Hba1c monoclonal antibody and application thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The application relates to the field of monoclonal antibody technology, in particular to an HbA1c monoclonal antibody and application. BACKGROUND

[0002] Glycosylated hemoglobin HbA1c is one of the key indicators for evaluating the blood glucose control level of diabetes and is an important basis for diagnosing and monitoring diabetes. HbA1c is the product of the non-enzymatic glycation reaction of glucose with hemoglobin in red blood cells and can stably reflect the average blood glucose level in the 120 days before measurement. At present, commonly used HbA1c detection methods include ion exchange chromatography, high performance liquid chromatography, latex immunoturbidimetry and the like, but these methods generally have 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 rapidity, simplicity and sensitivity. However, existing immunological methods still face some challenges such as large influence of temperature on detection results, complicated operation steps, insufficient detection accuracy and the like. In particular, in the latex immunoturbidimetry method, the reagent composition and ratio are complex, long-term storage can easily lead to a decrease in antibody activity, and the stability of the detection results is affected.

[0004] Therefore, it is urgent to develop an HbA1c detection method capable of overcoming the above-mentioned defects. An ideal detection method should have the characteristics of rapidity, accuracy and low cost, be able to work stably under various experimental conditions, and the detection results should not be disturbed by external factors. In addition, developing HbA1c specific antibodies with high specificity and sensitivity is also an important direction for improving detection accuracy. SUMMARY

[0005] The application aims to provide an HbA1c monoclonal antibody and application.

[0006] To achieve the above-mentioned purpose, the application provides the following technical scheme.

[0007] 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.

[0008] The HbA1c monoclonal antibody provided by the application can be used for preparing a latex immunoturbidimetry detection kit. The kit comprises reagent R1 and reagent R2.

[0009] The reagent R1 is a 25mM glycine buffer solution with pH 8.2 and latex particles.

[0010] The reagent R2 is 25 mM glycine buffer, 0.5 M NaCl, 0.06% Tween 20, 0.1% NaN3, HbA1c monoclonal antibody 0.01 g / L, pH 6.2.

[0011] The HbA1c monoclonal antibody provided by the application can be used for preparing an immunochromatographic detection reagent kit, and the preparation method is as follows: 5 mg of colored microspheres are taken in a centrifuge tube, 1 mL of 50 mM MES buffer solution with pH 6.5 is added, and vortex mixing is performed; 10 μL of 25 mg / mL NHS is added, vortex mixing is performed, and then 10 μL of 25 mg / mL EDC is added, vortex mixing is performed, and then incubation is performed at room temperature for 15 min; the activated colored microspheres are centrifuged at 15000 rpm for 15 min, the supernatant is removed, the microspheres are resuspended in 1 mL of 50 mM MES buffer solution with pH 6.5, and the resuspension is repeated for 3 times.

[0012] 0.5 mg of HbA1c monoclonal antibody is added, vortex mixing is performed, and then incubation is performed at room temperature for 2 h; 100 μL of 10% w / v BSA is added, incubation is performed at room temperature for 30 min; centrifugation is performed at 15000 rpm for 15 min, the supernatant is removed, the microspheres are resuspended in 1 mL of 50 mM PBS with pH 7.4, and the resuspension is repeated for 3 times, and the obtained product is stored at 4 DEG C for standby use.

[0013] The HbA1c monoclonal antibody provided by the application can also be used for preparing an ELISA detection reagent kit.

[0014] Compared with the prior art, the application has the beneficial effects that:

[0015] (1) The HbA1c monoclonal antibody provided by the application has high specificity and high sensitivity, can maintain stability under various experimental conditions, is not easily disturbed by external factors, and guarantees the accuracy and reliability of detection results.

[0016] (2) The HbA1c monoclonal antibody provided by the application can be used in the fields of biochemical immunoturbidimetric detection, immunochromatographic detection and ELISA, and realizes rapid, efficient and high-sensitivity clinical detection of diabetes antigens. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a structural diagram of the HbA1c monoclonal antibody.

[0018] Figure 2 It is a clinical correlation diagram of the HbA1c reagent (immunoturbidimetric method).

[0019] Figure 3 It is a room temperature bottle opening stability diagram of the HbA1c reagent (immunoturbidimetric method). DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be clearly and completely described with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of the present application.

[0021] Example 1 Preparation of HbA1c monoclonal antibody

[0022] (1) Immunization of animals

[0023] The immunization scheme is selected according to the characteristics of the antigen. For a soluble antigen, the immunogenicity is weak, and an adjuvant is generally added. Commonly used adjuvants are Freund's complete adjuvant and Freund's incomplete adjuvant. The antigen and the adjuvant are required to be mixed together in equal volume and ground into a chyliform of water-in-oil.

[0024] The first immunization is performed by subcutaneous multi-point injection of 50 μg of antigen plus Freund's complete adjuvant; the second immunization (same dose of antigen plus Freund's incomplete adjuvant) is performed after 3 weeks; the third immunization is performed without adjuvant by intraperitoneal injection, and the titer is detected after 7 days; and the final booster immunization (50 μg of antigen by intraperitoneal injection) is performed, and the spleen cells are taken for fusion after 3 days.

[0025] (2) Cell fusion

[0026] Myeloma cell suspension: a myeloma cell strain is selected, and myeloma cells are separated from a tumor growing in vivo to prepare a cell suspension;

[0027] Spleen cell suspension: the mouse immunized by the booster immunization 3 days ago is subjected to orbital blood collection, and the serum is separated and stored for later use. The mouse is sacrificed and soaked in 75% alcohol for 3-5 min. The spleen is taken out under sterile conditions, washed in a dish containing 5 mL of incomplete culture solution, and the surrounding connective tissue is cut off. The spleen is moved to a steel mesh in another dish containing 5 mL of incomplete culture solution, cut into 3-5 small pieces with scissors, and then ground with a syringe core. The spleen cell suspension is moved to a 50 mL centrifuge tube, 50 mL of incomplete culture solution is added, and centrifugation is performed at 1000 r / min for 5 min. The supernatant is discarded, and the same washing and centrifugation are performed once. Then the cell pellet is resuspended in 10 mL of incomplete culture solution, and the viable cell count is determined. One mouse can obtain 0.5-2 x 10 8 spleen cells.

[0028] Feeding cells: After the mice were killed, surface sterilized and fixed, the abdominal skin was lifted up from the back with sterilized tweezers to expose the peritoneum. The peritoneum was sterilized with an alcohol cotton ball. 10 mL of incomplete medium was injected into the abdominal cavity with a syringe, taking care not to penetrate the intestine. The right hand held the syringe with the needle remaining in the abdominal cavity, and the left hand held an alcohol cotton ball to gently massage the abdomen for 1 min, after which the injected medium was sucked out. Centrifugation was performed at 1000 r / min for 10 min, and the supernatant was discarded. The precipitated cells were suspended in 5 mL of HAT medium, and according to the cell count, HAT medium was added to make the cell concentration 2 x 10 5 / mL, ready for use.

[0029] Cell fusion: The prepared myeloma cells and mouse spleen cells were mixed at a ratio of 1:5, and 20 mL of PRMI-1640 liquid was added. Centrifugation was performed at 1000 r / min for 10 min, and the supernatant was discarded. The bottom of the centrifuge tube was gently tapped with the fingers to disperse the precipitated cells, and the centrifuge tube was placed in a 37°C water bath. 1 mL of 37°C pre-warmed 50% PEG was slowly added to the centrifuge tube, and it was added for about 45 s, with gentle stirring during the addition. 20 mL of incomplete complete medium (37°C pre-warmed) was added within 5 min, 1 mL in the first minute, 2 mL in the second minute, 5 mL in the third minute, and 6 mL in the fourth and fifth minutes. At the same time, the centrifuge tube should be gently rotated during the addition, and the liquid should be added along the wall of the tube, not directly onto the precipitated cells, to prevent the freshly fused cells from being washed away. Then add 1640 liquid to 50 mL to terminate the PEG action. Centrifugation was performed at 800 r / min for 10 min, and the supernatant was discarded. The precipitated cells were gently suspended in the required volume of HAT medium, and a 96-well culture plate was inoculated (0.10-0.15 mL per well). After inoculation, the culture plate was placed in a 37°C 5% CO2 incubator for culture.

[0030] (3) Selective culture

[0031] After 5 days of inoculation, 1 / 2 of the medium was replaced with HAT medium, and after 7-10 days, HAT medium was replaced with HT medium. After 14 days, ordinary complete medium can be used.

[0032] (4) Screening and cloning of hybridoma positive clones

[0033] Coating: The antigen was coated with Na2CO3-NaHCO3 buffer at pH 9.6, 20 μg of antigen was added to each enzyme-labeled plate, and the coating volume per well was 50 μL, and the coating was performed at 37°C for 2 h.

[0034] Blocking: The antigen was removed, and the liquid in the wells of the enzyme-labeled plate was patted dry. Blocking was performed at 37°C for 2 h with 1% BSA.

[0035] Primary antibody: After blocking, discard the liquid in the hole, pat dry, add the supernatant of the cells to be tested, 100 μL per hole, incubate at 37°C for 1 h.

[0036] Secondary antibody: After incubation of the primary antibody, discard the liquid in the hole, pat dry, wash with PBST for 3 times, 5 min each time. Dilute the secondary antibody according to the instructions, add 100 μL per hole, incubate at 37°C for 1 h.

[0037] Color development: After incubation of the secondary antibody, discard the liquid in the hole, pat dry, add the color developing agent, and terminate the reaction with the termination liquid when the color is the darkest. Read the OD value of each hole with an enzyme marker, select positive holes, expand culture and freeze.

[0038] (5) Purification of monoclonal antibody

[0039] The HbA1c monoclonal antibody was purified by affinity purification method, staphylococcal protein A was cross-linked with the carrier to prepare an affinity chromatography column to bind and elute the antibody, and the antibody was concentrated to 5 mg / mL. The heavy chain sequence of the HbA1c monoclonal antibody obtained by purification is shown as SEQ ID NO: 1, and the light chain sequence is shown as SEQ ID NO: 2. The structure diagram of the HbA1c monoclonal antibody is shown as Figure 1 .

[0040] Example 2 Preparation of HbA1c detection kit (latex immunoturbidimetry) by HbA1c monoclonal antibody

[0041] Reagent R1: 25 mM glycine buffer (pH 8.2), latex particles (LD-PS2116, Nanjing Liding Medical Biotechnology Co., Ltd.).

[0042] Reagent R2: 25 mM glycine buffer (pH 6.2), 0.5 M NaCl, 0.06% Tween 20, 0.1% NaN3, 0.01 g / L of HbA1c monoclonal antibody prepared in Example 1.

[0043] Detection instrument: Hitachi 7180 automatic biochemical analyzer

[0044] Parameters: sample volume 4 μL, reagent R1 150 μL, reagent R2 50 μL, fitting mode Spline, main wavelength 660 nm, secondary wavelength none.

[0045] Example 3 Preparation of HbA1c detection kit (chromatography latex method) by HbA1c monoclonal antibody

[0046] Take 5 mg of color microspheres (PR300, Nanjing Liteng Medical Biotechnology Co., Ltd.) in a centrifuge tube, add 1 mL of 50 mM pH 6.5 MES buffer, vortex mix; add 10 μL of 25 mg / mL NHS, vortex mix, then add 10 μL of 25 mg / mL EDC, vortex mix, then incubate at room temperature for 15 min; centrifuge the activated color microspheres at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres in 1 mL of 50 mM pH 6.5 MES buffer, repeat 3 times; add 0.5 mg of HbA1c monoclonal antibody prepared in Example 1, vortex mix, then incubate at room temperature for 2 h; add 100 μL of 10% (w / v) BSA, 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.

[0047] To highlight the beneficial effects of the present application, the following comparative example experiments are exemplified.

[0048] Comparative Example 1: Preparation of HbA1c test kit (latex immunoturbidimetry method) using HbA1c mAb (Beijing Bai'oleibo Technology Co., Ltd., Catalog No.: ARB13293)

[0049] Compared with Example 2, the HbA1c monoclonal antibody in reagent R2 in Example 2 is replaced with HbA1c mAb (Beijing Bai'oleibo Technology Co., Ltd., Catalog No.: ARB13293), and the rest of the reagents and parameters remain unchanged.

[0050] Comparative Example 2: Preparation of HbA1c test kit (chromatography latex method) using HbA1c mAb (Beijing Bai'oleibo Technology Co., Ltd., Catalog No.: ARB13293)

[0051] Compared with Example 3, the HbA1c monoclonal antibody in Example 3 is replaced with HbA1c mAb (Beijing Bai'oleibo Technology Co., Ltd., Catalog No.: ARB13293), and the rest of the reagents and parameters remain unchanged.

[0052] Using the kit in Example 2, 44 clinical samples were tested, and compared with the HPLC measured values, the correlation coefficient R 2 = 0.995, which has very good correlation, while the correlation coefficient R 2 in Comparative Example 1 is 0.99, as shown in Figure 2 The reagents R1 and R2 were placed at room temperature, and the low and high value room temperature bottle opening stability was tested, and the deviation of the HbA1c test kit (immunoturbidimetry method) in Example 2 within 14 days was within 5%, as shown in Figure 3As shown, the HbA1c kit is better than that in Comparative Example 1.

[0053] Table 1 HbA1c calibrator detection

[0054]

[0055] Note: (1) The signal value is read by a card reader, and the higher the value represents the more obvious color development; (2) S1, S2, S3, S4 are 3%, 6%, 12%, 15%, respectively.

[0056] Table 1 is the detection of HbA1c calibrator by chromatography latex method, wherein S1, S2, S3, S4 are gradient dilutions of 3%, 6%, 12%, 15%, respectively, all of which can be detected, and the signal value is higher than that of Comparative Example 2, indicating that Example 3 has better sensitivity.

[0057] Although embodiments of the present application have been shown and described, it is to be understood that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An HbAlc monoclonal antibody, characterized by: The heavy chain sequence of which is shown as SEQ ID NO: 1, and the light chain sequence of which is shown as SEQ ID NO:

2.

2. The use of the HbA1c monoclonal antibody in claim 1 in the preparation of a latex immunoturbidimetric detection kit.

3. The use of the HbA1c monoclonal antibody according to claim 2 in the preparation of a latex immunoturbidimetric test kit, characterized by the fact that it is a latex immunoturbidimetric test kit for the determination of HbA1c in human blood serum. The kit comprises reagent R1 and reagent R2; The reagent R1 is 25mM glycine buffer of pH 8.2, latex particles; The reagent R2 is 25mM glycine buffer of pH 6.2, 0.5M NaCl, 0.06% Tween 20, 0.1% NaN3, 0.01g / L of the HbA1c monoclonal antibody in claim 1.

4. The use of the HbA1c monoclonal antibody in claim 1 in the preparation of an immunochromatographic detection kit.

5. The use of the HbA1c monoclonal antibody according to claim 4 for the preparation of an immunochromatographic test kit, characterized by the fact that: The preparation method of the kit is as follows: 5 mg of colored microspheres are taken in a centrifuge tube, 1 mL of 50mM MES buffer of pH 6.5 is added, and vortexed to mix well; 10 μL of 25mg / mL NHS is added, vortexed to mix well, and then 10 μL of 25mg / mL EDC is added, vortexed to mix well, and 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 50mM MES buffer of pH 6.5, and the operation is repeated for 3 times; 0.5 mg of the HbA1c monoclonal antibody in claim 1 is added, vortexed to mix well, and then incubated at room temperature for 2 h; 100 μL of 10% w / v BSA is added, and incubated at room temperature for 30 min; centrifuged at 15000 rpm for 15 min, the supernatant is removed, the microspheres are resuspended in 1 mL of 50mM PBS of pH 7.4, and the operation is repeated for 3 times, and stored at 4°C for standby use.

6. The use of the HbA1c monoclonal antibody in claim 1 in the preparation of an ELISA detection kit.

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