Kit for preparing glycated hemoglobin control product and preparation method

Abstract

The present application relates to the field of biotechnology, and specifically discloses a kit for preparing a glycated hemoglobin control product, a method for preparing a glycated hemoglobin control product, and a kit. The kit for preparing a glycated hemoglobin control product comprises: a cell washing solution comprising: a buffer, an osmotic pressure maintaining agent; a saccharification solution comprising: a buffer, an osmotic pressure maintaining agent, and glucose; a hemolytic agent comprising: a buffer, a first surfactant; and a lyophilization protectant comprising: a buffer, an osmotic pressure maintaining agent, a stabilizer, and a second surfactant. Through the above-mentioned method, the glycated hemoglobin control product has the characteristics of low cost, high accuracy, good uniformity, and strong stability. The use of the glycated hemoglobin control product can meet the purpose of quality control of the adapted glycated hemoglobin meter system, and the obtained results are accurate and reliable.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a kit for preparing a glycated hemoglobin control product, a method for preparing a glycated hemoglobin control product, and a kit. Background Art

[0002] Glycated hemoglobin HbA1c is the diabetes diagnostic standard recommended by the World Health Organization (2011), the "gold standard" for diabetic blood sugar control, and an effective indicator for assessing diabetic microvascular and macrovascular complications.

[0003] Accurate measurement of glycated hemoglobin requires a stable quality control product to monitor instrument status and reagent quality. However, many manufacturers currently utilize incomplete quality control product production processes, resulting in poor stability, matrix effects, and unstable performance. These products are unable to meet the quality control requirements of glycated hemoglobin testing systems, limiting their application and impacting their sales and use. Summary of the Invention

[0004] The present application aims to solve one of the technical problems in the related art at least to a certain extent.

[0005] In the first aspect of the present application, the present application proposes a kit for preparing a glycated hemoglobin control product, comprising: a cell washing solution, comprising: a buffer, an osmotic pressure maintaining agent; a saccharification solution, comprising: a buffer, an osmotic pressure maintaining agent, and glucose; a hemolytic agent, comprising: a buffer, a first surfactant; a lyoprotectant, comprising: a buffer, an osmotic pressure maintaining agent, a stabilizer, and a second surfactant.

[0006] In the second aspect of the present application, the present application proposes a method for preparing a glycated hemoglobin control product, which is based on the aforementioned kit and includes the following steps: weighing a cell washing solution according to a formula, and using the cell washing solution to wash the red blood cells; weighing a saccharification solution according to a formula, and using the saccharification solution to perform in vitro glycosylation treatment on the washed red blood cells until the glycated hemoglobin concentration in the red blood cells reaches a preset concentration; weighing a cell washing solution according to a formula, and using the cell washing solution to wash the red blood cells after in vitro glycosylation treatment and deglycosylate them; weighing a hemolytic agent according to a formula, and using the hemolytic agent to treat the red blood cells to obtain glycated hemoglobin; weighing a lyophilization protectant according to a formula, and using the lyophilization protectant to lyophilize the glycated hemoglobin to obtain a glycated hemoglobin control product.

[0007] In the third aspect of the present application, the present application proposes a test kit, which includes: a box body; a calibration product area, a quality control product area and a reagent area are provided in the box body, wherein the quality control product area is provided with a reagent bottle for storing quality control products, and the quality control products are quality control products prepared as described above.

[0008] The technical solutions provided in the embodiments of the present application can bring the following beneficial effects:

[0009] Compared with the prior art, the kit for preparing a glycated hemoglobin control product in the present application includes: a cell washing solution, a saccharification solution, a hemolytic agent, and a lyophilization protective agent. The saccharification solution can undergo an in vitro glycosylation reaction with anticoagulated whole blood containing a low concentration of glycated hemoglobin, thereby preparing glycated hemoglobin controls of different concentrations. This avoids the use of anticoagulated whole blood samples from diabetic patients that are difficult to obtain in the past to prepare glycated hemoglobin controls, reduces the restrictions on preparation raw materials to a certain extent, and reduces the preparation cost. At the same time, the lyophilization protective agent can improve the transportation convenience and storage stability of the glycated hemoglobin control product. The glycated hemoglobin control product has the characteristics of low cost, high accuracy, good uniformity, and strong stability. The use of the glycated hemoglobin control product can meet the purpose of quality control of the adapted glycated hemoglobin meter system, and the results obtained are accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without inventive efforts. Among them:

[0011] Figure 1 The first process diagram of the preparation method of the glycated hemoglobin control product of the present application is shown;

[0012] Figure 2 The second flow chart of the preparation method of the glycated hemoglobin control product of the present application is shown. DETAILED DESCRIPTION

[0013] The following will be combined with the accompanying drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0014] The present application provides a kit for preparing a glycated hemoglobin control product, which comprises independently packaged cell washing solution, saccharification solution, hemolytic agent and lyophilization protective agent.

[0015] Wherein, the cell washing solution includes: phosphate buffer and sodium chloride.

[0016] Cell washing solution, including: buffer, osmotic pressure maintaining agent;

[0017] Saccharification solution, including: buffer, osmotic pressure maintaining agent, glucose;

[0018] A hemolytic agent, comprising: a buffer, a first surfactant;

[0019] Freeze-drying protective agents include: buffers, osmotic pressure maintaining agents, stabilizers, and second surfactants.

[0020] Specifically, the saccharification solution is used to undergo an in vitro glycosylation reaction with hemoglobin within red blood cells to produce glycated hemoglobin. A hemolytic agent is used to treat red blood cells to release glycated hemoglobin. A lyoprotectant is used to extend the transport and shelf life of glycated hemoglobin at room temperature.

[0021] Buffers are used to adjust the pH of a solution. Osmotic pressure maintainers are used to adjust the ionic strength of a solution to avoid changes in cellular osmotic pressure. Glucose is used to undergo an in vitro glycosylation reaction with hemoglobin within red blood cells to produce glycated hemoglobin.

[0022] The first surfactant can be an anionic surfactant or a nonionic surfactant. In other embodiments, the first surfactant can also be other types of surfactants. Among them, anionic surfactants have the function of rupturing cells and lysing red blood cells, and can also bind to hemoglobin to form a stable complex. Anionic surfactants can be selected from one of alkyl sulfonates, alkylbenzene sulfonates, and alkyl sulfates. Nonionic surfactants can participate in the dissolution of red blood cells and 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. Nonionic surfactants can be polyoxyethylene ethers, specifically Tween-20 and Span-40.

[0023] The stabilizer is used to stabilize the glycated hemoglobin to prevent oxidation during storage of the glycated hemoglobin. The stabilizer can be selected from at least one of BSA, trehalose, sucrose, and HSA.

[0024] Mannitol and polyethylene glycol can be used as the second surfactant. Mannitol acts as a filler and contains multiple hydroxyl groups, which can replace water and form hydrogen bonds with the hydrophilic groups of glycated hemoglobin, protecting the spatial structure of glycated hemoglobin and preventing aggregation. Polyethylene glycol provides a framework, improving toughness after freeze-drying and helping to maintain the freeze-dried shape.

[0025] Compared with the existing technology, the kit for preparing glycated hemoglobin control products in the present application includes: cell washing solution, saccharification solution, hemolytic agent and lyophilization protective agent. The saccharification solution can undergo in vitro glycosylation reaction with anticoagulated whole blood containing low concentrations of glycated hemoglobin, thereby preparing glycated hemoglobin control products of different concentrations, avoiding the use of anticoagulated whole blood samples of diabetic patients that are difficult to obtain in previous technologies to prepare glycated hemoglobin control products, reducing the restrictions on preparation raw materials to a certain extent, and reducing preparation costs; at the same time, the lyophilization protective agent can improve the transportation convenience and storage stability of the glycated hemoglobin control products.

[0026] In one embodiment, at least one of the saccharification solution, cell washing solution, hemolytic agent, and lyophilization protectant further comprises 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.

[0027] In one embodiment, the buffer is selected from at least one of glycine buffer, phosphate buffer, borate buffer, Tris buffer, and HEPES buffer. The osmotic pressure maintaining agent is selected from at least one of NaCl, KCl, sodium dihydrogen phosphate, and disodium hydrogen phosphate. The first surfactant is selected from at least one of Tween-20 and Span-40. The stabilizer is selected from at least one of BSA, trehalose, sucrose, and HSA. The second surfactant is selected from at least one of polyethylene glycol and glycine.

[0028] In one embodiment, the cell washing solution comprises: 50-100 mmol / L Tris(hydroxymethyl)aminomethane, 0.05-0.1% (mass percent concentration) of sodium chloride, 0.03-0.05% (mass percent concentration) of Proclin-300, and the balance being purified water, wherein the pH value of the cell washing solution is 7.2-7.4. Preferably, the cell washing solution comprises: 100 mmol / L Tris(hydroxymethyl)aminomethane, 0.09% (mass percent concentration) of sodium chloride, 0.03% (mass percent concentration) of Proclin-300, and the balance being purified water, wherein the pH value of the cell washing solution is 7.4.

[0029] In one embodiment, the saccharification liquid comprises: 50-100 mmol / L tris(hydroxymethyl)aminomethane, 0.05-0.1% (mass percent concentration) of sodium chloride, 2-6% (mass percent concentration) of glucose, 0.03-0.05% (mass percent concentration) of Proclin-300, and the balance being purified water, wherein the pH value of the saccharification liquid is 7.2-7.4. Preferably, the saccharification liquid comprises: 100 mmol / L tris(hydroxymethyl)aminomethane, 0.09% (mass percent concentration) of sodium chloride, 3% (mass percent concentration) of glucose, 0.03% (mass percent concentration) of Proclin-300, and the balance being purified water, wherein the pH value of the saccharification liquid is 7.4.

[0030] In one embodiment, the hemolytic agent comprises: 20-50 mmol / L tris(hydroxymethyl)aminomethane, 0.03-0.05% (mass percent concentration) of Proclin-300, 0.1-0.5% (mass percent concentration) of Tween-20, 0.1-0.5% (mass percent concentration) of Span-40, and the balance being purified water, wherein the pH value of the hemolytic agent is 7.2-7.4. Preferably, the hemolytic agent comprises: 50 mmol / L tris(hydroxymethyl)aminomethane, 0.03% (mass percent concentration) of Proclin-300, 0.2% (mass percent concentration) of Tween-20, 0.1% (mass percent concentration) of Span-40, and the balance being purified water, wherein the pH value of the hemolytic agent is 7.4.

[0031] In one embodiment, the lyoprotectant comprises: tris(hydroxymethyl)aminomethane 50-100 mmol / L, sodium chloride 0.05-0.1% (mass percentage concentration), trehalose 1-10% (mass percentage concentration), Proclin-300 0.03-0.05% (mass percentage concentration), glycine 3-5% (mass percentage concentration), polyethylene glycol-8000 1-2% (mass percentage concentration), BSA 1-5% (mass percentage concentration), and the balance is purified water. Preferably, the lyoprotectant comprises: tris(hydroxymethyl)aminomethane 100 mmol / L, sodium chloride 0.09% (mass percentage concentration), trehalose 5% (mass percentage concentration), Proclin-300 0.03% (mass percentage concentration), glycine 5% (mass percentage concentration), polyethylene glycol-8000 1% (mass percentage concentration), BSA 1% (mass percentage concentration), and the balance is purified water.

[0032] The present application also proposes a method for preparing a glycated hemoglobin control product, which is based on the aforementioned kit and comprises the following steps:

[0033] S10: Weigh the cell washing solution according to the recipe, and use the cell washing solution to wash the red blood cells.

[0034] In the above step 10, the number of washing times is 3 to 6 times. During each washing, the volume ratio of cell washing solution to red blood cells is 2:1 to 8:1. After each washing, centrifugation is performed and the supernatant is removed. The centrifugal speed is 2000 to 3000 rpm, the centrifugal temperature is 2 to 8°C, and the centrifugation time is 5 to 10 minutes.

[0035] Specifically, the red blood cells were washed three times at a volume ratio of 4:1 between the cell washing solution and the red blood cells, using centrifugation at 3000 rpm for 5 minutes at 4°C to remove impurities and obtain red blood cells with higher purity.

[0036] S20: Saccharification liquid is weighed according to the formula, and the washed red blood cells are subjected to in vitro glycosylation treatment using the saccharification liquid until the glycated hemoglobin concentration in the red blood cells reaches a preset concentration.

[0037] In the above step 20, the volume ratio of the saccharified liquid to the red blood cells is 1:1 to 3:1, the temperature of the saccharification treatment is 35 to 45°C, the reaction time of the saccharification treatment is 24 hours to 72 hours, and the DM saccharified hemoglobin analysis system is used to monitor the saccharified hemoglobin concentration in the red blood cells in real time.

[0038] Specifically, a saccharification solution is added to the washed red blood cells for in vitro glycation treatment, with a volume ratio of saccharification solution to red blood cells of 1:1. The glycation treatment temperature is 37°C. During the glycation treatment, the glycated hemoglobin concentration in the red blood cells can be monitored in real time using a DM glycated hemoglobin analysis system (HPLC method) at 12 hours, 24 hours, 36 hours, 42 hours, and 48 hours. When the glycated hemoglobin concentration reaches a preset concentration, step S30 is entered. The preset concentration can be a glycated hemoglobin concentration of 8% to 11% by weight.

[0039] S30: Weigh the cell washing solution according to the formula, and use the cell washing solution to wash the red blood cells after in vitro glycosylation treatment to perform deglycosylation.

[0040] In the above step 30, the number of washing times is 3 to 6 times. During each washing, the volume ratio of the cell washing solution to the red blood cells is 2:1 to 6:1. After each washing, centrifugation is performed and the supernatant is removed. The centrifugal speed is 2000 to 3000 rpm, the centrifugal temperature is 2 to 8°C, and the centrifugal time is 5 to 10 minutes.

[0041] Specifically, the red blood cells were washed three times with a cell washing solution to red blood cell volume ratio of 4:1 and centrifuged at 3000 rpm for 5 min at 4°C to remove glucose and prevent further glycation of hemoglobin.

[0042] S40: Weighing a hemolytic agent according to a recipe, and treating red blood cells with the hemolytic agent to obtain glycated hemoglobin.

[0043] In the above step 40, the volume ratio of the hemolytic agent to the red blood cells is 1:1.

[0044] Specifically, a hemolytic agent having an equal volume to that of red blood cells is added, mixed and allowed to stand for 5 to 10 minutes, and centrifuged at 3000 rpm for 5 minutes at 4°C to retain the supernatant containing glycated hemoglobin.

[0045] S50: Weigh a lyophilization protectant according to the formula, and lyophilize the glycated hemoglobin using the lyophilization protectant to obtain a glycated hemoglobin control product.

[0046] Specifically, a freeze-drying protectant is added to the supernatant obtained in step S40, and then freeze-drying is performed. After freeze-drying, a glycated hemoglobin control product can be obtained.

[0047] The freeze-drying process comprises the following steps:

[0048] S501: Pre-freezing: In this step, the pre-freezing time is set to 60 to 90 minutes, the plate temperature is -30 to 50°C, and the duration is 180 to 240 minutes.

[0049] S502: Primary drying: In this step, the primary drying time is set to 20 to 40 minutes, the plate temperature is -30 to 20°C, the vacuum degree is 0.10 to 0.20 mBar, and the duration is 900 to 1200 minutes.

[0050] S503: Desorption and drying: In this step, the desorption and drying time is set to 300 minutes, the plate temperature is 20-30°C, the vacuum degree is 0.10-0.20 mBar, and the duration is 240-360 minutes.

[0051] The present application also proposes a method for preparing a glycated hemoglobin control product, which is based on the aforementioned kit and comprises the following steps:

[0052] S60: Providing an anticoagulated whole blood sample, wherein the mass percentage concentration of glycated hemoglobin in the anticoagulated whole blood sample is less than the mass percentage concentration of glycated hemoglobin in a glycated hemoglobin control sample.

[0053] Among them, in the anticoagulated whole blood sample, the red blood cell count was 3.5×10 12~5.5×10 12 / L, the hemoglobin concentration is 12-16 g / L, and the mass percentage concentration of glycosylated hemoglobin is 4.0-5.0%.

[0054] S70: centrifuging the anticoagulated whole blood at a centrifugal speed of 2000-3000 rpm, a centrifugal temperature of 2-8° C., and a centrifugal time of 10-15 min to obtain red blood cells.

[0055] Specifically, after the anticoagulated whole blood sample passes the biosafety test, it is centrifuged at 3000 rpm for 5 minutes at 4°C, and the red precipitate at the bottom is retained to obtain red blood cells.

[0056] S10: Weigh the cell washing solution according to the recipe, and use the cell washing solution to wash the red blood cells.

[0057] S20: Saccharification liquid is weighed according to the formula, and the washed red blood cells are subjected to in vitro glycosylation treatment using the saccharification liquid until the glycated hemoglobin concentration in the red blood cells reaches a preset concentration.

[0058] S30: Weigh the cell washing solution according to the formula, and use the cell washing solution to wash the red blood cells after in vitro glycosylation treatment to perform deglycosylation.

[0059] S40: Weighing a hemolytic agent according to a recipe, and treating red blood cells with the hemolytic agent to obtain glycated hemoglobin.

[0060] S50: Weigh a lyophilization protectant according to the formula, and lyophilize the glycated hemoglobin using the lyophilization protectant to obtain a glycated hemoglobin control product.

[0061] The present application is described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present application, but are not intended to limit the present application in any form. It should be noted that those skilled in the art may make several variations and improvements without departing from the scope of the present application. These all fall within the scope of protection of the present application.

[0062] Experimental Example 1

[0063] Prepare a low-concentration HbA1c control as follows:

[0064] Step 101: Collect EDTA anticoagulated whole blood samples (EDTA anticoagulated whole blood samples are stored at 2-8°C for no more than 24 hours, and the red blood cell count in the anticoagulated whole blood sample is 3.5×10 12 ~5.5×10 12 / L, hemoglobin concentration of 12-16 g / L, and mass percentage concentration of glycosylated hemoglobin of 4.0-5.0%), after the anticoagulated whole blood sample passed the biosafety test, it was centrifuged at 2500 rpm at 6°C for 15 minutes, and the red precipitate at the bottom was retained to obtain red blood cells.

[0065] Step 102: The red blood cells are washed five times by centrifugation at 3500 rpm for 10 minutes at a cell washing solution to red blood cell volume ratio of 5:1 at 6°C to remove impurities and obtain red blood cells with higher purity.

[0066] Step 103: Add a hemolytic agent in an equal volume to the red blood cells, mix well, and let it stand for 15 minutes. Use a DM glycated hemoglobin analysis system (HPLC method) to measure the concentration of glycated hemoglobin. The mass percentage concentration of glycated hemoglobin should be in the range of 4.0-6.0%. Centrifuge at 2500 rpm for 10 minutes at 6°C, and retain the supernatant containing glycated hemoglobin.

[0067] Step 104: After adding a freeze-drying protectant to the supernatant, freeze-drying is performed. After the freeze-drying is completed, a low-concentration glycated hemoglobin control product can be obtained.

[0068] Table 1 Composition and concentration of cell washing solution, saccharification solution, hemolytic agent and lyophilization protective agent used in Example 1

[0069]

[0070]

[0071] Note: “ / ” means this ingredient is not included.

[0072] The freeze-drying process parameters in step 104 are shown in Table 2.

[0073] Table 2 Freeze-drying process parameters

[0074]

[0075]

[0076] Example 2

[0077] Prepare a high-concentration HbA1c control using the following method:

[0078] Step 201: Collect EDTA anticoagulated whole blood samples (EDTA anticoagulated whole blood samples are stored at 2-8°C for no more than 24 hours, and the red blood cell count in the anticoagulated whole blood sample is 3.5×10 12 ~5.5×10 12 / L, hemoglobin concentration of 12-16 g / L, and mass percentage concentration of glycosylated hemoglobin of 4.0-5.0%), after the anticoagulated whole blood sample passed the biosafety test, it was centrifuged at 2500 rpm at 6°C for 15 minutes, and the red precipitate at the bottom was retained to obtain red blood cells.

[0079] Step 202: The red blood cells are washed five times by centrifugation at 3500 rpm for 10 minutes at a cell washing solution to red blood cell volume ratio of 5:1 at 6°C to remove impurities and obtain red blood cells with higher purity.

[0080] Step 203: Add saccharification solution to the washed red blood cells for in vitro saccharification treatment. The in vitro saccharification temperature is 40°C, and the volume ratio of saccharification solution to red blood cells is 1:1. During the saccharification process, the DM saccharified hemoglobin analysis system (HPLC method) is used to monitor the saccharified hemoglobin concentration in the red blood cells in real time. The saccharified hemoglobin concentration in the red blood cells is monitored at 12 hours, 24 hours, 36 hours, 42 hours, and 48 hours respectively. At 42 hours, the mass percentage concentration of saccharified hemoglobin should be in the range of 8-11%.

[0081] Step 204: Wash the red blood cells five times with a cell washing solution to red blood cell volume ratio of 5:1 and centrifuge at 2500 rpm for 10 minutes at 6°C to remove glucose and prevent further glycation of hemoglobin.

[0082] Step 205: Add a hemolytic agent in an equal volume to the red blood cells, mix well, let stand for 10 minutes, centrifuge at 3000 rpm at 6°C for 10 minutes, and retain the supernatant containing glycated hemoglobin.

[0083] Step 206: After adding a freeze-drying protectant to the supernatant obtained in step 205, freeze-drying is performed. After freeze-drying, a high-concentration glycated hemoglobin control product can be obtained.

[0084] Table 3 Composition and concentration of cell washing solution, saccharification solution, hemolytic agent and lyophilization protective agent used in Example 2

[0085]

[0086] Note: “ / ” means this ingredient is not included.

[0087] The freeze-drying process parameters in step 206 are shown in Table 4.

[0088] Table 4 Freeze-drying process parameters

[0089]

[0090]

[0091] Example 3

[0092] Performance parameter testing of quality control products at low and high concentration levels

[0093] 1. Accuracy

[0094] After calibration using the national standard for normal values ​​and the national standard for abnormal values, the low-concentration quality control product and the high-concentration quality control product were measured on the glycated hemoglobin detection system. The accuracy test results are shown in Table 5.

[0095] Table 5 Accuracy test results

[0096]

[0097] As can be seen from the results in Table 5, the measured concentrations of the low-concentration and high-concentration glycated hemoglobin control products prepared in Examples 1 and 2, respectively, are both within the reference range, thus meeting the accuracy requirements.

[0098] 2. Uniformity

[0099] The coefficient of variation (CV) of the homogeneity between bottles is required 瓶间 ) should not be greater than 3.0%, CV bottle uniformity coefficient of variation (CV 瓶内 The results of the homogeneity test of the low-concentration glycated hemoglobin control product are shown in Table 6, and the results of the homogeneity test of the high-concentration glycated hemoglobin control product are shown in Table 7.

[0100] Table 6 Homogeneity test results of low concentration level of glycated hemoglobin control

[0101]

[0102] Table 7 Homogeneity test results of high concentration level of glycated hemoglobin control

[0103]

[0104]

[0105] It can be seen from the results in Tables 6 and 7 that the low-concentration and high-concentration glycated hemoglobin control products prepared in Examples 1 and 2, respectively, both meet the homogeneity requirements.

[0106] 3. Stability

[0107] Under accelerated conditions at 37°C, place for 16 days; after reconstitution, place at 2-8°C for 40-60 days. The relative deviation between the test results and the labeled value must be within ±10%. The accelerated stability test results for the low-concentration glycated hemoglobin control are shown in Table 8, and the accelerated stability test results for the high-concentration glycated hemoglobin control are shown in Table 9. The open-vial stability test results for the low-concentration glycated hemoglobin control after reconstitution are shown in Table 10, and the open-vial stability test results for the high-concentration glycated hemoglobin control after reconstitution are shown in Table 11.

[0108] Table 8 Accelerated stability test results of low concentration level of glycated hemoglobin control

[0109]

[0110] Table 9 Accelerated stability test results of high concentration level of glycated hemoglobin control

[0111]

[0112]

[0113] Table 10 Results of stability test of low concentration level of glycated hemoglobin control after reconstitution

[0114]

[0115] Table 11 Results of stability test of high concentration level of glycated hemoglobin control after reconstitution

[0116]

[0117]

[0118] It can be seen from the results in Tables 8 to 11 that the glycated hemoglobin control products at low and high concentration levels prepared in Examples 1 and 2 all meet the stability requirements.

[0119] Compared with the existing technology, the kit of the present application includes: cell washing solution, saccharification solution, hemolytic agent and lyophilization protective agent. The saccharification solution can undergo in vitro glycosylation reaction with red blood cells in anticoagulated whole blood samples of healthy normal people, and then prepare glycated hemoglobin control products of different concentrations, avoiding the use of anticoagulated whole blood samples of diabetic patients that are difficult to obtain in previous technologies to prepare glycated hemoglobin control products, reducing the restrictions on preparation raw materials to a certain extent, and reducing preparation costs.

[0120] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0121] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A kit for preparing a glycated hemoglobin control product, characterized in that: include: Cell washing solution, including: buffer, osmotic pressure maintaining agent; Saccharification solution, including: buffer, osmotic pressure maintaining agent, glucose; A hemolytic agent, comprising: a buffer, a first surfactant; Lyoprotectants, including: buffers, osmotic pressure maintainers, stabilizers, and second surfactants; The lyoprotectant comprises: tris(hydroxymethyl)aminomethane 50-100 mmol / L, sodium chloride 0.05-0.1% (mass percentage concentration), trehalose 1-10% (mass percentage concentration), Proclin-300 0.03-0.05% (mass percentage concentration), glycine 3-5% (mass percentage concentration), polyethylene glycol-8000 1-2% (mass percentage concentration), BSA 1-5% (mass percentage concentration), and the balance is purified water.

2. The kit according to claim 1, wherein At least one of the cell washing solution, the saccharification solution, the hemolytic agent, and the lyophilization protectant further comprises a preservative.

3. The kit according to claim 2, wherein The preservative is at least one of sodium benzoate, sodium azide, potassium sorbate, 2-methyl-4-isothiazoline-3-one, and Proclin-300.

4. The kit according to claim 1, wherein The buffer is selected from at least one of glycine buffer, phosphate buffer, borate buffer, Tris buffer, and HEPES buffer; The osmotic pressure maintaining agent is selected from at least one of NaCl, KCl, sodium dihydrogen phosphate, and disodium hydrogen phosphate; The first surfactant is selected from at least one of Tween-20 and Span-40; The stabilizer is selected from at least one of BSA, trehalose, sucrose or HSA; The second surfactant is selected from at least one of polyethylene glycol and glycine.

5. The kit according to claim 2, wherein The cell washing solution comprises: 50-100 mmol / L of tris(hydroxymethyl)aminomethane, 0.05-0.1% (mass percent concentration) of sodium chloride, 0.03-0.05% (mass percent concentration) of Proclin-300, and the balance being purified water, wherein the pH value of the cell washing solution is 7.2-7.4; The saccharification liquid comprises: 50-100 mmol / L of tris(hydroxymethyl)aminomethane, 0.05-0.1% (mass percentage concentration) of sodium chloride, 2-6% (mass percentage concentration) of glucose, 0.03-0.05% (mass percentage concentration) of Proclin-300, and the balance is purified water, wherein the pH value of the saccharification liquid is 7.2-7.4; The hemolytic agent includes: tris(hydroxymethyl)aminomethane 20-50 mmol / L, Proclin-300 0.03-0.05% (mass percentage concentration), Tween-20 0.1-0.5% (mass percentage concentration), Span-40 0.1-0.5% (mass percentage concentration), and the balance is purified water, wherein the pH value of the hemolytic agent is 7.2-7.

4.

6. A method for preparing a glycated hemoglobin control product, characterized in that: The preparation method is based on the kit according to any one of claims 1 to 5, and the preparation method comprises the following steps: Weigh the cell washing solution according to the formula, and use the cell washing solution to wash the red blood cells; Weighing a saccharification solution according to a formula, and using the saccharification solution to perform in vitro glycosylation treatment on the washed red blood cells until the glycated hemoglobin concentration in the red blood cells reaches a preset concentration; Weighing the cell washing solution according to the formula, and using the cell washing solution to wash the red blood cells after in vitro glycosylation treatment to perform deglycosylation; Weighing a hemolytic agent according to a formula, and treating the red blood cells with the hemolytic agent to obtain glycated hemoglobin; The lyophilization protectant is weighed according to the formula, and the lyophilization protectant is used to lyophilize the glycated hemoglobin to obtain a glycated hemoglobin control product.

7. The method according to claim 6, characterized in that Before the step of weighing the cell washing solution according to the formula and using the cell washing solution to wash the red blood cells, the method further comprises: Providing an anticoagulated whole blood sample, wherein the mass percentage concentration of glycated hemoglobin in the anticoagulated whole blood sample is less than the mass percentage concentration of glycated hemoglobin in a glycated hemoglobin control sample; The anticoagulated whole blood is centrifuged at a speed of 2000 to 3000 rpm, a temperature of 2 to 8° C., and a time of 10 to 15 minutes to obtain the red blood cells; Wherein, in the anticoagulated whole blood sample, the red blood cell count is 3.5×10 12 to 5.5×10 12 / L, the hemoglobin concentration is 12 to 16 g / L, and the mass percentage concentration of glycosylated hemoglobin is 4.0 to 5.0%.

8. The method according to claim 6, characterized in that In the step of weighing the cell washing fluid according to the formula and using the cell washing fluid to wash the red blood cells, the number of washes is 3 to 6 times, and during each wash, the volume ratio of the cell washing fluid to the red blood cells is 2:1 to 8:

1. After each wash, centrifugation is performed and the supernatant is removed, wherein the centrifugal speed is 2000 to 3000 rpm, the centrifugal temperature is 2 to 8°C, and the centrifugal time is 5 to 10 minutes.

9. The method according to claim 6, characterized in that In the step of weighing the saccharification liquid according to the formula and using the saccharification liquid to saccharify the washed red blood cells until the glycated hemoglobin concentration in the red blood cells reaches a preset concentration, the volume ratio of the saccharification liquid to the red blood cells is 1:1 to 3:1, the temperature of the in vitro saccharification treatment is 35 to 45° C., the reaction time of the saccharification treatment is 24 hours to 72 hours, and the DM glycated hemoglobin analysis system is used to monitor the glycated hemoglobin concentration in the red blood cells in real time.

10. The method according to claim 6, characterized in that In the step of weighing the cell washing fluid according to the formula and using the cell washing fluid to wash the red blood cells to remove glucose and prevent further glycation of hemoglobin, the number of washes is 3 to 6 times, and during each wash, the volume ratio of the cell washing fluid to the red blood cells is 2:1 to 6:

1. After each wash, centrifugation is performed and the supernatant is removed, wherein the centrifugal speed is 2000 to 3000 rpm, the centrifugal temperature is 2 to 8°C, and the centrifugal time is 5 to 10 minutes.

11. The method according to claim 6, characterized in that In the step of weighing the lyoprotectant according to the formula and freeze-drying the glycated hemoglobin with the lyoprotectant to obtain the glycated hemoglobin control product, the volume ratio of the hemolytic agent to the red blood cells is 1:

1.

12. A kit, characterized in that The kit comprises: Box body; The box body is provided with a calibration product area, a quality control product area and a reagent area, wherein the quality control product area is provided with a reagent bottle for storing quality control products, and the quality control products are quality control products prepared by the method according to any one of claims 6 to 11.

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