A lyophilized detection reagent for glycated hemoglobin HbA1c and a preparation method and application thereof

Abstract

The application relates to a glycosylated hemoglobin HbA1c freeze-drying detection reagent and a preparation method and application thereof, the preparation method comprising the following steps: preparing surface-aminoated polystyrene microspheres through an emulsion polymerization method, then introducing double bonds through a Schiff base reaction with methyl acrolein, and then preparing anion polystyrene microspheres with hydrophilic chains through free radical polymerization, preparing HbA1c detection reagent R1 freeze-dried powder by using the anion polystyrene microspheres; preparing a hydrophilic crosslinking agent 2,5-bis(2,4-dihydroxy-6-formyl phenoxy) p-phenylenedimethylene, and then preparing HbA1c oligomers by a Schiff base reaction with HbA1c mAb, and preparing HbA1c detection reagent R2 freeze-dried powder by using the HbA1c oligomers. The glycosylated hemoglobin HbA1c freeze-drying detection reagent prepared by the application has high antigen binding efficiency, excellent detection sensitivity, sufficient antibody activity reservation and strong detection specificity.

Description

Technical Field

[0001] This invention relates to the field of detection kit technology, specifically to a lyophilized detection kit for glycated hemoglobin (HbA1c) and its preparation method. Background Technology

[0002] Glycated hemoglobin (HbA1c) is a stable product formed by the non-enzymatic binding of glucose to the N-terminal amino group of the β-chain of hemoglobin in red blood cells. Its content can objectively reflect the average blood glucose level of the test subject over the past 2-3 months, and is not affected by momentary factors such as a single meal or exercise. Currently, commonly used HbA1c detection technologies in clinical practice mainly include high-performance liquid chromatography (HPLC), immunoassay, and enzymatic methods. Among them, HPLC has the advantages of high detection accuracy and strong specificity, but the required testing equipment is expensive (usually exceeding 500,000 yuan), the operation procedure is complex, and the professional skills required for operators are high, making it difficult to widely apply in primary healthcare institutions and on-site testing scenarios. Immunoassay, with its simple operation and relatively low detection cost, has become one of the mainstream choices for clinical batch testing. However, this method relies on imported core raw materials for a long time, resulting in high technical barriers, unstable supply chains, and difficulties in further reducing detection costs. At the same time, some immunoassay reagents have defects such as insufficient detection accuracy and susceptibility to interference, making it difficult to fully meet the needs of precise clinical diagnosis and treatment. Enzymatic detection technology has gradually emerged in recent years, with balanced detection performance, but it also faces practical challenges such as insufficient reagent stability and harsh storage and transportation conditions.

[0003] From the perspective of reagent formulation, most HbA1c test kits currently on the market are liquid reagents. These reagents have significant performance limitations: Firstly, the bioactive components (such as enzymes, antibodies, and proteins) in liquid reagents are easily degraded and inactivated by environmental factors such as temperature and humidity, leading to a decrease in reagent detection performance. They need to be stored and transported under refrigerated conditions of 2-8℃, which not only increases logistics and warehousing costs but also limits their application in areas with inadequate cold chain facilities. Secondly, liquid reagents have a short shelf life (usually 6-12 months), which is not conducive to long-term storage. Furthermore, multi-component liquid reagents require cumbersome dilution and mixing operations during use, increasing the risk of operational errors and reducing detection efficiency. Therefore, developing an HbA1c test kit that is highly stable, convenient to store and transport, and easy to use has become a key requirement for overcoming current technological bottlenecks.

[0004] Freeze-drying technology, as a highly efficient method for preserving biological agents, removes moisture by sublimation after freezing the material in a vacuum environment. This maximizes the preservation of the structure and function of bioactive components and significantly improves the stability of the formulation. Freeze-dried reagents can be stored and transported at room temperature, with a shelf life extended to 18-24 months or even longer. Furthermore, freeze-dried reagents are typically prepared as pre-formed reagent beads or powders, requiring only reconstitution with buffer solution before use, greatly simplifying the process and reducing operational errors. Currently, lyophilization technology has been gradually applied in the field of in vitro diagnostic (IVD) reagents. However, its application in HbA1c detection kits still faces many technical challenges, such as: how to optimize the lyophilization formulation (e.g., the selection and ratio of stabilizers and fillers) to ensure the activity retention of HbA1c-related bioactive components during lyophilization and reconstitution; how to optimize lyophilization process parameters (e.g., freezing temperature, sublimation pressure, drying time) to avoid problems such as lyophilization collapse, excessive residual moisture, and poor reconstitution performance; and how to achieve the technical goal of making the detection performance (accuracy, specificity, linear range, etc.) of lyophilized kits comparable to or even better than that of liquid reagents.

[0005] In summary, addressing the technical challenges and market demands of current HbA1c detection kits, such as insufficient stability, inconvenient storage and transportation, reliance on imported core technologies, and the need to improve detection efficiency, this application aims to provide a lyophilized HbA1c detection kit and its preparation method. By optimizing the lyophilization formula and process, the kit achieves high stability, high detection accuracy, and ease of use, meeting the needs of clinical medical institutions at all levels and diverse testing scenarios. Summary of the Invention

[0006] The purpose of this invention is to provide a lyophilized detection reagent for glycated hemoglobin (HbA1c), its preparation method, and its application.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing a lyophilized detection reagent for glycated hemoglobin (HbA1c) includes the following steps:

[0009] Aminated polystyrene microspheres were prepared by emulsion polymerization, and then double bonds were introduced by reacting with methacrolein via Schiff base reaction. Anionic polystyrene microspheres with hydrophilic chains were then prepared by free radical polymerization. HbA1c detection reagent R1 lyophilized powder was prepared using the anionic polystyrene microspheres.

[0010] 2,5-bis(2,4-dihydroxy-6-carboxyphenoxy)terephthalaldehyde, a hydrophilic crosslinking agent, was prepared by affinity substitution reaction. Then, HbA1c oligomers were prepared by reacting HbA1c mAb with HbA1c mAb via Schiff base reaction. The HbA1c detection reagent R2 lyophilized powder was prepared using the HbA1c oligomers.

[0011] The preparation method of the anionic polystyrene microspheres is as follows: pure water is added to the reaction vessel, nitrogen gas is purged for 20 minutes to remove oxygen from the system, sodium dodecyl sulfate is added and dissolved, methacrylamide is added, and styrene is added after stirring for 3 minutes. The mixture is stirred at high speed for 30 minutes, the water bath temperature is set to 80°C, and potassium persulfate is added as an initiator at 60°C. The reaction is carried out overnight to obtain amino polystyrene microspheres, which are then dialyzed against pure water for 3 days and finally stored in pure water.

[0012] Amino polystyrene microspheres were dispersed in an ethanol aqueous solution, methacrolein was added, acetic acid was added dropwise, and the reaction was carried out at room temperature for 30 min to prepare amino polystyrene microspheres with double bonds on the surface. The microspheres were dialyzed with pure water for 3 days and finally stored in pure water.

[0013] Amino polystyrene microspheres with double bonds on their surface were dispersed in pure water. Nitrogen gas was purged for 20 min to remove oxygen from the system. Sodium dodecyl sulfate and sodium methacrylate were added, and the mixture was stirred at high speed for 30 min. The water bath temperature was set to 80℃. Potassium persulfate, the initiator, was added at 60℃. The reaction was carried out overnight to obtain anionic polystyrene microspheres. The microspheres were dialyzed against pure water for 3 days and finally stored in pure water.

[0014] The volume ratio of styrene to methacrylamide is 1:0.01-0.1.

[0015] The specific method for preparing 2,5-bis(2,4-dihydroxy-6-formylphenoxy)-terephthalaldehyde involves sequentially adding 2,5-dibromo-terephthalaldehyde, 2,4,6-trihydroxybenzaldehyde, potassium carbonate, and N,N-dimethylformamide (DMF) to a reaction vessel. The reaction vessel is placed in an oil bath, and magnetic stirring is turned on to completely dissolve the system. The temperature is raised to 90°C and maintained overnight. After the reaction is completed, the mixture is cooled to room temperature, and the reaction solution is purified by neutral alumina column chromatography. The target eluent is collected, concentrated under reduced pressure, and the product 2,5-bis(2,4-dihydroxy-6-formylphenoxy)-terephthalaldehyde is obtained.

[0016] The molar ratio of 2,5-dibromo-terephthalaldehyde to 2,4,6-trihydroxybenzaldehyde is 1:2.0-2.6.

[0017] The specific method for preparing the HbA1c oligomer is as follows: Dilute HbA1c mAb to a concentration of 2 mg / mL with PBS buffer. Take 5 mL of this solution into an EP tube and slowly add a 1% (w / w) aqueous solution of 2,5-bis(2,4-dihydroxy-6-formylphenoxy)terephthalaldehyde, gently inverting to mix. Place the reaction tube in a 25°C constant temperature shaker and incubate at 200 rpm for 6 hours. After incubation, add a 5 mg / mL sodium borohydride solution to the reaction system, gently mix, and let stand at room temperature for 60 min. Transfer the reaction solution to a dialysis bag and dialyze with 10 mM pH 7.4 PBS at 4°C for 48 h, changing the dialysate 4 times during this period to obtain the HbA1c oligomer.

[0018] The volume ratio of the HbA1c mAb diluent to the 2,5-bis(2,4-dihydroxy-6-formylphenoxy) terephthalaldehyde aqueous solution is 1:0.01-0.1, and the volume ratio of the sum of the volumes of the HbA1c mAb diluent and the 2,5-bis(2,4-dihydroxy-6-formylphenoxy) terephthalaldehyde aqueous solution to the sodium borohydride solution is 10-15:1.

[0019] The preparation method of the HbA1c detection reagent R1 lyophilized powder is as follows: Tris buffer solution is prepared, and anionic polystyrene microspheres, trehalose, mannitol, and Proclin 300 are added sequentially. The solution is then dispensed into vials, which are placed in a vacuum freeze dryer. The vacuum pump is started, and the freeze-drying program is set as follows: the vacuum degree is controlled to ≤10 Pa, and the solution is pre-frozen at -50℃ for 6 h, followed by three repeated freeze-thaw cycles. Each freeze-thaw cycle involves holding the solution at -20℃ for 1 h, then holding it at 20℃ for 1 h. After this, the remaining freeze-drying process continues until the program ends. The freeze dryer is then turned off to obtain the R1 lyophilized powder. The Tris buffer solution concentration is 10-20 mmol / L, the content of the anionic polystyrene microspheres is 0.08-0.15%, the content of the trehalose is 5-20%, the content of the mannitol is 2-10%, and the content of the Proclin 300 is 0.04-0.1%.

[0020] The preparation method of the HbA1c detection reagent R2 lyophilized powder is as follows: prepare Tris buffer solution, and add NaCl, sucrose, PVP K30, Tween 80, BSA, HbA1c oligomer and Proclin 300 to it in sequence; then dispense the reagent into a vacuum freeze dryer, start the vacuum pump, and set the freeze-drying program: control the vacuum degree ≤10 Pa, pre-freeze at -50℃ for 6 h, then perform 3 repeated freeze-thaw treatments, each freeze-thaw process is -20℃ for 1 h and then at 20℃ for 1 h, and after completion, continue the remaining freeze-drying process until the program ends, then turn off the freeze dryer to obtain R2 lyophilized powder. The Tris buffer solution has a concentration of 10-20 mmol / L, the NaCl content is 1-5%, the sucrose content is 1-5%, the PVP K30 content is 1-2%, the Tween 80 content is 0.01-0.05%, the BSA content is 0.1-1%, the HbA1c oligomer concentration is 50-80 mg / L, and the Proclin 300 content is 0.04-0.1%.

[0021] The lyophilized HbA1c detection reagent described in this invention can be used to prepare a lyophilized HbA1c detection kit.

[0022] During the testing process, R1 lyophilized powder and R2 lyophilized powder are first reconstituted into working solutions. Then, the reconstituted reagents and the sample to be tested are loaded into the matching testing instrument. The instrument automatically draws the reagents into the reaction vessel. The anionic polystyrene microspheres first bind to the glycated hemoglobin (HbA1c) antigen in the sample through physical adsorption, and then specifically bind to the glycated hemoglobin oligomers, thus completing the entire testing process.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] The lyophilized HbA1c detection reagent prepared by this invention exhibits high antigen binding efficiency, excellent detection sensitivity, sufficient antibody activity retention, strong detection specificity, and a simple, efficient process route, making it easy to scale up industrially. Reagent kits prepared using this lyophilized reagent demonstrate strong stability, convenient storage and transportation, and a wide range of applications. Attached Figure Description

[0025] Figure 1 This is a route diagram for the preparation of HbA1c oligomers.

[0026] Figure 2 This is a flowchart illustrating the preparation process of anionic polystyrene microspheres.

[0027] Figure 3 The image shows the test results for the HbA1c calibrator.

[0028] Figure 4 The graph shows the results of the bottle-opening stability test at 4℃.

[0029] Figure 5 The graph shows the results of the accelerated stability test at 37℃. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1: Preparation of 2,5-bis(2,4-dihydroxy-6-formylphenoxy)-terephthalaldehyde

[0032] To a dry, clean reaction flask, add 1 mmol of 2,5-dibromo-terephthalaldehyde, 2.2 mmol of 2,4,6-trihydroxybenzaldehyde, 0.2 mmol of potassium carbonate (K₂CO₃), and 50 mL of N,N-dimethylformamide (DMF). Place the flask in an oil bath and turn on magnetic stirring to completely dissolve the substances. Heat to 90°C and maintain the temperature overnight. After the reaction is complete, cool to room temperature and purify the reaction solution by neutral alumina column chromatography. Collect the target eluent, concentrate under reduced pressure, and obtain the product 2,5-bis(2,4-dihydroxy-6-formylphenoxy)-terephthalaldehyde.

[0033] Example 2 Preparation of HbA1c oligomers

[0034] like Figure 1 As shown, HbA1c mAb (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD-mAb-HbA1c-06) was diluted to a concentration of 2 mg / mL with 10 mM pH 7.4 PBS buffer. 5 mL of this solution was transferred to an EP tube, and 200 μL of 1% 2,5-bis(2,4-dihydroxy-6-formylphenoxy)terephthalaldehyde aqueous solution from Example 1 was slowly added, followed by gentle inversion to mix. The reaction tube was placed in a 25°C constant temperature shaker and incubated with gentle shaking at 200 rpm for 6 hours. After incubation, 520 μL of 5 mg / mL sodium borohydride solution was added to the reaction system, and the mixture was gently mixed and allowed to stand at room temperature for 60 min. The reaction solution was then transferred to a dialysis bag and dialyzed against 10 mM pH 7.4 PBS at 4°C for 48 h, with the dialysate changed 4 times during the process, to obtain HbA1c oligomers.

[0035] Example 3 Preparation of anionic polystyrene microspheres

[0036] like Figure 2 As shown, 100 mL of pure water was added to a three-necked flask, and nitrogen gas was purged for 20 min to remove oxygen from the system. 48 mg of sodium dodecyl sulfate was added and dissolved. Then, 200 µL of methacrylamide was added, and the mixture was stirred for 3 min. 10 mL of styrene was then added, and the mixture was stirred at high speed for 30 min. The water bath temperature was set to 80 °C. At 60 °C, 100 mg of potassium persulfate (dissolved in 1 mL of pure water) was added as an initiator, and the reaction was allowed to proceed overnight to obtain aminopolystyrene microspheres with a particle size of 120 nm and a PDI < 0.05. The microspheres were dialyzed against pure water for 3 days and finally stored in pure water.

[0037] 1 g of aminopolystyrene microspheres were dispersed in a 50% ethanol aqueous solution, 100 µL of methacrolein was added, 3 drops of acetic acid were added, and the mixture was reacted at room temperature for 30 min to prepare aminopolystyrene microspheres with double bonds on the surface. The microspheres were dialyzed against pure water for 3 days and finally stored in pure water.

[0038] The above-mentioned amino-polystyrene microspheres were dispersed in 50 mL of pure water, and nitrogen gas was purged for 20 min to remove oxygen from the system. 20 mg of sodium dodecyl sulfate and 200 µL of sodium methylpropene sulfonate were added, and the mixture was stirred at high speed for 30 min. The water bath temperature was set to 80 °C, and 10 mg of potassium persulfate (dissolved in 0.1 mL of pure water) was added at 60 °C. The reaction was carried out overnight to obtain anionic polystyrene microspheres with a particle size of 150 nm and a PDI < 0.05. The microspheres were dialyzed against pure water for 3 days and finally stored in pure water.

[0039] Example 4: Preparation of HbA1c detection reagent by biochemical immunoturbidimetric assay

[0040] Preparation of HbA1c detection reagent R1: Prepare 12 mmol / L Tris buffer (pH 7.2), and add 0.1% (w / v) of the anionic polystyrene microspheres prepared in Example 3, 10% (w / v) trehalose, 5% (w / v) mannitol, and 0.1% (v / v) Proclin 300 sequentially. Dispense 2 mL / bottle. Place the dispensed reagent bottles into a vacuum freeze dryer (Dongfulong Technology Group Co., Ltd., model: LYO-0.5), start the vacuum pump, and set the freeze-drying program: control the vacuum degree ≤10 Pa, pre-freeze at -50℃ for 6 h, then perform 3 repeated freeze-thaw cycles (each freeze-thaw cycle involves holding at -20℃ for 1 h, then holding at 20℃ for 1 h). After completion, continue the remaining freeze-drying process until the program ends, then turn off the freeze dryer. Obtain R1 freeze-dried powder.

[0041] Preparation of HbA1c detection reagent R2: Prepare 15 mmol / L Tris buffer (pH 7.2), and add 2% (w / v) NaCl, 5% (w / v) sucrose, 2% (w / v) PVP K30, 0.1% (v / v) Tween 80, 0.1% (w / v) BSA, 50 mg / L HbA1c oligomer prepared in Example 2, and 0.1% (v / v) Proclin 300 in sequence. Dispense reagents at 2 mL / bottle; place the dispensed vials into a vacuum freeze dryer (Dongfulong Technology Group Co., Ltd., model: LYO-0.5), start the vacuum pump, and set the freeze-drying program: control the vacuum degree ≤10 Pa, pre-freeze at -50℃ for 6 h, then perform 3 repeated freeze-thaw cycles (each freeze-thaw cycle involves holding at -20℃ for 1 h, then holding at 20℃ for 1 h), and continue the remaining freeze-drying process until the program ends, then turn off the freeze dryer. R2 freeze-dried powder is obtained.

[0042] Detection instrument: Specific protein analyzer (Shenzhen Jinrui Biotechnology Co., Ltd.), instrument model: PA120.

[0043] Figure 3 For HbA1c calibrator testing, R 2 >0.99, indicating a high correlation between the test concentration and the target sample concentration.

[0044] Figure 4 The stability of low-value (L) and high-value (H) samples after opening was tested at 4℃. The relative deviation between the low-value and high-value samples after 14 days was within 10%, indicating good stability after opening.

[0045] Figure 5 The accelerated stability of low-value (L) and high-value (H) samples was tested at 37℃. The relative deviation between low-value and high-value samples after 14 days was within 10%, indicating good accelerated stability.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing a lyophilized detection reagent for glycated hemoglobin (HbA1c), characterized in that, Includes the following steps: Aminated polystyrene microspheres were prepared by emulsion polymerization, and then double bonds were introduced by reacting with methacrolein via Schiff base reaction. Anionic polystyrene microspheres with hydrophilic chains were then prepared by free radical polymerization. HbA1c detection reagent R1 lyophilized powder was prepared using the anionic polystyrene microspheres. 2,5-bis(2,4-dihydroxy-6-carboxyphenoxy)terephthalaldehyde, a hydrophilic crosslinking agent, was prepared by affinity substitution reaction. Then, HbA1c oligomers were prepared by reacting HbA1c mAb with HbA1c mAb via Schiff base reaction. The HbA1c detection reagent R2 lyophilized powder was prepared using the HbA1c oligomers.

2. The method for preparing the lyophilized detection reagent for glycated hemoglobin (HbA1c) according to claim 1, characterized in that: The specific preparation method of the anionic polystyrene microspheres is as follows: pure water is added to the reaction vessel, nitrogen gas is passed through to remove oxygen from the system, sodium dodecyl sulfate is added, and after dissolution, methacrylamide is added. Styrene is added after stirring, and the mixture is stirred at high speed. The water bath temperature is set to 80°C. Potassium persulfate, the initiator, is added at 60°C. The reaction is carried out overnight to obtain amino polystyrene microspheres, which are then dialyzed with pure water and finally stored in pure water. Amino polystyrene microspheres were dispersed in an ethanol-water solution, methacrolein was added, acetic acid was added dropwise, and the reaction was carried out at room temperature to prepare amino polystyrene microspheres with double bonds on the surface. The microspheres were then dialyzed with pure water and finally stored in pure water. Amino polystyrene microspheres with double bonds on their surface were dispersed in pure water. Nitrogen gas was purged to remove oxygen from the system. Sodium dodecyl sulfate and sodium methacrylate were added, and the mixture was stirred at high speed. The water bath temperature was set to 80°C. Potassium persulfate, the initiator, was added at 60°C. The reaction was carried out overnight to obtain anionic polystyrene microspheres. The microspheres were dialyzed with pure water and finally stored in pure water. The volume ratio of styrene to methacrylamide is 1:0.01-0.

1.

3. The method for preparing the lyophilized HbA1c detection reagent according to claim 1, characterized in that: The specific method for preparing 2,5-bis(2,4-dihydroxy-6-formylphenoxy)-terephthalaldehyde is as follows: 2,5-dibromo-terephthalaldehyde, 2,4,6-trihydroxybenzaldehyde, potassium carbonate, and N,N-dimethylformamide are added sequentially to a reaction vessel. The reaction vessel is placed in an oil bath, and magnetic stirring is turned on to completely dissolve the system. The temperature is raised to 90°C and maintained overnight. After the reaction is completed, the mixture is cooled to room temperature, and the reaction solution is purified by neutral alumina column chromatography. The target eluent is collected, concentrated under reduced pressure, and the product 2,5-bis(2,4-dihydroxy-6-formylphenoxy)-terephthalaldehyde is obtained. The molar ratio of 2,5-dibromo-terephthalaldehyde to 2,4,6-trihydroxybenzaldehyde is 1:2.0-2.

6.

4. The method for preparing the lyophilized HbA1c detection reagent according to claim 1, characterized in that: The specific method for preparing the HbA1c oligomer is as follows: dilute HbA1c mAb with PBS buffer, place it in an EP tube, add an aqueous solution of 2,5-bis(2,4-dihydroxy-6-formylphenoxy)terephthalaldehyde, and mix well; place the reaction tube in a constant temperature shaker at 25°C and gently shake and incubate; after incubation, add sodium borohydride solution to the reaction system, mix well, and let stand at room temperature; put the reaction solution into a dialysis bag and dialyze with PBS at 4°C to obtain the HbA1c oligomer; The volume ratio of the HbA1c mAb diluent to the 2,5-bis(2,4-dihydroxy-6-formylphenoxy) terephthalaldehyde aqueous solution is 1:0.01-0.1, and the volume ratio of the sum of the volumes of the HbA1c mAb diluent and the 2,5-bis(2,4-dihydroxy-6-formylphenoxy) terephthalaldehyde aqueous solution to the sodium borohydride solution is 10-15:

1.

5. The method for preparing the lyophilized detection reagent for glycated hemoglobin (HbA1c) according to claim 1, characterized in that: The preparation method of the HbA1c detection reagent R1 lyophilized powder is as follows: Tris buffer solution is prepared, and anionic polystyrene microspheres, trehalose, mannitol and Proclin 300 are added to it in sequence; then it is dispensed, the dispensed reagent bottles are placed in a vacuum freeze dryer, the vacuum pump is started, and the freeze-drying program is set: the vacuum degree is controlled to be ≤10 Pa, the pre-freeze is first at -50℃ for 6 h, and then three repeated freeze-thaw treatments are performed. Each freeze-thaw treatment is carried out at -20℃ for 1 h and then at 20℃ for 1 h; after completion, the remaining freeze-drying process is continued until the program ends, the freeze dryer is turned off, and R1 lyophilized powder is obtained.

6. The method for preparing the lyophilized detection reagent for glycated hemoglobin (HbA1c) according to claim 5, characterized in that: In the preparation method of the HbA1c detection reagent R1 lyophilized powder, the Tris buffer concentration is 10-20 mmol / L, the content of the anionic polystyrene microspheres is 0.08-0.15%, the content of the trehalose is 5-20%, the content of the mannitol is 2-10%, and the content of Proclin 300 is 0.04-0.1%.

7. The method for preparing the lyophilized detection reagent for glycated hemoglobin (HbA1c) according to claim 1, characterized in that: The preparation method of the HbA1c detection reagent R2 lyophilized powder is as follows: Prepare Tris buffer solution, and add NaCl, sucrose, PVP K30, Tween 80, BSA, HbA1c oligomer and Proclin 300 to it in sequence; then dispense the solution into a vacuum freeze dryer, start the vacuum pump, and set the freeze-drying program: control the vacuum degree ≤10 Pa, pre-freeze at -50℃ for 6 h, then perform 3 repeated freeze-thaw treatments, each freeze-thaw process is carried out at -20℃ for 1 h and then at 20℃ for 1 h. After completion, continue the remaining freeze-drying process until the program ends, turn off the freeze dryer, and obtain R2 lyophilized powder.

8. The method for preparing the lyophilized detection reagent for glycated hemoglobin (HbA1c) according to claim 7, characterized in that: In the preparation method of the HbA1c detection reagent R2 lyophilized powder, the concentration of the Tris buffer is 10-20 mmol / L, the content of NaCl is 1-5%, the content of sucrose is 1-5%, the content of PVP K30 is 1-2%, the content of Tween 80 is 0.01-0.05%, the content of BSA is 0.1-1%, the concentration of HbA1c oligomer is 50-80 mg / L, and the content of Proclin 300 is 0.04-0.1%.

9. A lyophilized detection reagent for glycated hemoglobin HbA1c obtained by the preparation method according to any one of claims 1-8.

10. The use of the lyophilized hemoglobin HbA1c detection reagent according to claim 9 in the preparation of a lyophilized hemoglobin HbA1c detection kit.

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