU/count) is measured to be 1483IU.
Example 4
Example 4 differs from example 1 in that potassium hyaluronate was used, the molecular weight of potassium hyaluronate was 490kDa, the substrate solution concentration of potassium hyaluronate was 0.5mg/ml, the corresponding buffer solutions were potassium dihydrogen phosphate and dipotassium hydrogen phosphate, the standard curve was shown in FIG. 4, and the enzyme activity of the sample Shanghai biochemical hyaluronidase (1500 IU/min) was 1547IU.
Example 5
Example 5 differs from example 1 in that 0.6% bovine serum albumin was used, the standard curve obtained is shown in FIG. 5, and the enzyme activity of the sample Shanghai biochemical hyaluronidase (1500 IU/min) tested was 1533IU.
Example 6
Example 6 differs from example 1 in that 0.2% bovine serum albumin was used, the standard curve obtained is shown in FIG. 6, and the enzyme activity of the sample Shanghai biochemical hyaluronidase (1500 IU/min) tested was 1497IU.
Example 7
Example 7 differs from example 1 in that sodium hyaluronate with a molecular weight of 220kDa was used, the standard curve obtained is shown in FIG. 7, and the enzyme activity of the Shanghai biochemical hyaluronidase test sample (1500 IU/min) was 1519IU.
EXAMPLE 8 determination of reproducibility
The same lot number as in example 1 was sampled and prepared in parallel in the same manner as in example 1, and the standard solutions and the measurement conditions were the same as in example 1, and the measurement results are shown in Table 2.
Comparative example 1
Comparative example 1 differs from example 1 in that sodium hyaluronate has a molecular weight of 940kDa, giving a standard curve as shown in FIG. 8.
As can be seen, sodium hyaluronate with high molecular weight forms flocculent precipitate with acidic bovine serum albumin, thereby reducing turbidity of the initial suspension. When the substrate sodium hyaluronate is added with hyaluronidase, the molecular weight is reduced, and then the sodium hyaluronate and the acidic bovine serum albumin can form stable suspension. Therefore, it is not feasible to use the sodium hyaluronate with the molecular weight of 940kDa as a substrate solution to prepare a standard curve for detecting the enzyme activity of the hyaluronidase.
Comparative example 2
Comparative example 2 differs from example 1 in that sodium hyaluronate has a molecular weight of 150kDa, giving a standard curve as fig. 9.
As can be seen, due to the use of low molecular weight sodium hyaluronate, when hyaluronidase is added to 4IU, A640 is already close to 0, and the enzyme activity-A640 standard curve cannot be made.
Comparative example 3
Comparative example 3 differs from example 1 in that the standard solution is a solution containing 15 enzyme activity units per 1ml, and the standard curve is obtained as shown in FIG. 10.
As can be seen from the figure, due to the excessive enzyme activity of the added hyaluronidase, when the hyaluronidase is added to 6IU, the A640 is close to 0, and the enzyme activity-A640 standard curve can not be made.
TABLE 1 Process conditions used in examples 1 to 7 and comparative examples 1 to 3 and enzyme activity tables tested
TABLE 2 example 8 determination of hyaluronidase enzyme Activity
As can be seen from Table 2, the stability of the test using the method of the present invention is good.
Experimental example 1
The hyaluronidase enzyme preparation (standard 10000 IU/ml) produced by Bacillus (Bacillus sp.) A50 CGMCC NO.5744 was measured by the method of example 1, and the measurement result was 10254IU/ml.
Experimental example 2
Sigma cat number H3757 bovine testicular enzyme (designated 300-1000U/mg) was measured by the method of example 1, and the measurement result was 869IU/mg.
In conclusion, the enzyme activity of the hyaluronidase can be accurately measured according to the turbidity change of the solution before and after enzymolysis by adopting the hyaluronate with a specific molecular weight as the substrate solution and using the acidic bovine serum albumin to react with the solution before and after enzymolysis.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention will still fall within the protection scope of the technical solution of the present invention.
Claims (7)
1. A hyaluronidase activity detection method, which uses hyaluronate with a molecular weight of 220-770 kDa as a substrate to detect hyaluronidase activity;
the method comprises the following steps:
preparing a substrate solution: preparing a hyaluronic acid salt solution with the molecular weight of 220kDa-770kDa by using a first buffer solution;
preparing an acidic bovine serum albumin solution: preparing a bovine serum albumin solution by using a second buffer solution, and adjusting the pH range to be 3.0 to 4.0;
preparing a standard solution: adding the first buffer solution into a hyaluronidase standard to obtain a standard solution;
preparing a test solution: adding the first buffer solution into a hyaluronidase test sample to obtain a test sample solution;
and (3) determination: performing enzymolysis on the substrate solution by using a hyaluronidase standard solution and a hyaluronidase test solution respectively, reacting the solution before and after the enzymolysis with acid bovine serum albumin, and measuring the absorbance at 550-640 nm to obtain the enzyme activity of the test solution;
the concentration of the acidic bovine serum albumin is 0.2-0.6wt%;
the concentration of the substrate solution is 0.4-0.6mg/ml;
the hyaluronidase standard solution is a solution containing 8-12 enzyme activity units in each 1 ml.
2. The test method according to claim 1, wherein in the steps of preparing the standard solution and preparing the test solution, hydrolyzed gelatin is added to the first buffer solution.
3. The method according to claim 1, wherein the pH of the acidic bovine serum albumin solution is 3.1.
4. The detection method according to claim 1, wherein the absorbance in the enzyme activity measurement is 640nm.
5. The detection method according to claim 1, wherein the first buffer is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer.
6. The assay of claim 1 wherein the hyaluronic acid salt is sodium hyaluronate or potassium hyaluronate.
7. The test method according to any one of claims 1 to 6, wherein the range of the enzyme activity of the test sample to be tested is 8 to 12 enzyme activity units per 1ml of the solution.
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