HbA1c detection kit and preparation method thereof

Abstract

The invention relates to the technical field of detection kits, in particular to an HbA1c detection kit and a preparation method thereof, and the kit comprises latex microsphere freeze-dried powder DP-1, HbA1c monoclonal antibody freeze-dried powder DP-2, buffer solution dry powder H-1 and buffer solution dry powder H-2. A composite preparation scheme of combining a microsphere and antibody freeze-drying process and a buffer reagent drying process is adopted, on one hand, the traditional biochemical reagent freeze-drying process is greatly simplified through process optimization, and the production time consumption and the cost investment of the freeze-drying link are effectively reduced; and on the other hand, the cold chain dependence in the product storage and transportation process is remarkably reduced due to the dry reagent form, and the overall transportation cost and loss risk are greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of detection kit technology, specifically to an HbA1c detection kit and its preparation method. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia. Its pathological mechanisms primarily involve insulin resistance (decreased cellular sensitivity to insulin) and relative insulin insufficiency. This core abnormality leads to metabolic disorders of carbohydrates, lipids, and proteins, causing progressive damage to multiple systems and organs throughout the body. As one of the world's leading chronic diseases, diabetes is the leading cause of many serious complications in adults, including kidney damage, retinopathy, and limb injuries. Furthermore, diabetes significantly increases the risk of other fatal diseases.

[0003] During glucose metabolism, hemoglobin undergoes an important non-enzymatic chemical reaction to form its specific form—glycated hemoglobin (HbA1c). Biochemical latex immunoturbidimetry is a commonly used technique for clinical determination of HbA1c. It offers rapid detection, requires no complex pretreatment, and provides results for a single sample within 5-15 minutes. It also supports batch testing, quickly meeting the high-reporting-time demands of outpatient and emergency departments, thus shortening patient waiting times. Operationally, it boasts a high degree of automation, strong specificity, a wide linear range, and controllable costs. The instrument and reagent costs are lower than the "gold standard" high-performance liquid chromatography (HPLC). Reagent storage and usage conditions are less stringent, making it easily accessible in primary healthcare institutions. Furthermore, some reagents can reduce the influence of common matrix effects, exhibiting strong anti-interference capabilities.

[0004] However, with the continued rise in market demand for glycated hemoglobin testing reagents (latex turbidimetric method), especially the growth in overseas markets, reagent production has entered a ton-scale industrial production phase. Against this backdrop, the reliance on cold chain logistics and storage and transportation losses during export have become increasingly prominent, significantly driving up overall logistics costs and becoming a key bottleneck restricting the industry's development.

[0005] CN107255727B discloses a latex turbidimetric lyophilized reagent and its preparation method, comprising the following steps: conjugating latex particles with a monoclonal antibody, and then adding a protective agent to obtain a latex antibody reagent, wherein the protective agent is composed of bovine serum albumin, vitamin C, and glycine; subjecting the latex antibody reagent to low-temperature vacuum lyophilization to obtain a lyophilized product, wherein the low-temperature vacuum lyophilization is achieved by repeated freeze-thaw cycles; and sealing the lyophilized product in an inert gas atmosphere to obtain the latex lyophilized reagent. CN112798791B discloses a serum amyloid A detection kit and its preparation and application, the kit comprising at least one of reagent R1 and reagent R2, wherein reagent R2 contains a latex antibody complex, and the antibody is selected from antibodies that can specifically bind to serum amyloid A; the preparation method includes lyophilizing at least one of reagent R1 and reagent R2.

[0006] The aforementioned literature employs lyophilization technology to process the R1 component and / or R2 component of biochemical immunoturbidimetric reagents, with one key component being a latex-antibody complex. However, this type of lyophilization method has significant limitations: (1) it is not suitable for large-scale production of reagents and is difficult to adapt to industrial-scale mass production scenarios; (2) the lyophilization process of latex-antibody complexes has extremely stringent requirements on process parameters—it must ensure that the lyophilization operation does not destroy the binding morphology of latex and antibody, maintain the stable composite structure of the two, and ensure that the activity of the complex is not impaired. Any deviation in the process may lead to reagent performance failure.

[0007] In summary, developing an HbA1c detection kit based on latex turbidimetry that can overcome the above-mentioned defects has become an urgent need in this field. Summary of the Invention

[0008] The purpose of this invention is to provide an HbA1c detection kit and its preparation method. This detection kit has the core performance advantages of strong anti-interference ability, high specificity, excellent sensitivity and outstanding stability, and can accurately meet the stringent requirements of clinical testing for the reliability of results.

[0009] Specifically, the present invention provides the following technical solution: An HbA1c detection kit includes latex microsphere lyophilized powder DP-1, HbA1c monoclonal antibody lyophilized powder DP-2, buffer powder H-1, and buffer powder H-2.

[0010] The preparation method of the HbA1c detection kit of the present invention includes the following steps: (1) Preparation of DP-1 lyophilized latex microsphere powder: Latex microspheres were loaded into a container and concentrated initially using evaporation technology. Then, sucrose, glycerol and Proclin 300 were added. The concentrated microspheres were dispensed into 2 mL / bottles. The dispensed reagent bottles were placed into a vacuum freeze dryer, the vacuum pump was started, and the freeze-drying program was set: the vacuum degree was controlled to be ≤10 Pa, the pre-freezing was carried out at -0℃ for 6 h, and then the freeze-thaw process was repeated 3 times. After completion, the remaining freeze-drying process was continued until the program ended. The freeze dryer was then turned off to obtain DP-1 lyophilized latex microsphere powder. (2) Preparation of HbA1c monoclonal antibody lyophilized powder DP-2: Prepare 10 mM glycine buffer at pH 6.2, and add sucrose, glycerol, hydroxypropyl-β-cyclodextrin and Proclin 300. Filter the buffer solution aseptically through a 0.22 μm filter membrane. Concentrate the HbA1c monoclonal antibody to a concentration of 10 mg / mL by changing the buffer solution. Dispense the concentrated antibody into 1 mL / bottle. Place the dispensed reagent bottles 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 three repeated freeze-thaw cycles. After completion, continue the remaining freeze-drying process until the program ends. Turn off the freeze dryer to obtain HbA1c monoclonal antibody lyophilized powder DP-2. (3) Preparation of buffer powder H-1: Prepare glycine buffer, add Proclin300, and filter the buffer through a 0.22μm filter membrane under sterile conditions; dispense the buffer into 10 mL / bottle, and dry the dispensed reagent bottles in a forced-air drying oven to obtain buffer powder H-1; (4) Preparation of buffer powder H-2: Prepare glycine buffer, add NaCl, Tween 20 and Proclin 300, and filter the buffer through a 0.22μm filter membrane under sterile conditions. Dispense the buffer into 10 mL / bottle, and dry the dispensed reagent bottles in a forced-air drying oven to obtain buffer powder H-2.

[0011] In step (1), the latex microspheres are polystyrene nanospheres. The initial concentration temperature is set to 60°C, the rotation speed is 120 rpm, and the microsphere solid content is concentrated to 20% by open stirring for 6-10 hours.

[0012] In step (1), the mass fraction of sucrose is 2-8%, the mass fraction of glycerol is 0.1%, and the mass fraction of Proclin300 is 0.02-0.08%.

[0013] In the repeated freeze-thaw treatments of steps (1) and (2), each freeze-thaw cycle involves maintaining the temperature at -20°C for 1 hour, followed by maintaining the temperature at 20°C for another hour.

[0014] In step (2), a hollow fiber column is used to concentrate the antibody for the first time.

[0015] In step (2), the sucrose has a mass fraction of 8-15%, the glycerol has a mass fraction of 1-2%, the hydroxypropyl-β-cyclodextrin has a mass fraction of 1-2%, and the Proclin300 has a mass fraction of 0.02-0.08%.

[0016] In step (3), the pH of the glycine buffer is 7.8-8.2, and the mass fraction of Proclin300 is 0.02-0.08%.

[0017] In step (4), the pH of the glycine buffer is 6.0-6.8, the concentration of NaCl is 15-30 mol / L, the mass fraction of Tween 20 is 2-4%, and the mass fraction of Proclin 300 is 0.02-0.08%.

[0018] In steps (3) and (4), the drying temperature is 60°C and the drying time is 1-2 days.

[0019] Compared with the prior art, the beneficial effects of the present invention are: This invention innovatively employs a composite preparation scheme combining microsphere and antibody lyophilization with buffer reagent drying. On one hand, process optimization significantly simplifies the traditional lyophilization process for biochemical reagents, effectively reducing production time and costs in the lyophilization stage. On the other hand, the dried reagent form significantly reduces reliance on the cold chain during product storage and transportation, substantially lowering overall transportation costs and the risk of spoilage. This invention balances performance enhancement with industrial adaptability, fully meeting the diverse practical needs of large-scale industrial production. Attached Figure Description

[0020] Figure 1 The results are the 14-day stability test results of the HbA1c reagent in Example 1 under open bottle conditions of 2-8℃. Detailed Implementation

[0021] 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.

[0022] Example 1 (1) Preparation of DP-1 lyophilized latex microsphere powder 100 mL of latex microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD-PS2116) were loaded into a three-necked flask. The temperature was set to 60℃, the stirring speed was 120 rpm, and the mixture was stirred openly for 6-10 hours to concentrate the solid content of the microspheres to 20% (approximately 50 mL). Then, 4% sucrose, 0.1% glycerol, and 0.08% Proclin 300 were added. The concentrated microspheres were aliquoted into 2 mL vials. The aliquoted vials were placed in a vacuum freeze dryer (Dongfulong Technology Group Co., Ltd., model: LYO-0.5). The vacuum pump was started, and the freeze-drying program was set as follows: the vacuum degree was controlled to ≤10 Pa, and the microspheres were pre-frozen at -50℃ for 6 hours, followed by three repeated freeze-thaw cycles (each freeze-thaw cycle was -20℃ for 1 hour, then at 20℃ for 1 hour). After these cycles, the remaining freeze-drying process was continued until the program ended, and then the freeze dryer was turned off. The HbA1c monoclonal antibody lyophilized powder DP-1 was obtained.

[0023] (2) Preparation of HbA1c monoclonal antibody lyophilized powder DP-2 Prepare a 10 mM glycine buffer (pH 6.2) and add 12% sucrose, 2% glycerol, 1% hydroxypropyl-β-cyclodextrin (HP-β-CD), and 0.04% Proclin 300. Filter the buffer aseptically through a 0.22 μm filter membrane. Concentrate 0.1 g of HbA1c monoclonal antibody (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD-HbA1c-mab-06) to a concentration of 10 mg / mL using this buffer. The concentrated antibody was aliquoted into 1 mL vials. The aliquoted vials were placed in a vacuum freeze dryer (Dongfulong Technology Group Co., Ltd., model: LYO-0.5). The vacuum pump was started, and the freeze-drying program was set: vacuum degree ≤10 Pa, pre-freezing at -50℃ for 6 h, followed by three repeated freeze-thaw cycles (each freeze-thaw cycle consisting of holding at -20℃ for 1 h, then holding at 20℃ for 1 h). After completion, the remaining freeze-drying process was continued until the program ended, at which point the freeze dryer was turned off. The resulting HbA1c monoclonal antibody lyophilized powder DP-2 was obtained.

[0024] (3) Preparation of buffer dry powder H-1 Prepare a 10M pH 8.0 glycine buffer solution, add 0.04% Proclin 300, and sterilely filter the buffer solution through a 0.22μm filter membrane. Aliquot the buffer solution into 10 mL vials, and dry the aliquoted vials in a forced-air drying oven at 60℃ for 1-2 days to obtain buffer solution powder H-1.

[0025] (4) Preparation of buffer dry powder H-2 Prepare a 10M pH 6.2 glycine buffer solution by adding 20M NaCl, 2.5% Tween 20, and 0.04% Proclin 300, and then sterilely filter the buffer solution through a 0.22μm filter membrane. Aliquot the buffer solution into 10 mL vials and dry the vials in a forced-air drying oven at 60℃ for 1-2 days to obtain buffer powder H-2.

[0026] Example 2 (1) Preparation of DP-1 lyophilized latex microsphere powder 200 mL of latex microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD-PS2116) were loaded into a three-necked flask. The temperature was set to 60℃, the stirring speed was 120 rpm, and the mixture was stirred openly for 6-10 hours to concentrate the solid content of the microspheres to 20% (approximately 100 mL). Then, 4% sucrose, 0.1% glycerol, and 0.08% Proclin 300 were added. The concentrated microspheres were dispensed into 2 mL vials. The dispensed vials were placed in a vacuum freeze dryer (Dongfulong Technology Group Co., Ltd., model: LYO-0.5). The vacuum pump was started, and the freeze-drying program was set as follows: the vacuum degree was controlled to ≤10 Pa, and the microspheres were pre-frozen at -50℃ for 6 hours, followed by three repeated freeze-thaw cycles (each freeze-thaw cycle consisting of holding at -20℃ for 1 hour, then holding at 20℃ for 1 hour). After these cycles, the remaining freeze-drying process was continued until the program ended, and then the freeze dryer was turned off. The HbA1c monoclonal antibody lyophilized powder DP-1 was obtained.

[0027] (3) Preparation of HbA1c monoclonal antibody lyophilized powder DP-2 Prepare a 10 mM glycine buffer solution at pH 6.2, and add 12% sucrose, 2% glycerol, 1% hydroxypropyl-β-cyclodextrin (HP-β-CD), and 0.04% Proclin 300. Filter the buffer solution aseptically through a 0.22 μm filter membrane. Take 1 g of HbA1c monoclonal antibody (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD-HbA1c-mab-06) and concentrate it to a concentration of 10 mg / mL using this buffer solution. The concentrated antibody was aliquoted into 1 mL vials. The aliquoted vials were placed in a vacuum freeze dryer (Dongfulong Technology Group Co., Ltd., model: LYO-0.5). The vacuum pump was started, and the freeze-drying program was set: vacuum degree ≤10 Pa, pre-freezing at -50℃ for 6 h, followed by three repeated freeze-thaw cycles (each freeze-thaw cycle consisting of holding at -20℃ for 1 h, then holding at 20℃ for 1 h). After completion, the remaining freeze-drying process was continued until the program ended, at which point the freeze dryer was turned off. The resulting HbA1c monoclonal antibody lyophilized powder DP-2 was obtained.

[0028] (3) Preparation of buffer dry powder H-1 Prepare a 10M pH 8.0 glycine buffer solution, add 0.04% Proclin 300, and sterilely filter the buffer solution through a 0.22μm filter membrane. Aliquot the buffer solution into 10 mL vials, and dry the aliquoted vials in a forced-air drying oven at 60℃ for 1-2 days to obtain buffer solution powder H-1.

[0029] (4) Preparation of buffer dry powder H-2 Prepare a 10M pH 6.2 glycine buffer solution by adding 20M NaCl, 2.5% Tween 20, and 0.04% Proclin 300, and then sterilely filter the buffer solution through a 0.22μm filter membrane. Aliquot the buffer solution into 10 mL vials and dry the vials in a forced-air drying oven at 60℃ for 1-2 days to obtain buffer powder H-2.

[0030] Example 3 Performance Testing (1) Reagent preparation R1 reagent preparation: Add ultrapure water to buffer powder H-1 to prepare glycine buffer with a final concentration of 10 mM, add 0.1% DP-1 to prepare R1 reagent; R2 reagent preparation: Add ultrapure water to buffer powder H-2 to prepare glycine buffer with a final concentration of 25mM, and add 0.001% DP-2 to prepare R2 reagent.

[0031] (2) Detection instrument: Specific protein analyzer (Instrument manufacturer: Shenzhen Jinrui Biotechnology Co., Ltd.; Instrument model: PA120).

[0032] Table 1. Examples of HbA1c reagent detection in clinical whole blood samples Table 1 shows the clinical whole blood sample test results of HbA1c reagent in Examples 1-2, and compares them with HPLC measurements, with correlation coefficient R. 2 >0.98, indicating a good correlation. Figure 1 The results show the 14-day stability test of the HbA1c reagent in Example 1 under open-bottle conditions at 2-8°C. The data indicate that the relative deviation (CV) over 14 days is <10%, demonstrating the good stability of the reagent.

[0033] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An HbA1c detection kit, characterized in that: This includes latex microsphere lyophilized powder DP-1, HbA1c monoclonal antibody lyophilized powder DP-2, buffer powder H-1, and buffer powder H-2.

2. The method for preparing the HbA1c detection kit according to claim 1, characterized in that, Includes the following steps: (1) Preparation of DP-1 lyophilized latex microsphere powder: Latex microspheres were loaded into a container and concentrated initially using evaporation technology. Then, sucrose, glycerol and Proclin 300 were added. The concentrated microspheres were dispensed. The dispensed reagent bottles were placed in a vacuum freeze dryer, the vacuum pump was started, and the freeze-drying program was set: the vacuum degree was controlled to be ≤10 Pa, the pre-freezing was carried out at -0℃ for 6 hours, and then the freeze-thaw process was repeated 3 times. After completion, the remaining freeze-drying process was continued until the program ended. The freeze dryer was then turned off to obtain DP-1 lyophilized latex microsphere powder. (2) Preparation of HbA1c monoclonal antibody lyophilized powder DP-2: Prepare 10 mM glycine buffer at pH 6.2, and add sucrose, glycerol, hydroxypropyl-β-cyclodextrin and Proclin 300. Filter the buffer solution aseptically through a 0.22 μm filter membrane. Concentrate the HbA1c monoclonal antibody to a concentration of 10 mg / mL by changing the buffer solution. Dispense the concentrated antibody into vials. Place the dispensed vials 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 three repeated freeze-thaw cycles. After completion, continue the remaining freeze-drying process until the program ends. Turn off the freeze dryer to obtain HbA1c monoclonal antibody lyophilized powder DP-2. (3) Preparation of buffer powder H-1: Prepare glycine buffer, add Proclin 300, and filter the buffer through a 0.22μm filter membrane under sterile conditions; dispense the buffer, and dry the dispensed reagent bottles in a forced-air drying oven to obtain buffer powder H-1; (4) Preparation of buffer powder H-2: Prepare glycine buffer, add NaCl, Tween 20 and Proclin 300, and filter the buffer through a 0.22μm filter membrane under sterile conditions. Dispense the buffer into individual reagent bottles and dry them in a forced-air drying oven to obtain buffer powder H-2.

3. The method for preparing the HbA1c detection kit according to claim 2, characterized in that: In step (1), the latex microspheres are polystyrene nanospheres. The initial concentration temperature is set to 60°C, the rotation speed is 120 rpm, and the microsphere solid content is concentrated to 20% by open stirring for 6-10 hours.

4. The method for preparing the HbA1c detection kit according to claim 2, characterized in that: In step (1), the mass fraction of sucrose is 2-8%, the mass fraction of glycerol is 0.1%, and the mass fraction of Proclin300 is 0.02-0.08%.

5. The method for preparing the HbA1c detection kit according to claim 2, characterized in that: In the repeated freeze-thaw treatments of steps (1) and (2), each freeze-thaw cycle involves holding the temperature at -20°C for 1 hour, followed by holding it at 20°C for another hour.

6. The method for preparing the HbA1c detection kit according to claim 2, characterized in that: In step (2), a hollow fiber column is used to concentrate the antibody for the first time.

7. The method for preparing the HbA1c detection kit according to claim 2, characterized in that: In step (2), the sucrose has a mass fraction of 8-15%, the glycerol has a mass fraction of 1-2%, the hydroxypropyl-β-cyclodextrin has a mass fraction of 1-2%, and the Proclin300 has a mass fraction of 0.02-0.08%.

8. The method for preparing the HbA1c detection kit according to claim 2, characterized in that: In step (3), the pH of the glycine buffer is 7.8-8.2, and the mass fraction of Proclin300 is 0.02-0.08%.

9. The method for preparing the HbA1c detection kit according to claim 2, characterized in that: In step (4), the pH of the glycine buffer is 6.0-6.8, the concentration of NaCl is 15-30 mol / L, the mass fraction of Tween 20 is 2-4%, and the mass fraction of Proclin 300 is 0.02-0.08%.

10. The method for preparing the HbA1c detection kit according to claim 2, characterized in that: In steps (3) and (4), the drying temperature is 60°C and the drying time is 1-2 days.

Images