Method for determining binding activity of epidermal growth factor in sodium hyaluronate composite gel

Abstract

The invention discloses a method for determining the binding activity of epidermal growth factors in sodium hyaluronate composite gel. The method comprises the following steps: S1, weighing HE and carrying out enzymolysis to obtain a pre-diluent of a test sample; s2, weighing a human epidermal growth factor activity determination standard substance and redissolving to obtain a linear standard substance solution; s3, diluting the pre-diluent to enable the pre-diluent to be within a detection linear range to obtain a test solution; s4, incubating the standard solution and the test solution; s5, adding a color developing solution after incubation, incubating in a dark place, and adding a stop solution after color development; s6, putting into a microplate reader, measuring absorbance, and recording a result; and S7, drawing a standard curve and analyzing the sample concentration according to the result, and calculating the percentage of the sample concentration in the theoretical value. According to the invention, sodium hyaluronate enzyme is added, so that sodium hyaluronate in the gel can be subjected to enzymolysis, the constraint of the sodium hyaluronate hydrogel with a net structure on EGF in a test sample is relieved, the influence of a double-antibody one-step sandwich method ELISA test on the detection of the EGF content in HE is reduced, and the accuracy of a detection result is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical protein detection, and in particular to a method for determining epidermal growth factor binding activity in a sodium hyaluronate composite gel. Background Art

[0002] Sodium hyaluronate (HA) is the sodium salt of hyaluronic acid, a non-species-specific glucuronic acid composed of the disaccharide units sodium glucuronide and N-acetylglucosamine. Sodium hyaluronate itself is a component of human skin and the most widely distributed acidic mucosaccharide in the body. It is present in the matrix of connective tissue and has excellent moisturizing properties. This substance is primarily extracted from cockscombs and can also be produced through fermentation by lactic acid bacteria. It appears as white or off-white granules or powder, with a nitrogen content of 2.8-4.0% and a glucuronic acid content of 37.0-51.0% when dry. Sodium hyaluronate is primarily used in pharmaceuticals, cosmetics, and personal care products. It forms a viscoelastic solution in water; a 1% aqueous solution is gel-like, providing thickening and stabilizing properties. The solution's high viscosity provides mechanical protection for tissues (iris, retina) and cell layers (cornea, endothelium, and epithelium). The solution's elasticity helps absorb mechanical stress and provide a protective buffer for tissues. Furthermore, it promotes wound healing by acting as a protective transporter, delivering peptide growth factors and other structural proteins to the site of action. It is then enzymatically degraded and releases active proteins to promote tissue repair. Because it is non-immunogenic, non-rejection, biodegradable, and absorbable, its effects are natural and gentle. It is widely used clinically as a soft tissue filler and holds a key position in the field of facial micro-plastic surgery.

[0003] Epidermal growth factor (EGF) is a small polypeptide composed of 53 amino acids with a molecular weight of approximately 6,000 daltons. It contains three pairs of disulfide bonds, making it relatively stable to physical and chemical factors such as acid, alkali, and heat. EGF has a wide range of biological effects, promoting the growth of various epidermal tissues. It is used medically to treat burns, ulcers, various types of trauma, and corneal injuries. EGF is present in large quantities within human epithelial cells. It stimulates the growth of epithelial and endothelial cells, enabling the continuous formation of new epidermal cells and the gradual shedding of dead skin, maintaining smooth and tender skin. It promotes normal epidermal cell metabolism and enhances cellular absorption of nutrients. It also promotes the synthesis of collagen and collagenase, secretes collagen, hyaluronic acid, and glycoproteins, and regulates collagen fibers. It moisturizes the skin, enhances skin elasticity, reduces wrinkles, and prevents skin aging. Studies have shown that extremely small amounts of EGF can strongly stimulate cell growth, inhibit the expression of aging genes, prevent skin aging, and keep the various components of the skin in the best physiological state. Therefore, it is also known as the "beauty factor" in the beauty industry. Adding it to beauty and skin care products can achieve the effects of whitening, anti-wrinkle, and anti-aging.

[0004] Sodium hyaluronate composite gel for injection uses sodium hyaluronate as the gel matrix. The sodium hyaluronate hydrogel is a three-dimensional dense network structure. EGF is dissolved in it to form a highly hydrophilic gel. This can make the sodium hyaluronate composite gel more stable and the drug can be released slowly. When sodium hyaluronate and EGF are used together, hyaluronic acid can achieve instant filling with immediate effect. At the same time, epidermal growth factor can stimulate the body's own tissue to form new collagen and hyaluronic acid. The combination of the two combines instant filling with continuous stimulation of skin collagen regeneration, and has the dual functions of "physical space occupation + active regeneration", achieving the effect of "1+1>2".

[0005] Biological activity is a key attribute of biological preparations, and its accurate determination is essential for ensuring drug safety and efficacy. In vitro, immunochemical methods are the primary method for measuring EGF binding activity. Enzyme-linked immunosorbent assay (ELISA) is widely used due to its high sensitivity, high throughput, and wide linear dynamic range. Immunochemical assays can be developed using either homemade reagents or commercially available kits. The reticular structure of sodium hyaluronate hydrogels plays a crucial role in stabilizing EGF activity. However, due to the difficulty in fully releasing the trace amount of drug protein trapped within the three-dimensional reticular structure of sodium hyaluronate hydrogels, accurate determination of drug protein content and bioactivity in such gel-based drugs is limited. To address these issues, we conducted extensive experimental investigations addressing various factors and established an enzymatic hydrolysis method for sodium hyaluronate, which was used for sample pretreatment of sodium hyaluronate composite gels (HEs), facilitating accurate detection of EGF content in ELISA binding activity assays for HEs. Summary of the Invention

[0006] The purpose of the present invention is to address the problem that existing detection methods cannot accurately determine the drug protein content and biological activity in sodium hyaluronate composite gels. The present invention provides a method for determining the epidermal growth factor binding activity in sodium hyaluronate composite gels. The method of the present invention can effectively improve the accuracy of detection.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention provides a method for determining the epidermal growth factor binding activity in a sodium hyaluronate composite gel, the method comprising the following specific steps:

[0009] S1. Sample pretreatment: Accurately weigh the sodium hyaluronate composite gel and enzymatically hydrolyze it to obtain a pre-diluted solution of the sample;

[0010] S2. Preparation of linear standard solution: Weigh the recombinant human epidermal growth factor activity assay standard and reconstitute it under sterile conditions, then dilute it to obtain a linear standard solution;

[0011] S3. Preparation of test solution: Further dilute the pre-diluted solution of the test sample to obtain the test solution within the linear range of the assay;

[0012] S4, incubation: sandwich incubation of the diluted linear standard solution and the test solution;

[0013] S5, color development: After the incubation is completed, add color development solution and incubate in the dark. After the color development is completed, add stop solution;

[0014] S6. Signal detection: After mixing, place the mixture in a microplate reader to measure the absorbance and record the measurement results;

[0015] S7. Draw a standard curve: Draw a standard curve and analyze the sample concentration based on the results of the microplate reader, and calculate the percentage of the sample concentration to the theoretical value.

[0016] Specifically, the enzymatic hydrolysis of sodium hyaluronate in the assay method of the present invention can reduce the interference of sodium hyaluronate in the assay. The method of the present invention is suitable for detecting the binding activity of epidermal growth factor (EGF) in sodium hyaluronate composite gel (HE) for injection.

[0017] Furthermore, a method for determining the epidermal growth factor binding activity in sodium hyaluronate composite gel is as follows: Step S1, pretreatment of the test sample: accurately weigh the sodium hyaluronate composite gel, then add hyaluronidase for enzymatic hydrolysis, and then adjust the volume after enzymatic hydrolysis to obtain a pre-diluted solution of the test sample; wherein: the enzymatic hydrolysis temperature is 35-40°C, and the enzymatic hydrolysis time is 2-4 hours.

[0018] Furthermore, the method for determining the epidermal growth factor binding activity in the sodium hyaluronate composite gel is as follows: the concentration of added hyaluronidase is 100 to 200 IU / mL.

[0019] Furthermore, a method for determining the epidermal growth factor binding activity in the sodium hyaluronate composite gel: Step S6, signal detection: after mixing, place the mixture in a microplate reader, measure the absorbance at 450 nm as the detection wavelength and 540 nm as the reference wavelength, and record the measurement results.

[0020] The present method relates to the technical field of protein content determination in biological preparations, particularly a method for determining epidermal growth factor binding activity in a sodium hyaluronate composite gel for injection. The method comprises the steps of enzymatic hydrolysis of a test sample, preparation of the test sample, preparation of a standard curve solution, sample loading and incubation, detection, plotting a standard curve, and performing sample concentration analysis. The method exhibits high sensitivity, high throughput, and a wide linear dynamic range, resulting in accurate and stable data.

[0021] Beneficial effects of the present invention:

[0022] (1) The method of the present invention only requires the sample to be tested to be subjected to hyaluronic acid enzymatic hydrolysis, and the sodium hyaluronate in the composite gel is enzymatically hydrolyzed into small molecules, thereby avoiding the influence of sodium hyaluronate on the detection. The binding activity content of epidermal growth factor in the sodium hyaluronate composite gel for injection can be determined, and the detection sensitivity can be improved. The commercial kit used is of reliable source, stable quality, and batch control. The enzymatic hydrolysis method is combined with the double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA), which can be used to simultaneously determine the content of a large number of samples, truly achieving a high-throughput binding activity content determination.

[0023] (2) The present invention performs sample pretreatment on the test sample, and adds sodium hyaluronate enzyme to enzymatically hydrolyze the sodium hyaluronate in the gel, thereby releasing the binding of the reticular structure of the sodium hyaluronate hydrogel on the EGF in the test sample, reducing the effect of the double-antibody one-step sandwich enzyme-linked immunosorbent assay on the detection of the EGF content in the sodium hyaluronate composite gel, and improving the accuracy of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is the standard curve diagram after data processing in Example 1;

[0026] Figure 2 This is the standard curve diagram after data processing of Comparative Example 1. DETAILED DESCRIPTION

[0027] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] Example 1

[0029] Provided is a method for determining epidermal growth factor binding activity in a sodium hyaluronate composite gel, the method comprising the following steps:

[0030] S1. Sample pretreatment: Accurately weigh 1.1 g of sodium hyaluronate composite gel (HE), then add 150 IU / mL hyaluronidase and shake well in a shaking incubator at 37°C for 3 hours. After enzymatic hydrolysis, weigh 1.0 g of the enzymatic hydrolysis sample into a 10 mL volumetric flask and dilute to volume with PBS to obtain the sample pre-dilution solution;

[0031] S2. Preparation of linear standard solution: Weigh the national standard for recombinant human epidermal growth factor activity assay, reconstitute under sterile conditions according to the instructions, dilute to 1.0 μg / mL, dilute again to 250 pg / mL, and perform 1.5-fold serial dilutions for a total of six dilution steps to obtain a linear standard solution;

[0032] S3. Preparation of test solution: Dilute the test sample pre-dilution solution to 160 pg / mL with the kit diluent so that it is within the linear range of detection to obtain the test sample solution;

[0033] S4. Incubation: Transfer the diluted linear standard solution and test solution to the microwell strips provided with the kit, 200 μL per well, 2 replicates per sample, and perform sandwich incubation according to the kit instructions;

[0034] S5. Color development: After incubation, add 200 μL of color development solution to each well and incubate in the dark for 20 minutes. After color development, immediately add 50 μL of stop solution to each well, and the color in the well will change from blue to yellow.

[0035] S6. Signal detection: After mixing, place the sample in a microplate reader and measure the absorbance at 450 nm as the detection wavelength and 540 nm as the reference wavelength, and record the measurement results.

[0036] S7. Draw the standard curve: Draw the standard curve according to the results of the enzyme marker (such as Figure 1 Calculate the percentage of sample concentration to theoretical value based on the analysis of sample concentration (as shown) and sample concentration;

[0037] Specifically, the four-parameter calculation method on the microplate reader is used to fit the diluted concentration as the horizontal axis and the measured OD value as the vertical axis to display the standard curve and the fitting correlation coefficient R 2 and report the coefficient of variation (CV%); where: R 2 R is the ratio of the regression sum of squares to the total deviation sum of squares. 2 It is between 0 and 1. The closer it is to 1, the better the fitting effect. CV% is a percentage representing the coefficient of variation, which is used to measure the degree of dispersion of a set of data. In this example, R 2 =0.999, CV%≤10%, proving the data are credible.

[0038] The percentage of sample concentration to theoretical value is calculated according to the following formula:

[0039] hEGF binding activity in the test sample (μg / mL) = hEGF content in the solution after enzymatic hydrolysis (μg / g) × (sample weight + enzyme solution volume) / sample weight (g);

[0040] Where: hEGF content in the solution after enzymatic hydrolysis (μg / g) = (calculated value (pg / mL) × 6250 / 1000000) × 10 / enzymatic hydrolysis sample weight (g);

[0041] The calculated value is the concentration of the sample obtained by substituting the OD value of the sample into the standard curve. The theoretical value is the labeled amount of the sample. For specific test data and results, please refer to Table 1 below.

[0042] Example 2

[0043] The difference between Example 2 and Example 1 is that: in step S1 of Example 2, 200 IU / mL of hyaluronidase is added and the enzyme is shaken in a shaking incubator at 40°C for 2 hours; step S5 is incubated in the dark for 25 minutes, and the other conditions are the same as in Example 1.

[0044] Example 3

[0045] The difference between Example 3 and Example 1 is that: in step S1 of Example 3, 100 IU / mL of hyaluronidase is added and the enzyme is shaken in a shaking incubator at 35°C for 4 hours; step S5 is incubated in the dark for 15 minutes, and the other conditions are the same as in Example 1.

[0046] Comparative Example 1

[0047] Provided is a method for determining epidermal growth factor binding activity in a sodium hyaluronate composite gel, the method comprising the following steps:

[0048] (1) Preparation of linear standard solution A: Weigh the national standard for recombinant human epidermal growth factor activity assay, reconstitute under sterile conditions according to the instructions, and dilute to 1.0 μg / mL to obtain hEGF pre-dilution solution; weigh 1.0 g of sodium hyaluronate into a 10 mL volumetric flask, add 10 μL of 1.0 μg / mL hEGF pre-dilution solution to the volumetric flask, and make up to volume with PBS, then dilute again to 250 pg / mL and perform 1.5-fold serial dilutions, a total of six dilution gradients, to obtain linear standard solution A;

[0049] (2) Preparation of test solution A: Weigh 1.0 g of HE gel sample into a 10 mL volumetric flask and dilute to volume with PBS. Shake well to obtain test solution A for use. Then dilute the test solution A to 160 pg / mL with the kit diluent to obtain test solution A.

[0050] (3) Incubation: Transfer the linear standard solution A and the test solution A to the microwell strip provided with the kit, 200 μL per well, 2 replicates per sample, and perform sandwich incubation according to the kit instructions;

[0051] (4) Color development: After incubation, add 200 μL of color development solution to each well and incubate in the dark for 20 minutes. After color development, immediately add 50 μL of stop solution to each well, and the color in the well changes from blue to yellow.

[0052] (5) Signal detection: After mixing, place the sample in a microplate reader and measure the absorbance at 450 nm as the detection wavelength and 540 nm as the reference wavelength, and record the measurement results.

[0053] (6) Draw the standard curve: Draw the standard curve according to the results of the enzyme marker (such as Figure 2 Calculate the percentage of sample concentration to theoretical value based on the analysis of sample concentration (as shown) and sample concentration;

[0054] That is, the four-parameter calculation method on the microplate reader is used to fit the diluted concentration as the horizontal axis and the measured OD value as the vertical axis to display the standard curve and the fitting correlation coefficient R 2 and report the coefficient of variation (CV%); R 2 R is the ratio of the regression sum of squares to the total deviation sum of squares. 2 It is between 0 and 1. The closer it is to 1, the better the fitting effect. CV% is a percentage representing the coefficient of variation, which is used to measure the degree of dispersion of a set of data. In this comparative example 1, R 2 =0.955, CV%≥20%, the correlation coefficient is low and the coefficient of variation percentage is large, which proves that the data is inaccurate. For specific test data and results, please refer to Table 1 below.

[0055] The difference between the above-mentioned comparative example 1 and embodiment 1 is that the linear standard solution A in comparative example 1 is different from that in embodiment 1, and the test solution A in comparative example 1 is different from that in embodiment 1. The other conditions are the same as those in embodiment 1.

[0056] Comparative Example 2

[0057] The difference between Comparative Example 2 and Example 1 is that the linear standard solution in Comparative Example 2 is different from that in Example 1 (the linear standard solution in Comparative Example 2 is the same as that in Comparative Example 1),

[0058] The rest of Comparative Example 2 is the same as Example 1; the specific test data and results of Comparative Example 2 are shown in Table 1 below.

[0059] Comparative Example 3

[0060] The difference between Comparative Example 3 and Example 1 is that the test solution in Comparative Example 3 is different from that in Example 1 (the test solution in Comparative Example 3 is the same as that in Comparative Example 1), and the remaining conditions in Comparative Example 3 are the same as those in Example 1; the specific test data and results of Comparative Example 3 are shown in Table 1 below.

[0061] Table 1 shows the test results of Examples 1 to 3 and Comparative Examples 1 to 3

[0062]

[0063] As can be seen from the test data in Table 1 above, the present invention uses the national standard for recombinant human epidermal growth factor activity as the test standard, performs sample enzymatic hydrolysis on the test sample, and uses hyaluronidase to enzymatically hydrolyze the sodium hyaluronate in the composite gel before diluting and testing. This effectively reduces interference with EGF content detection and improves detection accuracy and sensitivity. The binding activity results are between 80% and 120% of the labeled amount, with a coefficient of variation (CV%) of ≤20%. However, the test results of Comparative Examples 1 to 3 are not within this range, and their test data are inaccurate.

[0064] The detection method of the present invention is simple. The sample to be tested is subjected to hyaluronic acid enzymatic hydrolysis, and the sodium hyaluronate in the composite gel is enzymatically hydrolyzed into small molecules to avoid the influence of sodium hyaluronate on the detection. The binding activity content of epidermal growth factor in the sodium hyaluronate composite gel for injection can be determined, effectively improving the detection sensitivity. The commercial kit used in the present invention has a reliable source, stable quality, and batch control. The enzymatic hydrolysis method is combined with the double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) to perform simultaneous content determination of a large number of samples, truly achieving a throughput binding activity content determination.

[0065] The above preferred embodiments of the present invention are only used to explain the present invention and are not used to limit the present invention. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for determining the epidermal growth factor binding activity in sodium hyaluronate composite gel, characterized in that: The method comprises the following steps: S1. Sample pretreatment: Accurately weigh the sodium hyaluronate composite gel and enzymatically hydrolyze it to obtain a pre-diluted solution of the sample; S2. Preparation of linear standard solution: Weigh the recombinant human epidermal growth factor activity assay standard and reconstitute it under sterile conditions, then dilute it to obtain a linear standard solution; S3. Preparation of test solution: Further dilute the pre-diluted solution of the test sample to obtain the test solution within the linear range of the assay; S4, incubation: sandwich incubation of the diluted linear standard solution and the test solution; S5, color development: After the incubation is completed, add color development solution and incubate in the dark. After the color development is completed, add stop solution; S6. Signal detection: After mixing, place the mixture in a microplate reader to measure the absorbance and record the measurement results; S7. Draw a standard curve: Draw a standard curve and analyze the sample concentration based on the results of the microplate reader, and calculate the percentage of the sample concentration to the theoretical value.

2. The method for determining the epidermal growth factor binding activity in the sodium hyaluronate composite gel according to claim 1, characterized in that: Step S1, sample pretreatment: accurately weigh the sodium hyaluronate composite gel, then add hyaluronidase for enzymatic hydrolysis, and then adjust the volume to obtain a sample pre-diluted solution; The enzymatic hydrolysis temperature is 35-40° C., and the enzymatic hydrolysis time is 2-4 hours.

3. The method for determining the epidermal growth factor binding activity in the sodium hyaluronate composite gel according to claim 1 or 2, characterized in that: The concentration of added hyaluronidase is 100-200 IU / mL.

4. The method for determining the epidermal growth factor binding activity in the sodium hyaluronate composite gel according to claim 1, characterized in that: Step S6, signal detection: after mixing, put it into the microplate reader, measure the absorbance with 450 nm as the detection wavelength and 540 nm as the reference wavelength, and record the measurement results.

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