Method for preparing specific glycosylated hemoglobin antibody based on glycosylated hemoglobin

Abstract

The invention provides a method for preparing a specific glycosylated hemoglobin antibody based on glycosylated hemoglobin, and belongs to the technical field of glycosylated hemoglobin antibody preparation. The method for preparing the specific glycosylated hemoglobin antibody based on glycosylated hemoglobin comprises the following steps: collecting blood of a diabetic patient, and purifying to obtain natural glycosylated hemoglobin; preparing a complete antigen based on the natural glycosylated hemoglobin; immunizing a mouse by adopting the complete antigen, and then carrying out cell fusion to obtain a positive hybridoma cell with a higher optical density value; and finally, carrying out intraperitoneal injection on a mouse by adopting the positive hybridoma cells with the higher optical density value to prepare ascites, thereby obtaining the specific glycosylated hemoglobin antibody. The antibody obtained by the method solves the technical bottlenecks that the domestic HbA1c antibody is poor in specificity and the product quality cannot meet clinical application, and brings important research value for prevention, diagnosis and treatment of diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the technical field of glycated hemoglobin antibody preparation, and in particular to a method for preparing specific glycated hemoglobin antibodies based on glycated hemoglobin. Background Art

[0002] The glycated hemoglobin (HbA1c) content in the blood of healthy adults is generally 4%-6%, a relatively low level. HbA1c levels in diabetic patients can reach as high as 15%-18%, but obtaining blood from diabetic patients is difficult. Therefore, purified Hb from healthy adults is subjected to in vitro glycosylation in the hope of increasing the glycation rate. In solution, a large amount of glucose exists in an inactive cyclic form, while the active, non-cyclic, chain-like glucose forms account for only 0.002-0.004%. Furthermore, Hb glycation is a spontaneous, non-enzymatic reaction, resulting in a slow, relatively low glycation rate. Research data from Yang Jinyun et al. demonstrates that by optimizing in vitro reaction conditions (e.g., glucose content, pH, reaction temperature, and time), the glycation rate can be increased to as high as 29%. Even though the glycation rate of in vitro glycosylated Hb can reach as high as 29%, its components are a mixture of HbA1c, HbA0, and other proteins. Therefore, in addition to crude Hb extraction from blood, further protein separation and purification is required. Existing methods for HbA1c separation and detection include: cation exchange, electrophoresis, and isoelectric focusing electrophoresis based on charge differences; affinity and immunoassays based on structural differences; and colorimetric and enzymatic methods based on chemical reaction properties.

[0003] The Bio-Rcx70 ion exchange method is the most commonly used laboratory and clinical method for detecting and separating HbA1c. This method separates Hb components based on their charge. The isoelectric point of HbA1c is 6.75, and that of HbA10 is 6.85. Therefore, Hb is positively charged at pH 6.4-6.6. Using eluents of varying pH (Pondus hydrogenii, pH) and ionic strength, the glycated components are first eluted using a low-salt sodium phosphate buffer at pH 6.6, followed by a high-salt sodium phosphate buffer at pH 6.4 to elute the non-glycosylated components. The elution order is HbA1(a+b), HbA1c, and HbA10. Other post-translationally modified Hb variants, such as HbF, HbS, HbC, formylated Hb, and acetylated Hb, may coelute with HbA1c due to their similar net surface charge to HbA1c, thus affecting HbA1c separation efficiency. In 2005, Goodall reported that the HbA1c peak separated by the Bio-Rex 70 ion exchange method contained only 65%-75% glycosylated moieties. In addition to the commonly used Bio-Rex 70 ion exchange method, Cai Haobin also reported using CM Sepharose Fast Flow to obtain HbA1c protein with a concentration greater than 99%. However, the resin exchange capacity is smaller than that of Bio-Rex 70 resin and can be used as a reference method. Another ion exchange method, published by Jeppsson et al., utilizes MonoSHR5 / 5 cation exchange to separate HbA1c. In its ion exchange operation manual, GE Healthcare details the operating steps for using Mono S high-performance liquid chromatography (HPLC) to detect HbA1c. Due to its superior detection accuracy, it can be used as a method for HbA1c purity analysis.

[0004] The basic principle of the affinity method is to crosslink aminophenylboronic acid with a stationary phase matrix as a separation medium. The hydroxyl groups carried by the boronic acid groups reversibly bind to the cis-diol formed by Hb glycosylation, binding total GHb to the separation medium and separating it from non-glycosylated Hb. The bound GHb fraction is then eluted with sorbitol. Colorimetric results indicate that the glycosylated portion bound to the boronic acid groups accounts for only 52% of HbA1c, making this method ineffective as a preliminary separation. However, the boronic acid groups on affinity chromatography media are unable to bind to Hb variants such as HbF, HbS, HbC, formylated Hb, and acetylated Hb. These variants do not coelute with HbA1c, so affinity chromatography does not affect the purity of HbA1c separation and can be used as a further protein purification step. Because the presence of variants does not interfere with HbA1c determination, affinity chromatography is more widely applicable. Therefore, boronic acid derivatives are often used as the immobilization matrix for HbA1c sensors for electrochemical detection of HbA1c.

[0005] In addition to the commonly used boric acid affinity method, sensors for HbA1c detection also utilize immobilized antibodies. Due to their advantages such as high specificity, compact size, high sensitivity, and minimal interference from other substances and the environment, these methods are often used in the design of micro-HbA1c sensors. However, due to their irreversibility, they are not suitable for protein separation and purification. Enzymatic methods involve enzymatic hydrolysis of HbA1c and then indirectly measure HbA1c levels through enzyme catalysis. However, these methods are also unsuitable for HbA1c separation and purification due to their integration with conventional medical equipment and the inability to recycle the sample.

[0006] Tu Guohua et al. used a combination of ion exchange and boric acid affinity to separate and purify Hb from normal adults, obtaining HbA1c with a purity of 99.73%. This method can serve as a foundation for the separation and purification of HbA1c and lays the foundation for the analysis and crystallization screening of HbA1c.

[0007] Since the rise of monoclonal technology in the 1980s, with the rapid development of cell biology and molecular biology techniques, monoclonal antibodies have been widely used in disease diagnosis, environmental monitoring, biomedical research and other fields due to their advantages such as strong specificity, high sensitivity, uniform properties and easy availability.

[0008] The technology for preparing monoclonal antibodies against small molecules, as opposed to large proteins, is based on molecular weights typically less than 5000. These molecules are considered haptens, lacking immunogenicity and possessing only immunoreactivity. Therefore, the key to developing this technology lies in the design and preparation of specific antibodies for immunoassays against these small molecules. Haptens are conjugated to immunogenic carrier proteins to further synthesize complete antigens. Immunogenicity is achieved through the T-cell epitopes of the carrier protein, thereby stimulating an immune response in animals and generating specific antibodies. The design and preparation of haptens is based on the molecular structure of the analyte to be detected. The appropriate conjugation carrier is typically selected based on the hapten's chemical structure and active groups. Commonly used carrier proteins include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), chicken ovalbumin (OVA), rabbit serum albumin (RSA), human serum albumin (HSA), and polylysine (PLL). BSA is a commonly used peptide carrier, but because it is often used as a blocking agent in laboratory assays, its use as a carrier protein to conjugate haptens to synthesize complete antigens for animal immunization can result in the production of BSA-specific antibodies, which can lead to false-positive results during antibody screening. KLH is often used as a peptide carrier due to its high antigenicity. However, due to its large molecular weight, it easily precipitates during hapten conjugation, resulting in reduced protein content. Furthermore, it can stimulate a large number of B lymphocyte clones specific for KLH's own antigenic determinants during animal immunization, making it less commonly used. OVA is often used as a secondary carrier for antibody screening when screening antibodies against the peptide rather than the carrier protein during monoclonal antibody preparation. OVA can minimize antibody reactivity against the carrier protein. Different preparation methods can be used to prepare complete antigens, depending on the reactive groups of the small molecule. Common methods for conjugating the carrier protein and small molecule include the carbodiimide method, glutaraldehyde method, mixed anhydride method, active ester method, diazotization method, and physical methods. To obtain a complete antigen of high purity and stability, the prepared artificial antigen must be purified and identified. Commonly used antigen identification methods include electrophoresis, mass spectrometry, UV scanning, thin-layer chromatography, spectroscopy, nuclear magnetic resonance, and animal immunization. Animal immunization, electrophoresis, spectroscopy, and mass spectrometry are often chosen for artificial antigen identification due to their ease of use, low cost, and high accuracy.

[0009] In recent years, with the advancement of disciplines and technologies such as bioinformatics, molecular biology, and artificial intelligence, new MAb production technologies have emerged. Monoclonal antibodies have evolved from murine antibodies to fully humanized antibodies, with significant improvements in immunogenicity and safety. Previously, the most widely used monoclonal antibody technologies included hybridoma technology, phage display technology, and single B cell antibody production technology. In addition, chimeric antibody technology, transgenic mouse technology, ribosome display technology, and yeast cell display technology can also be used for monoclonal antibody production. Single B cell antibody technology is currently a research hotspot due to its ability to rapidly produce humanized or fully humanized monoclonal antibodies with high specificity and affinity. Phage display technology has become the main experimental technique for humanized antibody applications because it does not require a cell fusion step and is not restricted by the internal environment or immunization methods. Compared with the above two methods, hybridoma technology remains the preferred technology for laboratory production of monoclonal antibodies due to its low cost, ease of use, and improved clinical diagnostics.

[0010] As a key raw material for HbA1c level testing, domestic HbA1c antibodies suffer from technical bottlenecks such as poor specificity and insufficient product quality for clinical application. This has led to long-term reliance on imports and restrictions from foreign countries. This testing program has placed significant pressure on diabetes prevention, diagnosis, and treatment in my country.

[0011] Therefore, providing a method for convenient and rapid diagnosis of HbA1C has important research value and social benefits. Summary of the Invention

[0012] The purpose of the present invention is to provide a method for preparing specific glycated hemoglobin antibodies based on glycated hemoglobin, aiming to solve the technical problems in the prior art that HbA1c antibodies have poor specificity and product quality cannot meet clinical application requirements.

[0013] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0014] The present invention provides a method for preparing a specific glycated hemoglobin antibody based on glycated hemoglobin, comprising the following steps:

[0015] Blood from diabetic patients is collected and purified to obtain natural glycosylated hemoglobin;

[0016] preparing a complete antigen based on the natural glycated hemoglobin;

[0017] Immunizing mice with the complete antigen, and then performing cell fusion to obtain positive hybridoma cells with a higher optical density value;

[0018] Finally, the positive hybridoma cells with higher optical density values are injected into the peritoneal cavity of mice to prepare ascites and obtain the specific glycated hemoglobin antibody.

[0019] Furthermore, the purification includes separation by cation exchange chromatography.

[0020] Furthermore, the preparation method of the complete antigen is to select the N-terminal 10 amino acid residues of the natural glycated hemoglobin β chain, artificially synthesize a glycosylated polypeptide, and use the carbodiimide method to activate the carboxyl group on the hapten with carbodiimide and N-hydroxysuccinimide and then couple it to the amino group on the carrier protein.

[0021] Furthermore, the complete antigen includes an immune antigen and a detection antigen.

[0022] Furthermore, the immunization method is to use the natural glycated hemoglobin-complete antigen-natural glycated hemoglobin-complete antigen four times immunization.

[0023] Furthermore, after the immunization, the mouse serum is titered, and mice with the same titer are used for further cell fusion.

[0024] Furthermore, the preparation of ascites includes using 1640 culture medium to adjust the positive hybridoma cells with a higher optical density value so that the cell number reaches 5×10 6 cells / mL, and then injecting the cells into the abdomen of mice.

[0025] Furthermore, the method further comprises purifying the ascites, wherein the purification adopts an octanoic acid ammonium sulfate precipitation method.

[0026] The present invention also provides a specific glycated hemoglobin antibody obtained by the method for preparing a specific glycated hemoglobin antibody based on glycated hemoglobin as described in the above technical solution.

[0027] The present invention also provides the use of the specific glycated hemoglobin antibody described in the above technical solution in the prevention, diagnosis and treatment of diabetes.

[0028] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0029] The antibodies obtained by the method of the present invention solve the technical bottleneck of poor specificity of domestic HbA1c antibodies and product quality that cannot meet clinical application requirements. There is no need to rely on imports for a long time and be subject to foreign restrictions, which brings important research value to the prevention, diagnosis and treatment of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 and Figure 2 This is a schematic diagram of the results of detecting complete antigens using ultraviolet spectroscopy in Example 1 of the present invention;

[0031] Figure 3This is a schematic diagram of the results of the DOT-ELISA experiment to identify complete antigens in Example 1 of the present invention;

[0032] Figure 4 This is a schematic diagram of the titer results of the serum of mice after four immunizations using the indirect ELISA method in Example 1 of the present invention;

[0033] Figure 5 Schematic diagram of the reactivity test results of four monoclonal antibodies (1C5, 3G5, 3F8, 1B5) with the detection antigen HbA1c-OVA using Western Blot in Example 1 of the present invention. DETAILED DESCRIPTION

[0034] The present invention provides a method for preparing a specific glycated hemoglobin antibody based on glycated hemoglobin, comprising the following steps:

[0035] Blood from diabetic patients is collected and purified to obtain natural glycosylated hemoglobin;

[0036] preparing a complete antigen based on the natural glycated hemoglobin;

[0037] Immunizing mice with the complete antigen, and then performing cell fusion to obtain positive hybridoma cells with a higher optical density value;

[0038] Finally, the positive hybridoma cells with higher optical density values are injected into the peritoneal cavity of mice to prepare ascites and obtain the specific glycated hemoglobin antibody.

[0039] Blood from diabetic patients is collected and purified to obtain natural glycosylated hemoglobin;

[0040] Fresh whole blood from a diabetic patient was collected and centrifuged at 1000 RPM for 20 minutes at 4°C. The upper plasma layer was carefully removed with a pipette. The lower red blood cell layer was then resuspended and washed three times with an equal volume of 0.9% NaCl solution, then resuspended and washed once with 1.0% NaCl solution. Finally, half the volume of lysis buffer was added, the cells were placed in an ice bath, and the red blood cells were disrupted by sonication. Lysis was complete when the lysate turned from viscous to a clear bright red. The lysate was transferred to a centrifuge tube and centrifuged at high speed (15000 RPM for 2 hours at 4°C) to remove cell debris in the lower layer. The supernatant was transferred to a beaker, and solid NaCl was slowly added (to a final concentration of 40-60 mg / mL). The supernatant was then centrifuged at high speed (15000 RPM for 1 hour at 4°C) to remove contaminating proteins in the lower layer. The upper protein layer was dialyzed against a buffer (10 mM NaH2PO4, pH 7.0). The desalted protein solution is packaged, quickly frozen with liquid nitrogen, and stored in a refrigerator at -80°C. In the present invention, the purification includes separation by cation exchange chromatography.

[0041] Before use, the ion exchange chromatography column should be equilibrated with ion exchange solution A. When the pH and conductivity of the influent are the same as those of the effluent, the column is properly equilibrated. Add the protein solution to the ion exchange column. Once the sample has completely entered the column bed, cover it with ion exchange solution A. Connect the tubing and wash the protein unbound to the column. This fraction is designated 100% A, and separate tubes are collected every 30 minutes. When the absorbance decreases (or the color fades) to essentially unchanged levels, switch to 75% ion exchange solution A and 25% ion exchange solution B to elute proteins weakly bound to the column. This fraction is designated 25% B and collected as before. Finally, elute proteins bound to the column with 100% ion exchange solution B. This fraction is designated 100% B and collected as before. Each collected eluate is concentrated separately, diluted with HPLC solution A, and purity assessed by MonoS HPLC.

[0042] After elution is completed, use 0.5M NaOH to regenerate the column. If the pH of the effluent is greater than 9, use ddH2O to wash away the NaOH in the column, which requires about 3 volumes. Finally, use ion exchange solution A to balance until the pH and conductivity of the influent and effluent are equal.

[0043] In the present invention, the preparation method of the complete antigen is to select the N-terminal 10 amino acid residues of the natural glycated hemoglobin β chain, artificially synthesize a glycosylated polypeptide, and use the carbodiimide method to activate the carboxyl group on the hapten with carbodiimide and N-hydroxysuccinimide and then couple it to the amino group on the carrier protein.

[0044] The method for preparing the complete antigen of the present invention is specifically as follows:

[0045] Immune antigen preparation

[0046] (1) BSA activation: Accurately weigh 4 mg of BSA and dissolve it in 4 mL of 0.02 mol / L, pH 6.0 MES. Add 100 μL of 10 mg / mL EDC and 1 mL of 20 mg / mL NHS and mix well. Slightly shake at 25°C to 30°C, 150 rpm, for 1 h.

[0047] (2) Coupling of peptide and BSA: Accurately weigh 2 mg of hapten peptide and dissolve it in 100 μL of sterile purified water, then add it to the above-mentioned activated BSA protein solution and mix thoroughly; control the temperature at 25℃~30℃, 150rpm, and shake gently for 2 hours. Then transfer the reactant into a dialysis bag, then place it on a magnetic stirrer and dialyze it in 10 mmol / L phosphate buffer (PBS) with a pH value of 7.4 at 4℃ for 72 hours; replace the dialysate once every 8~10 hours. After dialysis, centrifuge at 12000 rpm for 10 minutes at 4℃. Take the supernatant and discard the precipitate, divide it into 0.5 mL / vial, and store it at -80℃.

[0048] Detection antigen preparation

[0049] The specific method for preparing the detection antigen (conjugate: HbA1c-OVA) is the same as the above-mentioned immune antigen preparation.

[0050] In the present invention, the complete antigen includes an immunizing antigen and a detecting antigen.

[0051] Immunizing mice with the complete antigen, and then performing cell fusion to obtain positive hybridoma cells with a higher optical density value;

[0052] Three 6-8-week-old female BALB / c mice were immunized with a four-dose regimen of native glycated hemoglobin, complete antigen, and native glycated hemoglobin, complete antigen. For the first immunization, equal volumes of Freund's complete adjuvant and protein were emulsified. The emulsified solution was aspirated with a 1.0 mL syringe and injected intraperitoneally at a dose of 50 μg per mouse into the back and abdomen. For the second, third, and fourth immunizations, equal volumes of Freund's incomplete adjuvant and protein were administered. The immunization method and dosage were the same as for the first immunization. Two weeks after the fourth immunization, tail vein blood was collected and centrifuged, and the supernatant was collected for analysis. Immunization was stopped when the titer reached 1:105, and cell fusion was allowed to proceed. When the cell proliferation area reached approximately 1 / 5 of the well area, the cell supernatant was analyzed by indirect ELISA. Cells with high OD values and few cell clusters were isolated and cultured. Cloning was performed using limiting dilution until single cell clusters with high OD values were obtained and expanded.

[0053] In the present invention, the immunization method is to use the natural glycated hemoglobin-complete antigen-natural glycated hemoglobin-complete antigen four-time immunization method.

[0054] In the present invention, after the immunization, the mouse serum is titered, and mice with the same titer are used for further cell fusion.

[0055] Finally, the positive hybridoma cells with higher optical density values are injected into the peritoneal cavity of mice to prepare ascites and obtain the specific glycated hemoglobin antibody.

[0056] 8-10 week old BALB / c mice were selected for intraperitoneal injection to prepare ascites. Liquid paraffin was drawn up using a 1 mL syringe and injected into the mouse peritoneal cavity, with 0.5 mL of paraffin injected per mouse. One week later, the expanded hybridoma cells were adjusted with 1640 culture medium to a cell count of 5 × 106 cells / mL. 0.2 mL of cell suspension was injected into each mouse. The abdominal condition of the mouse was observed, and ascites were collected 8-10 days later. Centrifuge at 10,000 rpm at 4°C for 20 minutes, retaining the intermediate layer of ascites.

[0057] Purify ascites by caprylic acid ammonium sulfate precipitation method. Mix ascites with 60mmol / L acetate buffer (pH4.3) at a volume ratio of 1:3, add caprylic acid (ascites: caprylic acid volume ratio = 40:1), and shake at 25℃ for 30min. Remove, centrifuge at room temperature, filter with a filter membrane and retain the supernatant, and adjust the pH to 7.2 with NaOH. Add 0.28g of ammonium sulfate per milliliter of ascites, mix well, and let it stand for 30min. Centrifuge at room temperature, discard the supernatant, and suspend the precipitate with 0.01mol / L PBS. Dialyze overnight at 4℃, centrifuge at room temperature, and retain the supernatant.

[0058] In the present invention, the preparation of ascites includes using 1640 culture medium to adjust the positive hybridoma cells with higher optical density values so that the cell number reaches 5×10 6 After the concentration of 100 μg / mL was reached, it was injected into the abdomen of mice.

[0059] The present invention also includes purifying the ascites, and the purification adopts an octanoic acid ammonium sulfate precipitation method.

[0060] After the purification of the present invention is completed, the antibodies are identified and screened, specifically

[0061] Purity was determined by SDS-PAGE electrophoresis;

[0062] Western Blot was used to identify the reactivity of monoclonal antibodies to the complete antigen of glycated hemoglobin;

[0063] Based on the principle of indirect ELISA method, the purified monoclonal antibodies are tested for potency and specific monoclonal antibodies are screened;

[0064] The optimal monoclonal antibody was screened using an ELISA double-antibody sandwich assay. HRP-labeled glycated hemoglobin monoclonal antibodies were prepared using a modified sodium periodate method. Complete glycated hemoglobin antigen (100 ng / well) was cold-packed overnight and blocked at 37°C for 2 hours. 100 μL / well of the selected glycated hemoglobin monoclonal antibody was added, along with unimmunized mouse serum as a negative control. The plates were incubated at 37°C for 30 minutes, washed five times with 1% PBST buffer, and patted dry on absorbent paper. Monoclonal antibody-HRP was added and incubated at 37°C for 30 minutes. Using a multichannel pipette, 50 μL each of colorimetric reagents A and B were pipetted into a 96-well plate and incubated at 37°C for 20 minutes. The plate was analyzed immediately using a microplate reader with dual wavelengths of 450 nm and 630 nm. The highest P / N ratio was calculated, and the optimal monoclonal antibody was identified.

[0065] The present invention also provides a specific glycated hemoglobin antibody obtained by the method for preparing a specific glycated hemoglobin antibody based on glycated hemoglobin as described in the above technical solution.

[0066] The present invention also provides the use of the specific glycated hemoglobin antibody described in the above technical solution in the prevention, diagnosis and treatment of diabetes.

[0067] In the present invention, unless otherwise specified, the raw materials required for preparation are all commercially available products well known to those skilled in the art.

[0068] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0069] Example 1

[0070] Step 1: Prepare fresh whole blood (10 mL) from a diabetic patient. Centrifuge (1000 RPM, 20 min, 4°C) and carefully remove the upper plasma layer using a pipette. Resuspend and wash the lower red blood cells (RBCs) three times with an equal volume of 0.9% NaCl solution, then resuspend and wash once with 1.0% NaCl solution. Finally, add one-half volume of lysis buffer, incubate on ice, and disrupt the RBCs with sonication. Lysis is complete when the lysate changes from viscous to a clear, bright red color. Transfer the lysate to a centrifuge tube and centrifuge at high speed (15,000 RPM, 2 h, 4°C) to remove cell debris. Transfer the supernatant to a beaker, slowly add solid NaCl (to a final NaCl concentration of 40-60 mg / mL), and centrifuge at high speed (15,000 RPM, 1 h, 4°C) to remove contaminating proteins. Desalt the upper protein layer by dialyzing against a buffer (10 mM NaH₂PO₄, pH 7.0). The desalted protein solution was divided into aliquots, quickly frozen with liquid nitrogen, and stored in a refrigerator at -80°C;

[0071] Step 2: The solution is dialyzed against ion exchange solution A for 12 hours until it reaches the column environment. Finally, centrifuge (15,000 RPM, 4°C, 10 min) and pipette for sample loading. Before use, the ion exchange column is equilibrated with ion exchange solution A. When the pH and conductivity of the influent are the same as those of the effluent, the column is properly equilibrated. Add the protein solution to the ion exchange column. Once the sample has completely entered the column bed, cover it with ion exchange solution A. Connect the tubing and wash away proteins not bound to the column. This fraction is designated 100% A. Collect one tube every 30 minutes. When the absorbance decreases (or the color fades) to essentially unchanged levels, switch to 75% ion exchange solution A and 25% ion exchange solution B to elute proteins less bound to the column. This fraction is designated 25% B and collected as before. Finally, use 100% ion exchange solution B to elute proteins bound to the column. This fraction is designated 100% B and collected as before. The collected eluate from each tube was concentrated separately, diluted with HPLCA solution, and the purity was identified by MonoS HPLC.

[0072] After elution is completed, use 0.5M NaOH to regenerate the column. If the pH of the effluent is greater than 9, use ddH2O to wash away the NaOH in the column, which requires about 3 volumes. Finally, use ion exchange solution A to balance until the pH and conductivity of the influent and effluent are equal.

[0073] Step 3: The N-terminal 10 amino acid residues of the hemoglobin β chain (V-HLTPEEKSAC) were selected and artificially synthesized into a glycosylated polypeptide (glycosylated V-HLTPEEKSAC). The carbodiimide method was used to activate the carboxyl group on the hapten with carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and then couple it to the amino group on the carrier protein (BSA and OVA) to synthesize the complete antigen, namely the immune antigen (HbA1c-BSA) and the complete detection antigen (HbA1c-OVA).

[0074] Immune antigen preparation

[0075] (1) BSA activation: Accurately weigh 4 mg of BSA and dissolve it in 4 mL of 0.02 mol / L, pH 6.0 MES. Add 100 μL of 10 mg / mL EDC and 1 mL of 20 mg / mL NHS and mix well. Slightly shake at 25°C to 30°C, 150 rpm, for 1 h.

[0076] (2) Coupling of peptide and BSA: Accurately weigh 2 mg of hapten peptide and dissolve it in 100 μL of sterile purified water, then add it to the above-mentioned activated BSA protein solution and mix thoroughly; control the temperature at 25℃~30℃, 150rpm, and shake gently for 2 hours. Then transfer the reactant into a dialysis bag, then place it on a magnetic stirrer and dialyze it in 10 mmol / L phosphate buffer (PBS) with a pH value of 7.4 at 4℃ for 72 hours; replace the dialysate once every 8~10 hours. After dialysis, centrifuge at 12000 rpm for 10 minutes at 4℃. Take the supernatant and discard the precipitate, divide it into 0.5 mL / vial, and store it at -80℃.

[0077] Detection antigen preparation

[0078] The specific method for preparing the detection antigen (conjugate: HbA1c-OVA) is the same as the above immune antigen preparation

[0079] Step 4: Use UV spectroscopy to detect the complete antigen. The results are as follows: Figure 1 and Figure 2 As shown, based on Figure 1 It can be seen that BSA has a protein characteristic absorption peak at 287nm, and the conjugate BSA-GLU has an absorption peak at 283nm. By comparing the obvious difference in the absorption peaks of the two curves, it is determined that the glycosylated polypeptide is successfully coupled with BSA. Figure 2 It can be seen that OVA has a protein characteristic absorption peak at 286nm, and the conjugate OVA-GLU has an absorption peak at 284nm. Comparing the obvious difference in the absorption peaks of the two curves, it is determined that the glycosylated polypeptide is successfully coupled to OVA;

[0080] Step 5: Use DOT-ELISA to identify the complete antigen. The results are as follows: Figure 3 shown; based on Figure 3 It can be seen that when the coating protein is the immune protein (HbA1c-BSA), the primary antibody is a commercially available HbA1c monoclonal antibody, which can react with the immune protein but not with BSA; when the coating protein is the detection protein (HbA1c-OVA), the primary antibody is a commercially available HbA1c monoclonal antibody, which can react with the detection protein but not with OVA; when the coating protein is PBS, the commercially available HbA1c monoclonal antibody does not react, proving that the immune antigen and the detection antigen are successfully coupled, and also proving that the immune antigen has good immunogenicity and the detection antigen has good immunodetectability;

[0081] Step 6: Three 6-8-week-old female BALB / c mice were immunized with a four-dose regimen of native glycated hemoglobin, complete antigen, and native glycated hemoglobin, complete antigen. For the first immunization, equal volumes of Freund's complete adjuvant and protein were emulsified. A 1.0 mL syringe was used to draw up the emulsified solution and 50 μg / mouse was injected into the back and peritoneal cavity of the mouse. For the second, third, and fourth immunizations, equal volumes of Freund's incomplete adjuvant and protein were used. The immunization method and dosage were the same as for the first immunization. Two weeks after the fourth immunization, tail vein blood was collected and centrifuged, and the supernatant was collected for analysis. Immunization was stopped when the titer reached 1:105, and cell fusion was allowed to proceed. When the cell proliferation area reached approximately 1 / 5 of the well area, the cell supernatant was analyzed by indirect ELISA. Cells with high OD values and few cell clusters were cultured for monoclonal growth. Cloning was performed using limiting dilution until single cell clusters with high OD values were obtained and expanded.

[0082] Among them, the titer of the serum of mice after four immunizations was detected by indirect ELISA method. The results are shown in Figure 4 ,based on Figure 4 It can be seen that according to the optimized optimal ELISA detection conditions, the antibody levels of the two mice serum were tested three days after the fourth immunization. The titers of mice No. 1 and No. 2 were the same, both reaching 1.28×10 5 , more than 1×10 5 , so these two mice can be used for the next step of cell fusion experiment;

[0083] Step 7: Select 8-10 week old BALB / c mice and perform intraperitoneal injection to prepare ascites. Use a 1 mL syringe to draw up liquid paraffin and inject it into the mouse's peritoneal cavity, injecting 0.5 mL of paraffin per mouse. One week later, adjust the expanded hybridoma cells with 1640 culture medium to a cell count of 5 × 106 cells / mL. Inject 0.2 mL of the cell suspension per mouse. Observe the abdominal condition of the mouse. After 8-10 days, collect ascites. Centrifuge at 4°C, 10,000 rpm, for 20 minutes, retaining the intermediate layer of ascites.

[0084] (2) Purify ascites by caprylic acid ammonium sulfate precipitation method. Mix ascites with 60mmol / L acetate buffer (pH 4.3) at a volume ratio of 1:3, add caprylic acid (ascites: caprylic acid volume ratio = 40:1), and shake at 25℃ for 30min. Remove, refrigerate centrifuge, filter with filter membrane and retain the supernatant, and adjust the pH to 7.2 with NaOH. Add 0.28g ammonium sulfate per ml of ascites, mix well, and let it stand for 30min. Refrigerate centrifuge, discard the supernatant, and suspend the precipitate with 0.01mol / L PBS. Dialyze overnight at 4℃, refrigerate centrifuge, and retain the supernatant;

[0085] Step 8:

[0086] (1) Purity identification by SDS-PAGE electrophoresis;

[0087] (2) Western Blot was used to identify the reactivity of the monoclonal antibody to the complete glycated hemoglobin antigen;

[0088] (3) Based on the principle of indirect ELISA method, the purified monoclonal antibodies are tested for titer and specific monoclonal antibodies are screened;

[0089] (4) The best monoclonal antibody was screened using the ELISA double antibody sandwich method, and the HRP labeling of the glycated hemoglobin monoclonal antibody was performed using the improved sodium periodate method; the complete glycated hemoglobin antigen was cold packed at 100 ng / well overnight and blocked at 37°C for 2 hours. The glycated hemoglobin monoclonal antibody screened for analysis was added at 100 μL / well, and the serum of unimmunized mice was used as a negative control. The plate was incubated at 37°C for 30 minutes, washed 5 times with 1% PBST buffer, and patted dry on absorbent paper. Monoclonal antibody-HRP was added and placed at 37°C for 30 minutes. A multi-channel pipette was used to sequentially draw 50 μL of the color developer A solution and B solution and added to a 96-well plate, and placed at 37°C for 20 minutes. The dual wavelength parameters of the microplate reader were set to 450 nm and 630 nm, and the detection was performed immediately. The results of the monoclonal antibody content determination are shown in Table 1. The results of the monoclonal antibody specificity identification are shown in Table 2.

[0090] Table 1 Monoclonal antibody content determination

[0091]

[0092] Table 2 Monoclonal antibody specificity identification

[0093]

[0094]

[0095] The reactivity of four monoclonal antibodies (1C5, 3G5, 3F8, and 1B5) with the detection antigen HbA1c-OVA was analyzed by Western Blot. Figure 5 As shown, all four monoclonal antibodies showed obvious bands at approximately 44 kD, indicating that they reacted well with the fully detected HbA1c-OVA.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a specific glycated hemoglobin antibody based on glycated hemoglobin, characterized in that: The following steps are involved: Blood from diabetic patients is collected and purified to obtain natural glycosylated hemoglobin; preparing a complete antigen based on the natural glycated hemoglobin; Immunizing mice with the complete antigen, and then performing cell fusion to obtain positive hybridoma cells with a higher optical density value; Finally, the positive hybridoma cells with higher optical density values are injected into the peritoneal cavity of mice to prepare ascites and obtain the specific glycated hemoglobin antibody.

2. The method for preparing a specific glycated hemoglobin antibody based on glycated hemoglobin according to claim 1, characterized in that: The purification includes separation using cation exchange chromatography.

3. The method for preparing a specific glycated hemoglobin antibody based on glycated hemoglobin according to claim 1, characterized in that: The preparation method of the complete antigen comprises selecting the N-terminal 10 amino acid residues of the natural glycated hemoglobin β chain, artificially synthesizing a glycosylated polypeptide, and using the carbodiimide method to activate the carboxyl groups on the hapten with carbodiimide and N-hydroxysuccinimide and then couple them to the amino groups on the carrier protein.

4. The method for preparing a specific glycated hemoglobin antibody based on glycated hemoglobin according to claim 1, characterized in that: The complete antigen includes an immune antigen and a detection antigen.

5. The method for preparing specific glycated hemoglobin antibodies based on glycated hemoglobin according to claim 1, characterized in that: The immunization method is to use the natural glycated hemoglobin-complete antigen-natural glycated hemoglobin-complete antigen four times immunization.

6. The method for preparing specific glycated hemoglobin antibodies based on glycated hemoglobin according to claim 1, characterized in that: After the immunization, the mouse serum is titered, and mice with the same titer are used for further cell fusion.

7. The method for preparing specific glycated hemoglobin antibodies based on glycated hemoglobin according to claim 1, characterized in that: The preparation of ascites includes using 1640 culture medium to adjust the positive hybridoma cells with a higher optical density value so that the cell number reaches 5×10 6 After the concentration of 100 μg / mL was reached, it was injected into the abdomen of mice.

8. The method for preparing specific glycated hemoglobin antibodies based on glycated hemoglobin according to claim 1, characterized in that: The method also includes purifying the ascites, wherein the purification adopts an octanoic acid ammonium sulfate precipitation method.

9. A specific glycated hemoglobin antibody obtained by the method for preparing a specific glycated hemoglobin antibody based on glycated hemoglobin according to any one of claims 1 to 8.

10. Use of the specific glycated hemoglobin antibody according to claim 9 in the prevention, diagnosis and treatment of diabetes.

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