CN115372631A - Kit for preparing glycosylated hemoglobin calibrator and preparation method - Google Patents

Abstract

The application relates to the technical field of biology, and particularly discloses a kit for preparing a glycosylated hemoglobin calibrator and a preparation method thereof. The kit comprises: a cell stock solution comprising: phosphate buffer, sodium chloride; a saccharification liquid comprising: phosphate buffer, sodium chloride and glucose; a hemolytic agent comprising: phosphate buffer solution, sodium chloride and first surfactant; a lyoprotectant comprising: phosphate buffer solution, sodium chloride, antioxidant and second surfactant. Through the mode, the method can avoid the problem that a diabetic blood sample which is not easy to obtain is used for preparing the glycosylated hemoglobin calibrator in the prior art, so that the limitation of preparation raw materials is reduced to a certain extent, and the preparation cost is reduced.

Description

Kit and preparation method for preparing glycosylated hemoglobin calibrator

technical field

The present application relates to the field of biotechnology, in particular to a kit and a preparation method for preparing a glycosylated hemoglobin calibrator.

Background technique

Glycosylated hemoglobin HbA1c is the diagnostic standard for diabetes recommended by the World Health Organization (2011), and it is also the "gold standard" for diabetic blood sugar control, and it is also an effective indicator for evaluating microvascular and macrovascular complications of diabetes. Usually, fresh blood samples of diabetic patients are used as HbA1c calibrator, but there are many limitations in acquisition and use, so it is necessary to prepare a calibrator that can be stably used in daily work.

Contents of the invention

This application aims to solve one of the technical problems in the related art at least to a certain extent.

In the first aspect of the present application, the present application proposes a kit for preparing glycosylated hemoglobin calibrator, which kit includes: cell storage solution, including: phosphate buffer saline, sodium chloride; glycosylated solution, including : Phosphate buffer, sodium chloride, glucose; hemolytic agent, including: phosphate buffer, sodium chloride, first surfactant; lyoprotectant, including: phosphate buffer, sodium chloride, antioxidant , the second surfactant.

In the second aspect of the present application, the present application proposes a method for preparing a glycosylated hemoglobin calibrator. The preparation method of the glycosylated hemoglobin calibrator is based on the aforementioned kit, and includes the following steps: weighing the cell storage solution according to the formula, and Use the cell storage solution to clean the red blood cells; weigh the saccharification solution according to the formula, and use the saccharification solution to glycosylate the washed red blood cells until the concentration of glycosylated hemoglobin in the red blood cells reaches the preset concentration; weigh the cell storage solution according to the formula , and use the cell storage solution to wash the glycosylated red blood cells for deglycosylation; weigh the hemolytic agent according to the formula, and use the hemolytic agent to treat the red blood cells to obtain glycosylated hemoglobin; weigh the freeze-dried protective agent according to the formula , and freeze-dry the glycated hemoglobin with a freeze-drying protectant to obtain the glycated hemoglobin calibrator.

The technical solutions provided in the embodiments of the present application can bring the following beneficial effects:

Compared with the prior art, the kit of the present application includes: cell storage solution, saccharification solution, hemolysis agent and lyoprotectant, the saccharification solution can undergo in vitro glycosylation reaction with red blood cells in blood samples of healthy normal people, and then prepare different The concentration glycosylated hemoglobin calibrator avoids the preparation of the glycosylated hemoglobin calibrator using the blood samples of diabetic patients which is not easily obtained in the previous technology, reduces the limitation of preparation raw materials to a certain extent, and reduces the preparation cost.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort. in:

Fig. 1 shows the schematic flow chart of the preparation method of the glycosylated hemoglobin calibrator of the present application;

Fig. 2 shows the glycosylated hemoglobin detection system (assessment system) calibrated using the glycosylated hemoglobin calibrator of Example 1 of the present application, and the glycosylated hemoglobin sample of different concentrations tested with the Bio-Rad D-100 glycosylated hemoglobin analysis system (reference system) Time correlation test.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all of them. Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the scope of protection of this application.

The present application provides a kit for preparing a glycosylated hemoglobin calibrator. The kit includes an independently packaged cell storage solution, a saccharification solution, a hemolytic agent and a lyoprotectant.

Wherein, the cell storage solution includes: phosphate buffer saline and sodium chloride.

The saccharification solution includes: phosphate buffer saline, sodium chloride, and glucose.

Hemolytic agents include: phosphate buffer saline, sodium chloride, and the first surfactant.

Lyoprotectant: including: phosphate buffer saline, sodium chloride, antioxidant, second surfactant.

Specifically, the saccharification solution is used for glycosylation reaction with hemoglobin in red blood cells to obtain glycosylated hemoglobin. Hemolytic agents are used to treat red blood cells to release glycated hemoglobin. Lyoprotectants are used to improve the shipping and shelf life of glycated hemoglobin at room temperature.

Phosphate buffer is used to adjust the pH of the solution. Sodium chloride is used to adjust the ionic strength of the solution to avoid changes in the osmotic pressure of the cells. Glucose is used for glycosylation reaction with hemoglobin in red blood cells to obtain glycosylated hemoglobin.

The first surfactant may be an anionic surfactant or a nonionic surfactant, and in other embodiments, the first surfactant may also be other types of surfactants. Among them, the anionic surfactant not only has the effect of breaking cells and dissolving red blood cells, but also can form a stable complex with hemoglobin. The anionic surfactant can be selected from alkylsulfonate, alkylbenzenesulfonate, alkylsulfuric acid A kind of salt. Nonionic surfactants can not only participate in the dissolution of red blood cells, but also accelerate the formation of hemoglobin complexes, increase the solubility of anionic surfactants, prevent their precipitation at low temperatures, and enhance the stability of hemoglobin complexes. The nonionic surfactant can be polyoxyethylene ethers, specifically polyethylene glycol octylphenyl ether Triton X-100.

Antioxidants are used to prevent oxidation reactions during storage of glycated hemoglobin. The antioxidant may be selected from at least one of vitamin C and its derivatives, vitamin E and its derivatives, butylated hydroxyanisole (BIA), tea polyphenols, sesamol or guaiac resin.

Mannitol and polyethylene glycol can be selected as the second surfactant. Among them, mannitol can be used as a filler and also contains multiple hydroxyl groups, which can replace water and form hydrogen bonds with the hydrophilic groups of glycosylated hemoglobin to protect the space of glycosylated hemoglobin. structure and avoid agglomeration. Polyethylene glycol can provide a framework structure, improve the toughness after freeze-drying, and help maintain the shape after freeze-drying.

Compared with the prior art, the kit of the present application includes: cell storage solution, saccharification solution, hemolysis agent and lyoprotectant, the saccharification solution can undergo in vitro glycosylation reaction with red blood cells in blood samples of healthy normal people, and then prepare different The concentration glycosylated hemoglobin calibrator avoids the preparation of the glycosylated hemoglobin calibrator using the blood samples of diabetic patients which is not easily obtained in the previous technology, reduces the limitation of preparation raw materials to a certain extent, and reduces the preparation cost.

In one embodiment, at least one of the saccharification solution, the cell storage solution, the hemolytic agent, and the lyoprotectant further includes: a preservative.

Optionally, the preservative is at least one of sodium benzoate, sodium azide, potassium sorbate, 2-methyl-4-isothiazolin-3-one, and Proclin-300.

In one embodiment, the first surfactant at least includes: Triton X-100, lauryl polyethylene glycol ether (also known as Bianze 35). The second surfactant at least includes: polyethylene glycol and mannitol. Antioxidants include at least: sodium D-isoascorbate.

In one embodiment, the cell storage solution includes: disodium hydrogen phosphate 50-70mmol/L, sodium dihydrogen phosphate 50-70mmol/L, sodium chloride 0.05-0.1% (mass percentage concentration), Proclin-3000.03-0.05% (mass percentage concentration), the balance is purified water, wherein the pH value of the cell storage solution is 7.2-7.4.

Preferably, the cell storage solution includes: disodium hydrogen phosphate 50mmol/L, sodium dihydrogen phosphate 50mmol/L, sodium chloride 0.09% (mass percentage concentration), Proclin-300 0.03% (mass percentage concentration), and the balance is purified Water, wherein the cell storage solution has a pH value of 7.3.

In one embodiment, the saccharification solution includes: disodium hydrogen phosphate 50-70mmol/L, sodium dihydrogen phosphate 50-70mmol/L, sodium chloride 0.05-0.1% (mass percentage concentration), glucose 1-2% (mass percentage concentration) percentage concentration), Proclin-3000.03-0.05% (mass percentage concentration), and the balance is purified water, wherein the pH value of the saccharification solution is 7.2-7.4.

Preferably, the saccharification solution includes: disodium hydrogen phosphate 50mmol/L, sodium dihydrogen phosphate 50mmol/L, sodium chloride 0.09% (mass percentage concentration), glucose 1% (mass percentage concentration), Proclin-300 0.03% (mass percentage concentration) percentage concentration), and the balance is purified water, wherein the pH value of the saccharification solution is 7.3.

In one embodiment, the hemolytic agent includes: disodium hydrogen phosphate 30-50 mmol/L, sodium dihydrogen phosphate 30-50 mmol/L, sodium chloride 0.01-0.05% (mass percentage concentration), Proclin-3000.03-0.05% ( mass percentage concentration), Triton X-100 0.05～0.1% (mass percentage concentration), lauryl polyethylene glycol ether 0.05～0.1% (mass percentage concentration), and the balance is purified water, wherein, hemolytic agent The pH value is 7.2 to 7.4.

Preferably, the hemolytic agent includes: disodium hydrogen phosphate 30mmol/L, sodium dihydrogen phosphate 30mmol/L, sodium chloride 0.01% (mass percentage concentration), Proclin-300 0.03% (mass percentage concentration), Triton X- 100 0.05% (mass percentage concentration), 0.05% (mass percentage concentration) of lauryl polyethylene glycol ether, and the balance is purified water, wherein the pH value of the hemolytic agent is 7.3.

In one embodiment, the lyoprotectant includes: disodium hydrogen phosphate 50-70 mmol/L, sodium dihydrogen phosphate 50-70 mmol/L, sodium chloride 0.05-0.1% (mass percentage concentration), Proclin-3000.03-0.05 % (mass percentage concentration), mannitol 1-2% (mass percentage concentration), polyethylene glycol (6000) 1-2% (mass percentage concentration), D-sodium erythorbate 0.05-0.1% (mass percentage concentration) , The balance is purified water.

Preferably, the lyoprotectant includes: disodium hydrogen phosphate 50mmol/L, sodium dihydrogen phosphate 50mmol/L, sodium chloride 0.09% (mass percentage concentration), Proclin-300 0.03% (mass percentage concentration), mannitol 2 % (mass percentage concentration), polyethylene glycol (6000) 1% (mass percentage concentration), D-sodium erythorbate 0.05% (mass percentage concentration), and the balance is purified water.

The present application also proposes a method for preparing a glycosylated hemoglobin calibrator, the preparation method is based on the aforementioned kit, and the preparation method includes the following steps:

S10: Weigh the cell storage solution according to the formula, and use the cell storage solution to wash the red blood cells.

In the above step 10, the volume ratio of the cell storage solution to the red blood cells is 3:1-5:1 during each washing.

Specifically, according to the volume ratio of the cell storage solution to the red blood cells of 4:1, the red blood cells were washed three times at 4°C by centrifugation at 3000 rpm for 5 minutes to remove impurities and obtain red blood cells with higher purity.

S20: Weighing the saccharification solution according to the formula, and using the saccharification solution to perform glycosylation on the washed red blood cells until the concentration of glycosylated hemoglobin in the red blood cells reaches a preset concentration.

In the above step 20, the volume ratio of the saccharification solution to the red blood cells is 1:1, the temperature of the glycosylation treatment is 36-38°C, the reaction time of the glycosylation treatment is 24h-72h, and the red blood cell is monitored in real time by the DM glycated hemoglobin analysis system glycosylated hemoglobin concentration.

Specifically, glycosylation solution was added to the washed red blood cells for in vitro glycosylation, the volume ratio of the saccharification solution to red blood cells was 1:1, and the temperature of the glycosylation treatment was 37°C. During the glycosylation treatment, the DM glycosylated hemoglobin analysis system (HPLC method) can be used to monitor the concentration of glycosylated hemoglobin in red blood cells in real time at 12 hours, 24 hours, 36 hours, 42 hours and 48 hours, until the glycosylated hemoglobin is formed When the density reaches the preset density, go to step S30. Wherein, the preset concentration may be: the mass percentage concentration of glycosylated hemoglobin is 9-11%.

S30: Weigh the cell storage solution according to the formula, and use the cell storage solution to wash the red blood cells to remove glucose and prevent further glycosylation of hemoglobin.

In the above step 30, the volume ratio of the cell storage solution to the red blood cells is 3:1-5:1.

Specifically, the red blood cells were washed three times at 4°C by centrifugation at 3000 rpm for 5 minutes to remove glucose and prevent further glycosylation of hemoglobin according to the volume ratio of cell storage solution to red blood cells of 4:1.

S40: Weighing the hemolyzing agent according to the formula, and treating the red blood cells with the hemolyzing agent to obtain glycosylated hemoglobin.

In the above step 40, the volume ratio of the hemolytic agent to the red blood cells is 1:1.

Specifically, add a hemolytic agent equal to the volume of red blood cells, mix well and let stand for 5-10 minutes, centrifuge at 3000 rpm for 5 minutes at 4°C, and retain the supernatant containing glycosylated hemoglobin.

S50: Weigh the lyoprotectant according to the formula, and use the lyoprotectant to freeze-dry the glycosylated hemoglobin to obtain the glycosylated hemoglobin calibrator.

Specifically, after adding a lyoprotectant to the supernatant obtained in step S40, lyophilization is performed, and after lyophilization, a glycosylated hemoglobin calibrator can be obtained.

Wherein, freeze-drying process comprises the following steps:

S501: Prefreezing. In this step, the pre-freezing time is set at 60-90 minutes, the layer temperature is -40°C, and the duration is 120-180 minutes.

S502: Dry once. In this step, the primary drying time is set at 20-40 minutes, the temperature of the plate layer is -25° C., the degree of vacuum is 0.15 mBar, and the duration is 720-900 minutes.

S503: Analysis and drying. In this step, the analytical drying time is set to 300 min, the plate layer temperature is -30° C., the vacuum degree is 0.15 mBar, and the duration is 300-360 min.

The present application also proposes a method for preparing a glycosylated hemoglobin calibrator, the preparation method is based on the aforementioned kit, and the preparation method includes the following steps:

S60: Provide a blood sample.

Wherein, in the blood sample, the red blood cell count is 3.5×10 ¹² -5.5×10 ¹² /L, the hemoglobin concentration is 120-160 g/L, and the mass percentage concentration of glycosylated hemoglobin is 4.5-5.5%.

S70: Perform centrifugation on the blood sample to obtain red blood cells.

Specifically, after the blood sample passed the biosafety test, it was centrifuged at 3000 rpm for 5 minutes at 4° C., and the red precipitate at the bottom was retained to obtain red blood cells.

The present application will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present application. These all belong to the protection scope of this application.

Experimental example 1

Prepare low-level HbA1c calibrator as follows:

Step 101: Collect a blood sample (in the blood sample, the red blood cell count is 3.5×10 ¹² to 5.5×10 ¹² /L, the hemoglobin concentration is 120 to 160 g/L, and the mass percentage concentration of glycated hemoglobin is 4.5 to 5.5%), in the After the blood sample passed the biosafety test, it was centrifuged at 3000 rpm for 5 minutes at 4°C, and the red precipitate at the bottom was retained to obtain red blood cells.

Step 102: According to the volume ratio of the cell storage solution to the red blood cells of 4:1, at 4°C, centrifuge at 3000 rpm for 5 minutes, and wash the red blood cells three times to remove impurities and obtain red blood cells with higher purity.

Step 103: Add a hemolytic agent equal to the volume of red blood cells, mix well and let it stand for 5-10 minutes, then measure the concentration of glycosylated hemoglobin using DM glycosylated hemoglobin analysis system (HPLC method), and the concentration of glycosylated hemoglobin should be 4.5-5.5% Within the range, centrifuge at 3000rpm for 5min at 4°C, and retain the supernatant containing glycosylated hemoglobin.

Step 104: After adding a lyoprotectant to the supernatant, perform lyophilization. After lyophilization, a low-concentration glycosylated hemoglobin calibrator can be obtained.

The composition and concentration of cell storage solution, saccharification solution, hemolytic agent and lyoprotectant used in Table 1 Example 1

Note: "/" means not containing this ingredient.

The freeze-drying process parameters in step 104 are shown in Table 2.

Table 2 freeze-drying process parameters

Example 2

Prepare the glycosylated hemoglobin calibrator with high concentration level HbA1c calibrator, the specific method is as follows:

Step 201: Collect a blood sample (in the blood sample, the red blood cell count is 3.5×10 ¹² to 5.5×10 ¹² /L, the hemoglobin concentration is 120 to 160 g/L, and the mass percentage concentration of glycated hemoglobin is 4.5 to 5.5%), in the After the blood sample passed the biosafety test, it was centrifuged at 3000 rpm for 5 minutes at 4°C, and the red precipitate at the bottom was retained to obtain red blood cells.

Step 202: According to the volume ratio of the cell storage solution to the red blood cells being 4:1, the red blood cells were washed 3 times by centrifugation at 3000 rpm for 5 minutes at 4°C to remove impurities and obtain red blood cells with higher purity.

Step 203: adding saccharification solution to the washed red blood cells for in vitro glycosylation, the volume ratio of saccharification solution to red blood cells is 1:1, during the glycosylation process, use DM glycated hemoglobin analysis system (HPLC method) to monitor red blood cells in real time The glycosylated hemoglobin concentration within the range until the mass percentage concentration of glycosylated hemoglobin should be in the range of 9-11%.

Step 204: According to the volume ratio of the cell storage solution to the red blood cells of 4:1, at 4°C, centrifuge at 3000 rpm for 5 minutes, and wash the red blood cells three times to remove glucose and prevent further glycosylation of hemoglobin.

Step 205: Add a hemolytic agent equal to the volume of the red blood cells, mix well and let stand for 5-10 minutes, centrifuge at 3000 rpm for 5 minutes at 4°C, and retain the supernatant containing glycosylated hemoglobin.

Step 206: After adding a lyoprotectant to the supernatant obtained in step 205, perform lyophilization. After lyophilization, a high-concentration glycosylated hemoglobin calibrator can be obtained.

The composition and concentration of cell storage solution, saccharification solution, hemolytic agent and lyoprotectant used in Table 3 Example 2

Note: "/" means not containing this ingredient.

The freeze-drying process parameters in step 206 are shown in Table 4.

Table 4 freeze-drying process parameters

Example 3

Performance parameter testing of low and high level calibrators

1. Accuracy

After using the normal value national standard product and the abnormal value national standard product for calibration, the low concentration level calibrator and the high concentration level calibrator were measured on the glycosylated hemoglobin detection system, and the accuracy test results are shown in Table 5.

Table 5 Accuracy test results

As can be seen from the results in Table 5, the relative deviation between the measured concentration and the marked value of the low-concentration level and high-concentration level glycosylated hemoglobin calibrator prepared respectively in Examples 1 and 2 is within ± 10%, therefore, the accuracy is satisfied. requirements.

2. Uniformity

It is required that the coefficient of variation of uniformity between bottles ( _{between CV bottles} ) should not be greater than 3.0%, and the coefficient of variation of uniformity within CV bottles ( _inside CV bottles) should not be greater than 1.5%. The homogeneity test results of the glycated hemoglobin calibrator at low concentration levels are shown in Table 6, and the homogeneity test results of the high concentration level glycated hemoglobin calibrator are shown in Table 7.

Table 6 The results of the homogeneity test of the glycosylated hemoglobin calibrator at low concentration levels

Table 7 The homogeneity test results of the HbA1c calibrator at high concentration levels

From the results in Tables 6 and 7, it can be seen that the low-concentration level and high-concentration level glycosylated hemoglobin calibrator prepared in Examples 1 and 2 respectively meet the requirement of uniformity.

3. Stability

Under accelerated conditions at 37°C, place it for 16 days; after reconstitution, place it at 2-8°C for 40 days. The relative deviation between the obtained test result and the marked value is required to be within ±10%. Table 8 shows the accelerated stability test results of the glycated hemoglobin calibrator at a low concentration level, and Table 9 shows the accelerated stability test results of the glycated hemoglobin calibrator at a high concentration level. Table 10 shows the stability test results after reconstitution of low-concentration glycosylated hemoglobin calibrator, and table 11 shows the results of reconstitution of high-concentration glycohemoglobin calibrator.

Table 8 Accelerated stability test results of glycosylated hemoglobin calibrator at low concentration levels

Table 9 Accelerated stability test results of high-concentration HbA1c calibrator

Table 10 The stability test results after reconstitution of the glycosylated hemoglobin calibrator at low concentration levels

Table 11 Stability test results after reconstitution of high-concentration HbA1c calibrator

From the results in Tables 8-11, it can be seen that the glycosylated hemoglobin calibration products prepared in Examples 1 and 2 at low and high concentrations all meet the stability requirements.

4. Relevance

When the glycated hemoglobin detection system (assessment system) calibrated with the glycated hemoglobin calibrator of Example 1 of the present application is tested with the Bio-Rad D-100 glycated hemoglobin analysis system (reference system) when testing different concentrations of glycated hemoglobin samples, test results are required It should be consistent, that is, the slope k=1.00±0.10, and the correlation coefficient r≥0.975. The correlation test results are shown in Table 12 and accompanying drawing 2 of the description.

Table 12 Correlation test results

It can be seen from the results in Table 12 and accompanying drawing 2 that both the low-concentration and high-concentration HbA1c calibration products prepared in Examples 1 and 2 can meet the correlation requirements.

Compared with the prior art, the kit of the present application includes: cell storage solution, saccharification solution, hemolysis agent and lyoprotectant, the saccharification solution can undergo in vitro glycosylation reaction with red blood cells in blood samples of healthy normal people, and then prepare different The concentration glycosylated hemoglobin calibrator avoids the preparation of the glycosylated hemoglobin calibrator using the blood samples of diabetic patients which is not easily obtained in the previous technology, reduces the limitation of preparation raw materials to a certain extent, and reduces the preparation cost.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only represent several implementation modes of the present application, and the description thereof is relatively specific and detailed, but it should not be construed as limiting the scope of the patent for the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the scope of protection of the patent application should be based on the appended claims.

Claims (14)

1. A kit for preparing a glycated hemoglobin calibrator, comprising:

a cell storage fluid comprising: phosphate buffer, sodium chloride;

a saccharification fluid comprising: phosphate buffer, sodium chloride and glucose;

a hemolytic agent comprising: phosphate buffer solution, sodium chloride and first surfactant;

a lyoprotectant comprising: phosphate buffer solution, sodium chloride, antioxidant and second surfactant.

2. The kit of claim 1, wherein at least one of the saccharification fluid, the cell stock solution, the hemolytic agent, and the lyoprotectant further comprises: and (4) a preservative.

3. The kit according to claim 2,

the preservative is at least one of sodium benzoate, sodium azide, potassium sorbate, 2-methyl-4-isothiazoline-3-ketone and Proclin-300.

4. The kit according to claim 1,

the first surfactant includes at least: triton X-100 and dodecyl polyglycol ether;

the second surfactant includes at least: polyethylene glycol, mannitol;

the antioxidant at least comprises: d-sodium erythorbate.

5. The kit according to claim 2,

the cell stock solution comprises: 50-70 mmol/L of disodium hydrogen phosphate, 50-70 mmol/L of sodium dihydrogen phosphate, 0.05-0.1 percent of sodium chloride (mass percent concentration), 0.03-0.05 percent of Proclin-300 (mass percent concentration) and the balance of purified water, wherein the pH value of the cell stock solution is 7.2-7.4.

6. The kit according to claim 2,

the saccharification liquid comprises: 50-70 mmol/L of disodium hydrogen phosphate, 50-70 mmol/L of sodium dihydrogen phosphate, 0.05-0.1 percent of sodium chloride (mass percentage concentration), 1-2 percent of glucose (mass percentage concentration), 0.03-0.05 percent of Proclin-300 (mass percentage concentration) and the balance of purified water, wherein the pH value of the saccharification liquid is 7.2-7.4.

7. The kit according to claim 2,

the hemolytic agent comprises: 30-50 mmol/L disodium hydrogen phosphate, 30-50 mmol/L sodium dihydrogen phosphate, 0.01-0.05% (mass percent concentration) sodium chloride, 0.03-0.05% (mass percent concentration) Proclin-300, 0.05-0.1% (mass percent concentration) Triton X-100, 0.05-0.1% (mass percent concentration) dodecyl polyglycol ether, and the balance of purified water, wherein the pH value of the hemolytic agent is 7.2-7.4.

8. The kit according to claim 2,

the lyoprotectant includes: 50-70 mmol/L of disodium hydrogen phosphate, 50-70 mmol/L of sodium dihydrogen phosphate, 0.05-0.1 percent (mass percentage concentration) of sodium chloride, 0.03-0.05 percent (mass percentage concentration) of Proclin-300, 1-2 percent (mass percentage concentration) of mannitol, 1-2 percent (mass percentage concentration) of polyethylene glycol (6000), 0.05-0.1 percent (mass percentage concentration) of D-sodium erythorbate and the balance of purified water.

9. A method for preparing a glycated hemoglobin calibrator, the method being based on the kit according to any one of claims 1 to 8, the method comprising the steps of:

weighing cell storage liquid according to a formula, and cleaning erythrocytes by using the cell storage liquid;

weighing a saccharification liquid according to a formula, and performing glycosylation treatment on the cleaned red blood cells by adopting the saccharification liquid until the concentration of the glycosylated hemoglobin in the red blood cells reaches a preset concentration;

weighing the cell stock solution according to a formula, and washing the glycosylated erythrocytes by using the cell stock solution for deglycosylation;

weighing a hemolytic agent according to a formula, and treating the red blood cells by using the hemolytic agent to obtain glycosylated hemoglobin;

weighing a freeze-drying protective agent according to a formula, and carrying out freeze-drying treatment on the glycosylated hemoglobin by adopting the freeze-drying protective agent to obtain a glycosylated hemoglobin calibrator.

10. The method of claim 9, wherein prior to the step of weighing the cell stock according to the formula and washing the red blood cells with the cell stock, the method further comprises:

providing a blood sample;

centrifuging the blood sample to obtain the red blood cells;

wherein in the blood sample, the red blood cell count is 3.5X 10 ¹² ～5.5×10 ¹² The concentration of the hemoglobin is 120-160 g/L, and the mass percentage concentration of the glycosylated hemoglobin is 4.5-5.5%.

11. The method of claim 9, wherein in the step of weighing the cell stock solution according to the formula and washing the red blood cells with the cell stock solution, the volume ratio of the cell stock solution to the red blood cells is 3.

12. The method according to claim 9, wherein in the step of weighing a saccharification liquid according to a formula and performing glycosylation treatment on the washed red blood cells by using the saccharification liquid until the concentration of the glycated hemoglobin in the red blood cells reaches a preset concentration, the volume ratio of the saccharification liquid to the red blood cells is 1.

13. The method of claim 9, wherein in the step of weighing the cell stock solution according to the formula and washing the red blood cells with the cell stock solution to remove glucose and prevent further glycosylation of hemoglobin, the volume ratio of the cell stock solution to the red blood cells is 3 to 5.

14. The method according to claim 9, wherein in the step of weighing the lyoprotectant according to the formulation and performing lyophilization treatment on the glycated hemoglobin by using the lyoprotectant to obtain the glycated hemoglobin calibrator, the volume ratio of the hemolytic agent to the red blood cells is 1.

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