HbA1c monoclonal antibody and application thereof

Abstract

The invention relates to the technical field of monoclonal antibodies, in particular to an HbA1c monoclonal antibody and application, the heavy chain sequence of the HbA1c monoclonal antibody is shown as SEQ ID NO: 1, and the light chain sequence of the HbA1c monoclonal antibody is shown as SEQ ID NO: 2. The HbA1c monoclonal antibody provided by the invention has high specificity and high sensitivity, can maintain stability under various experimental conditions, is not easily interfered by external factors, and ensures the accuracy and reliability of a detection result. The HbA1c monoclonal antibody can be used in a plurality of detection fields such as the biochemical immunoturbidimetric detection field, the immunochromatography detection field, the ELISA field and the like, and realizes rapid, efficient and high-sensitivity clinical detection of diabetes antigens.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, specifically to an HbA1c monoclonal antibody and its applications. Background Technology

[0002] Glycated hemoglobin (HbA1c) is a key indicator for assessing glycemic control in diabetes and an important basis for the diagnosis and monitoring of diabetes. HbA1c is a product of the non-enzymatic glycation reaction between glucose and hemoglobin in red blood cells, and it can stably reflect the average blood glucose level over the previous 120 days. Currently, commonly used methods for HbA1c detection include ion exchange chromatography, high-performance liquid chromatography, and latex immunoturbidimetry, but these methods generally suffer from problems such as complex operation, high cost, and long detection time.

[0003] In recent years, immunological methods have been widely used in HbA1c detection due to their advantages such as speed, simplicity, and sensitivity. However, existing immunological methods still face some challenges, such as the significant influence of temperature on test results, cumbersome operation procedures, and insufficient detection accuracy. In particular, in latex immunoturbidimetry, the reagent composition and ratio are complex, and long-term storage can easily lead to a decrease in antibody activity, affecting the stability of the test results.

[0004] Therefore, there is an urgent need to develop an HbA1c detection method that can overcome the aforementioned shortcomings. An ideal detection method should be rapid, accurate, and low-cost, capable of operating stably under various experimental conditions, and its results should be unaffected by external factors. Furthermore, developing HbA1c-specific antibodies with high specificity and sensitivity is also an important direction for improving detection accuracy. Summary of the Invention

[0005] The purpose of this invention is to provide an HbA1c monoclonal antibody and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: An HbA1c monoclonal antibody, the heavy chain sequence of which is shown in SEQ ID NO:1 and the light chain sequence of which is shown in SEQ ID NO:2.

[0007] The HbA1c monoclonal antibody described in this invention can be used to prepare a latex immunoturbidimetric assay kit. The kit includes reagent R1 and reagent R2. The reagent R1 is a 25mM glycine buffer solution at pH 8.2 and latex particles; The reagent R2 is a 25mM glycine buffer at pH 6.2, 0.5M NaCl, 0.06% Tween 20, 0.1% NaN3, and 0.01g / L HbA1c monoclonal antibody.

[0008] The HbA1c monoclonal antibody described in this invention can be used to prepare an immunochromatographic detection kit. The preparation method is as follows: Take 5 mg of colored microspheres into a centrifuge tube, add 1 mL of 50 mM pH 6.5 MES buffer, and vortex to mix; add 10 μL of 25 mg / mL NHS, vortex to mix, then add 10 μL of 25 mg / mL EDC, vortex to mix, and incubate at room temperature for 15 min; centrifuge the activated colored microspheres at 15000 rpm for 15 min, remove the supernatant, and resuspend the microspheres in 1 mL of 50 mM pH 6.5 MES buffer. Repeat this process three times. Add 0.5 mg of HbA1c monoclonal antibody, vortex to mix, and incubate at room temperature for 2 h; add 100 μL of 10% w / v BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres in 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 4 °C for later use.

[0009] The HbA1c monoclonal antibody of this invention can also be used to prepare ELISA detection kits.

[0010] Compared with the prior art, the present invention has the following beneficial effects: (1) The HbA1c monoclonal antibody of the present invention has high specificity and high sensitivity, can maintain stability under various experimental conditions, is not easily affected by external factors, and ensures the accuracy and reliability of the detection results.

[0011] (2) The HbA1c monoclonal antibody of the present invention can be used in multiple detection fields such as biochemical immunoturbidimetric detection, immunochromatographic detection and ELISA, realizing rapid, efficient and high-sensitivity clinical detection of diabetes antigen. Attached Figure Description

[0012] Figure 1 This is a structural diagram of an HbA1c monoclonal antibody.

[0013] Figure 2 This is a clinical correlation diagram of HbA1c reagent (immunoturbidimetric assay).

[0014] Figure 3 The stability of HbA1c reagent (immunoturbidimetric assay) at room temperature after opening is shown in the figure. Detailed Implementation

[0015] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0016] Example 1: Preparation of HbA1c monoclonal antibody (1) Immunized animals The immunization regimen is selected based on the characteristics of the antigen. For soluble antigens with weak immunogenicity, adjuvants are generally added. Commonly used adjuvants are Freund's complete adjuvant and Freund's incomplete adjuvant. The antigen and adjuvant should be mixed in equal volumes and ground into a water-in-oil emulsion.

[0017] The initial immunization was performed by subcutaneous injection of 50 μg antigen with Freund's complete adjuvant at multiple sites; the second immunization was performed 3 weeks later (same dose of antigen with Freund's incomplete adjuvant); the third immunization was performed by intraperitoneal injection without adjuvant, and the titer was tested 7 days later; the final booster immunization was performed (50 μg antigen intraperitoneal injection), and spleen cells were harvested for fusion 3 days later.

[0018] (2) Cell fusion Myeloma cell suspension: Select a good myeloma cell line, isolate myeloma cells from tumors growing in vivo, and prepare a cell suspension; Spleen cell suspension: Mice that received a booster 3 days prior were subjected to orbital bloodletting, and the serum was separated and frozen for later use. The mice were euthanized and immersed in 75% alcohol for 3-5 minutes. The spleen was aseptically removed and washed in a petri dish containing 5 mL of incomplete culture medium. The surrounding connective tissue was removed, and the spleen was transferred to a steel mesh in another petri dish containing 5 mL of incomplete culture medium. It was first cut into 3-5 small pieces with scissors, then ground with a syringe core. The spleen cell suspension was transferred to a 50 mL centrifuge tube, 50 mL of incomplete culture medium was added, and the mixture was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the mixture was washed and centrifuged again using the same method. The precipitated cells were then resuspended in 10 mL of incomplete culture medium, and the viable cell count was recorded. 0.5-2 × 10⁶ cells were obtained from one mouse. 8 One spleen cell.

[0019] Feeder Cells: After euthanizing, disinfecting, and fixing the mice, use sterile scissors to lift the abdominal skin from the posterior abdomen to expose the peritoneum. Disinfect the peritoneum with an alcohol swab. Inject 10 mL of incomplete culture medium into the peritoneal cavity using a syringe, taking care to avoid perforation of the intestine. Hold the syringe firmly with your right hand, leaving the needle in the peritoneal cavity, and gently massage the abdomen with an alcohol swab in your left hand for 1 minute. Then aspirate the injected culture medium. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Resuspend the pelleted cells in 5 mL of HAT medium. Based on the cell count, add more HAT medium to bring the cell concentration to 2 × 10⁶ cells / mL. 5 / mL, for later use.

[0020] Cell fusion: Mix the prepared myeloma cells and mouse spleen cells at a ratio of 1:5, add 20 mL of PRMI-1640 solution; centrifuge at 1000 r / min for 10 min, discard the supernatant, and aspirate as much supernatant as possible; gently tap the bottom of the centrifuge tube with your finger to disperse the precipitated cells, and place the centrifuge tube in a 37℃ water bath; slowly add 1 mL of 50% PEG pre-warmed at 37℃ into the centrifuge tube over about 45 seconds, stirring gently while adding, and let stand at 37℃ for 5 min; within 5 min, add 20 mL of incomplete complete culture medium (pre-warmed at 37℃), adding 1 mL in the first minute, 2 mL in the second minute, 5 mL in the third minute, and 6 mL each in the fourth and fifth minutes, while gently rotating the centrifuge tube as you add the medium. The medium should be added along the tube wall, not directly onto the precipitated cells, to prevent dislodging the newly fused cells. Then add 1640 solution to 50 mL to terminate the PEG effect; centrifuge at 800 r / min for 10 min, discard the supernatant, gently suspend the precipitated cells in the required volume of HAT culture medium, and seed 0.10-0.15 mL per well in a 96-well culture plate. After seeding, place the culture plate in a 37℃ 5% CO2 incubator for culture.

[0021] (3) Selective culture Five days after inoculating 96-well plates, replace half of the medium with HAT medium. After 7-10 days, replace the HAT medium with HT medium. After the 14th day, ordinary complete medium can be used.

[0022] (4) Screening and cloning of hybridoma-positive clones Coating: The antigen was coated with Na2CO3-NaHCO3 buffer at pH 9.6. 20 μg of antigen was added to each microplate, and the coating volume per well was 50 μL. Coating was carried out at 37°C for 2 h.

[0023] Blocking: Discard the antigen, blot dry the liquid in the wells of the ELISA plate, and block with 1% BSA at 37°C for 2 hours.

[0024] Primary antibody: After blocking, discard the liquid in the well, pat dry, add 100 μL of the supernatant of the cells to be tested to each well, and incubate at 37°C for 1 h.

[0025] Secondary antibody: After incubation with primary antibody, discard the liquid in the wells, pat dry, and wash three times with PBST for 5 min each time. Dilute the secondary antibody according to the instructions, add 100 μL to each well, and incubate at 37°C for 1 h.

[0026] Color development: After incubation with the secondary antibody, discard the liquid in the wells, pat dry, add the colorimetric reagent, and stop the reaction with the stop solution when the color is the deepest. Read the OD value of each well with a microplate reader, select the positive wells, expand the culture and freeze them.

[0027] (5) Purification of monoclonal antibodies HbA1c monoclonal antibody was purified using affinity purification. Staphylococcal A protein was cross-linked with a carrier, and an affinity chromatography column was prepared to bind the antibody before elution. The antibody was then concentrated to 5 mg / mL. The heavy chain sequence of the purified HbA1c monoclonal antibody is shown in SEQ ID NO:1, and the light chain sequence is shown in SEQ ID NO:2. The structural diagram of the HbA1c monoclonal antibody is shown below. Figure 1 As shown.

[0028] Example 2: Preparation of HbA1c Monoclonal Antibody for HbA1c Detection Kit (Latex Immunoturbidimetric Assay) Reagent R1: 25mM glycine buffer (pH 8.2), latex particles (LD-PS2116, Nanjing Liding Medical Biotechnology Co., Ltd.).

[0029] Reagent R2: 25mM glycine buffer (pH 6.2), 0.5M NaCl, 0.06% Tween 20, 0.1% NaN3, and 0.01g / L of HbA1c monoclonal antibody prepared in Example 1.

[0030] Testing instrument: Hitachi 7180 fully automated biochemical analyzer Parameters: Sample volume 4 μL, reagent R1 150 μL, reagent R2 50 μL, fitting method Spline, main wavelength 660 nm, secondary wavelength none.

[0031] Example 3: Preparation of HbA1c Monoclonal Antibody for HbA1c Detection Kit (Chromatographic Latex Method) Take 5 mg of colored microspheres (PR300, Nanjing Liding Medical Biotechnology Co., Ltd.) into a centrifuge tube, add 1 mL of 50 mM pH 6.5 MES buffer, and vortex to mix; add 10 μL of 25 mg / mL NHS, vortex to mix, then add 10 μL of 25 mg / mL EDC, vortex to mix, and incubate at room temperature for 15 min; centrifuge the activated colored microspheres at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres in 1 mL of 50 mM pH 6.5 MES buffer, and repeat 3 times; add 0.5 mg of HbA1c monoclonal antibody prepared in Example 1, vortex to mix, and incubate at room temperature for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres in 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 4 °C for later use.

[0032] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.

[0033] Comparative Example 1: An HbA1c detection kit (latex immunoturbidimetric assay) was prepared using HbA1c mAb (Beijing Biolabs Technology Co., Ltd., catalog number: ARB13293). Compared with Example 2, the HbA1c monoclonal antibody in reagent R2 in Example 2 was replaced with HbA1c mAb (Beijing Bio-Lab Technology Co., Ltd., catalog number: ARB13293), while all other reagents and parameters remained unchanged.

[0034] Comparative Example 2: An HbA1c detection kit (chromatographic latex method) was prepared using HbA1c mAb (Beijing Bio-Lab Technology Co., Ltd., catalog number: ARB13293). Compared with Example 3, the HbA1c monoclonal antibody in Example 3 was replaced with HbA1c mAb (Beijing Bio-Lab Technology Co., Ltd., catalog number: ARB13293), while all other reagents and parameters remained unchanged.

[0035] The kit described in Example 2 was used to test 44 clinical samples. The results were compared with HPLC measurements, and the correlation coefficient R was [value missing]. 2 =0.995, indicating a very good correlation, while the reagent correlation coefficient R in Comparative Example 1 is much lower. 2 =0.99, such as Figure 2 As shown, the kit in Example 2 is superior to the kit in Comparative Example 1. Reagents R1 and R2 were placed at room temperature, and the stability of low and high values ​​after opening at room temperature was tested. In Example 2, the deviation between low and high values ​​for the HbA1c kit (immunoturbidimetric assay) was within 5% over 14 days. Figure 3 As shown, it is superior to the HbA1c kit in Comparative Example 1.

[0036] Table 1 HbA1c Calibrator Testing Note: (1) The signal value is read by the card reader. The higher the value, the more obvious the color is; (2) S1, S2, S3 and S4 are 3%, 6%, 12% and 15% respectively.

[0037] Table 1 shows the detection of HbA1c calibrators by the chromatographic latex method. S1, S2, S3, and S4 are gradient dilutions of 3%, 6%, 12%, and 15%, respectively, and all can be detected. Moreover, the signal values ​​are higher than those of Comparative Example 2, indicating that Example 3 has better sensitivity.

[0038] 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 monoclonal antibody, characterized in that: Its heavy chain sequence is shown in SEQ ID NO:

1.

2. The HbA1c monoclonal antibody according to claim 1, characterized in that: Its light chain sequence is shown in SEQ ID NO:

2.

3. The use of the HbA1c monoclonal antibody as described in claim 1 or 2 in the preparation of a latex immunoturbidimetric assay kit.

4. The application of the HbA1c monoclonal antibody according to claim 3 in the preparation of a latex immunoturbidimetric assay kit, characterized in that: The kit includes reagent R1 and reagent R2; The reagent R1 is a 25mM glycine buffer solution at pH 8.2 and latex particles; The reagent R2 is a 25mM glycine buffer at pH 6.2, 0.5M NaCl, 0.06% Tween 20, 0.1% NaN3, and 0.01g / L HbA1c monoclonal antibody.

5. The use of the HbA1c monoclonal antibody as described in claim 1 or 2 in the preparation of an immunochromatographic detection kit.

6. The application of the HbA1c monoclonal antibody according to claim 5 in the preparation of an immunochromatographic detection kit, characterized in that: The kit is prepared as follows: 5 mg of colored microspheres are placed in a centrifuge tube, 1 mL of 50 mM pH 6.5 MES buffer is added, and the mixture is vortexed. 10 μL of 25 mg / mL NHS is added, and the mixture is vortexed. Then, 10 μL of 25 mg / mL EDC is added, and the mixture is vortexed. The mixture is then incubated at room temperature for 15 min. The activated colored microspheres are centrifuged at 15000 rpm for 15 min, the supernatant is removed, and the microspheres are resuspended in 1 mL of 50 mM pH 6.5 MES buffer. This process is repeated 3 times. Add 0.5 mg of HbA1C monoclonal antibody, vortex to mix, and incubate at room temperature for 2 h; add 100 μL of 10% w / v BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres in 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 4 °C for later use.

7. The use of the HbA1c monoclonal antibody as described in claim 1 or 2 in the preparation of an ELISA detection kit.

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