Anti-human GBM recombinant monoclonal antibody and preparation method and application thereof

Abstract

The application provides a recombinant monoclonal antibody against human GBM (glomerular basement membrane) and a preparation method and application thereof, the recombinant monoclonal antibody against human GBM has a heavy chain complementarity determining region as shown in SEQ ID NO. 1-3 and a light chain complementarity determining region as shown in SEQ ID NO. 4-6; the recombinant monoclonal antibody against human GBM has good binding capacity and specificity to human GBM, does not produce cross activity with other self-antigens, and has good thermal stability and freeze-thaw stability, thereby providing raw material guarantee for downstream development of high-quality diagnostic reagents.

Description

Technical Field

[0001] This application belongs to the field of biological detection technology, and in particular relates to an anti-human GBM recombinant monoclonal antibody, its preparation method and application. Background Technology

[0002] Anti-GBM (glomerular basement membrane) disease is a rare, rapidly progressive autoantibody-mediated disease that can simultaneously affect the kidneys and lungs. It is characterized by the production of autoantibodies against the α3 chain of type IV collagen (α3(IV)NC1), with the antigenic determinant located in the non-collagenous globular region NC1 at the carboxyl terminus of collagen IV. This antigen is a key structural component of the glomerular basement membrane (GBM) and alveolar basement membrane; therefore, antibody attack can lead to damage to both organs, potentially causing crescentic glomerulonephritis, diffuse alveolar hemorrhage, and acute respiratory failure. Its clinical progression is explosive, and the mortality rate in untreated patients is extremely high, making it one of the most critical and severe medical conditions.

[0003] Anti-human GBM antibody detection is a key technology for diagnosing rapidly progressive glomerulonephritis and pulmonary hemorrhage-nephritis syndrome, playing a vital role in assisting diagnosis, guiding treatment, assessing prognosis, and monitoring disease progression. In anti-human GBM antibody detection, the quality of the positive antibody control directly affects the reliability of the test results. Current human GBM antibody detection technologies mainly include enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IIFT), and chemiluminescence immunoassay (CLIA). As a core component of the detection system, the quality control is crucial for verifying the accuracy of the detection system, monitoring the stability of the detection process, and ensuring the comparability and interrecognition of results.

[0004] However, existing technologies primarily utilize polyclonal antibodies or monoclonal antibodies prepared using traditional hybridoma techniques, which generally suffer from insufficient antibody specificity and significant batch-to-batch variability. To address these shortcomings, this invention employs flow cytometry to sort anti-GBM memory B cells, combined with single-cell RT-PCR technology to directly obtain antibody genes. Furthermore, through antibody humanization, the risk of immunogenicity in clinical applications is reduced. Compared to traditional hybridoma techniques, the preparation cycle is shortened to within two months. Summary of the Invention

[0005] This application provides an anti-human GBM recombinant monoclonal antibody, its preparation method, and its application to solve the problems existing in related technologies. The technical solution is as follows: In a first aspect, embodiments of this application provide an anti-human GBM recombinant monoclonal antibody, comprising a light chain variable region and a heavy chain variable region; The heavy chain variable region includes complementarity-determining regions VH CDR1, VH CDR2, and VH CDR3; the light chain variable region includes complementarity-determining regions VL CDR1, VL CDR2, and VL CDR3. The amino acid sequence of VH CDR1 is SDFAWN (SEQ ID NO.1), the amino acid sequence of VH CDR2 is YINNRDTTSNNPSLKS (SEQ ID NO.2), and the amino acid sequence of VH CDR3 is GDYGYGYFDV (SEQ ID NO.3). The amino acid sequence of VL CDR1 is RASESIEYYGTSLMQ (SEQ ID NO.4), the amino acid sequence of VL CDR2 is AASNVES (SEQ ID NO.5), and the amino acid sequence of VL CDR3 is QQSRKVPYT (SEQ ID NO.6).

[0006] In one embodiment, the heavy chain variable region amino acid sequence of the anti-human GBM recombinant monoclonal antibody is shown in SEQ ID NO.7; SEQ ID NO.7: EVQLQESGPGLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYINNRDTTSNNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGDYGYGYFDVWGAGTTVTVSA.

[0007] The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8; SEQ ID NO.8: DVLMTQSPASLAVSLGQRATISCRASESIEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDITMYFCQQSRKVPYTFGGGTKLEIK.

[0008] In one embodiment, the anti-human GBM recombinant monoclonal antibody is any one of a mouse antibody, a chimeric antibody, a bispecific antibody, a multispecific antibody, a humanized antibody, or a fully human antibody.

[0009] Secondly, embodiments of this application provide a nucleic acid that encodes the aforementioned anti-human GBM recombinant monoclonal antibody.

[0010] In one embodiment, the nucleotide sequence used to encode the heavy chain variable region is shown in SEQ ID NO.9; SEQ ID NO.9: GAGGTACAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTTTGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAACAACAGGGATACCACTAGCAA CAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCCAGAGGGGACTACGGCTACGGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACTGTCTCTGCG.

[0011] The nucleotide sequence used to encode the variable region of the light chain is shown in SEQ ID NO.10; SEQ ID NO.10: GACGTTTTGATGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGAGCCACCATCTCCTGCAGAGCCAGTGAAAGTATTGAATATTATGGCACAAGTTTAATGCAGTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATC CAACGTAGAATCTGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTGTGGAGGAGGATGATATTACAATGTATTTCTGTCAGCAAAGTAGGAAGGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA.

[0012] Thirdly, embodiments of this application provide a biological material comprising the aforementioned nucleic acid; the biological material is an expression cassette, a vector, or a transgenic cell.

[0013] Fourthly, the embodiments of this application provide a method for preparing the above-mentioned anti-human GBM recombinant monoclonal antibody, wherein the above-mentioned nucleic acid is used for gene expression or the above-mentioned transgenic cells are cultured.

[0014] Fifthly, the application of anti-human GBM recombinant monoclonal antibodies in the preparation of reagents, drugs, or medical devices for detecting human GBM. Sixthly, embodiments of this application provide a reagent for in vitro assay of human GBM, the reagent comprising the above-mentioned anti-human GBM recombinant monoclonal antibody, or the above-mentioned nucleic acid, or the above-mentioned biological material.

[0015] In a seventh aspect, embodiments of this application provide a kit comprising the above-described anti-human GBM recombinant monoclonal antibody, or the above-described nucleic acid, or the above-described biological material, or the above-described reagent; the reagent or kit is used for in vitro detection of human GBM.

[0016] The advantages or beneficial effects of the above technical solutions include at least the following: This invention develops a recombinant monoclonal antibody against human GBM. This recombinant monoclonal antibody against human GBM has a heavy chain complementarity-determining region as shown in SEQ ID NO.1-3 and a light chain complementarity-determining region as shown in SEQ ID NO.4-6. It has excellent binding ability to human GBM protein, and still has a high signal when diluted to 0.06 ng / mL, indicating high sensitivity. It does not exhibit cross-activity with other autoimmune antigens and specifically binds to human GBM antigen. After accelerated treatment at 37°C for 1 day, 3 days, and 7 days, or after 1, 2, and 3 freeze-thaw cycles, the CV relative to the untreated control sample does not exceed 10%, and it has excellent thermal stability and freeze-thaw stability. This provides a raw material guarantee for the downstream development of high-quality diagnostic reagents.

[0017] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0018] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0019] Figure 1 This is an SDS-PAGE image of the recombinant monoclonal antibody against human GBM. Detailed Implementation

[0020] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0021] Example I. Antigen Preparation 1. Eukaryotic expression of human GBM protein The amino acid sequence of the α3 chain of type IV collagen was obtained from the Uniport database (UniPort ID: Q01955). The amino acid range of 1426-1670 amino acids was selected as the antigen protein for expression (this range contains the NC1 domain with an antigenic determinant). The expression sequence was codon-optimized for a eukaryotic expression system using Beijing Qingke Biotechnology Co., Ltd., and the gene was synthesized. The synthesized gene sequence was ligated into the pCAGGS vector using a seamless cloning method. The protein C-terminus has a 6×HIS tag. The specific amino acid sequence is shown in SEQ ID No. 12; the gene sequence is shown in SEQ ID No. 11.

[0022] SEQ ID No. 11: CCAGGTCTGAAGGGTAAACGCGGTGACTCTGGATCACCTGCTACCTGGACAACACGCGGTTTCGTGTTCACAAGGCACTCTCAGACCACCGCTATACCTAGTTGCCCTGAGGGTACTGTGCCTCTGTACAGCGGATTCAGCTTTCTGTTCGTGCAGGGAAACCAGAGGGCTCATGGTCAAGACCTCGGTACACTGGGTAGCTGTCTGCAGAGATTCACCACCATGCCATTTCTGTTCTGCAACGTCAACGACGTCTGCAACTTCGCTTCTCGCAACGACTACAGCTACTGGCTGTCTACACCAGCTCTGATGCCTATGAACATGGCACCTATCACTGGCAGAGCTCTCGAACCATACATCTCCAGATGCACAGTGTGTGAGGGACCTGCTATCGCCATCGCTGTCCATAGCCAGACAACCGACATACCTCCATGCCCTCATGGATGGATCAGCCTGTGGAAGGGCTTCAGCTTCATCATGTTCACCTCTGCTGGTTCAGAGGGTACAGGCCAGGCATTGGCCAGTCCAGGCAGCTGTCTGGAAGAGTTCAGGGCTTCTCCATTCTTGGAGTGTCACGGTAGAGGCACATGCAACTACTACAGCAACAGCTACTCTTTCTGGTTGGCAAGCCTGAACCCTGAGCGGATGTTTCGGAAGCCTATTCCAAGCACTGTGAAAGCTGGCGAATTGGAGAAGATCATCTCTAGATGCCAGGTGTGCATGAAGAAGCGCCAC; SEQ ID No.12: PGLKGKRGDSGSPATWTTRGFVFTRHSQTTAIPSCPEGTVPLYSGFSFLFVQGNQRAHGQDLGTLGSCLQRFTTMPFLFCNVNDVCNFASRNDYSYWLSTPALMPMNMAPITGRALEPYISRC TVCEGPAIAIAVHSQTTDIPPCPHGWISLWKGFSFIMFTSAGSEGTGQALASPGSCLEEFRASPFLECHGRGTCNYYSNSYSFWLASLNPERMFRKPIPSTVKAGELEKIISRCQVCMKKRH.

[0023] 2. Antigen protein purification The expression plasmid was transfected into 293F cells. After transfection, the cells were cultured at 37°C for 1 day, and then cultured for another 5 days with 4% feed. The supernatant was collected by centrifugation. The collected supernatant was purified by affinity chromatography using Ni-NTA affinity resin (Invitrogen™, R90115). The filtered cell supernatant was added to equilibrated packing material and incubated at 4°C for 2.5–3 hours. After loading onto a column, the supernatant was washed sequentially with 10 mL of 1X Native Wash Buffer (10 mM imidazole - 50 mM low-concentration imidazole) to remove contaminating proteins, and finally eluted with 10 mL of Native Elution Buffer (250 mM imidazole) to obtain the target protein. The eluent containing the target protein was added to a dialysis bag, which was then placed in a 1 L beaker containing PBS buffer and dialyzed three times at 4°C. SDS-PAGE was then performed for identification, and the electrophoresis results are shown below. Figure 1 As shown, the molecular weight of human GBM protein is approximately 80 kDa, and its purity is >90%.

[0024] II. Mouse Immunization The initial immunization used Freund's complete adjuvant, while subsequent booster immunizations used Freund's incomplete adjuvant. The initial antigen dose was 100 μg / mouse, and the booster dose was 50 μg / mouse, with an adjuvant-to-antigen volume ratio of 1:1. After emulsification, mice were injected subcutaneously at multiple sites on the nape of the neck, with booster immunizations every 3-4 weeks. Seven days after the third immunization, serum titers were measured using an indirect ELISA method. After 4-5 immunizations, mice with the highest titers were selected for a pulse immunization via intraperitoneal injection, with the antigen mixed with physiological saline at a dose of 150 μg / mouse.

[0025] III. Sorting of Individual B Cells 1. Obtaining mouse lymphocytes Four days after the initial shock immunization, mice were euthanized by cervical dislocation, disinfected by soaking in 70% ethanol for 5 minutes, and the spleens were removed in a biosafety cabinet, ground, and a sterile spleen cell suspension was prepared. The suspension was then centrifuged at 800g for 30 minutes at room temperature using mouse lymphocyte separation medium to obtain the intermediate lymphocyte layer cells.

[0026] 2. Fluorescent staining and sorting of individual B cells PBMCs were washed and resuspended using FACS buffer (10 mM PBS, 1 mM EDTA, 2% FBS). 100 μL of the resuspended cells (5 × 10⁻⁶ cells) were collected. 6 Fluorescent markers were added sequentially: DAPI, CD138-APC, IgG-PE, IgD-PB, IgM-FITC, and antigen protein-PE / cy7, and incubated on ice for 30 min. Lymphocytes were sorted using a BD FACSAria III flow cytometer. Cells with DAPI-, CD138-, IgG+, IgD-, IgM-, and antigen protein+ were selected. The selected B cells were sorted into 96-well PCR plates containing 5 µL of cell lysis buffer, with only one B cell per well, resulting in 88 specific B cells.

[0027] IV. Amplification of the variable region gene of single B cell antibodies 1. Reverse transcription to generate cDNA, The TaKaRa RNase reverse transcription kit was used to reverse transcribe RNA to synthesize cDNA. The specific reaction system is shown in Table 1.

[0028] Table 1 Reverse transcription reaction system

[0029] After thoroughly mixing the reagents according to the ratio in Table 1, add 4.5 μL of the mixed reagent to each well of the sorted 96-well plate. The reaction conditions are: 25℃ for 5 min, 37℃ for 45 min, and 85℃ for 5 s. The obtained cDNA is stored at -20℃.

[0030] 2. Amplification of antibody variable region genes The obtained first-strand cDNA was subjected to PCR amplification. The amplification primers and conditions were as described in the book "Recombinant Antibodies" edited by Shen Beifen (Science Press, 2005). dNTPs, buffer, and hot-start Taq DNA polymerase were added as usual, and finally, 1 μL of cDNA template was added.

[0031] The product can be stored at 4℃ for later use or directly electrophoresed. Take 5 μL of PCR product for electrophoretic analysis and verify on a 1% agarose gel. The length of the variable region of the light and heavy chains is about 400-500 bp.

[0032] 3. Construction and sequencing of humanized plasmids The positive light and heavy chain variable region PCR product was ligated into a human Fc fragment and cloned into a mammalian expression vector (pcDNA3.4(+)). The ligation product was transformed into E. coli competent cells (DH5α), plated on LB agar plates containing ampicillin, and incubated overnight at 37°C. Single colonies were picked, cultured in a shaking environment, and plasmids were extracted for restriction enzyme digestion identification and sequencing. Positive clones were sent to a sequencing company for sequencing.

[0033] 4. Small-batch expression of humanized antibodies The recombinant antibody plasmid with accurate sequencing results was transfected into 293T cells for low-level expression. 30 ml of DMEM medium was mixed with 500 μL of PEI (1 mg / ml), and then added to 96-well plates at 300 μL / well. 6 μL of light chain and 6 μL of heavy chain were added to each well, and the plates were incubated at room temperature for 15 min. The resulting inoculum was then added to the cells. After culturing at 37°C and 5% CO2 for 3 days, the cell supernatant was collected.

[0034] 5. Specific screening of humanized antibody ELISA Indirect ELISA was used to screen for antibodies that specifically bind to human GBM antigen. A protein antigen with the same expression system and label was used as a control group to screen out non-specific antibodies. The specific method is as follows: Dilute the coating antigen to 2 µg / mL with carbonate coating buffer and add 100 µL to each well of the ELISA plate. Incubate overnight at 4°C and wash once with PBST solution. Block with 200 µL of 5% skim milk to each well, incubate overnight at 4°C or 60 min at 37°C, discard the blocking solution, and wash once with PBST solution. Add 100 µL of cell supernatant to each well, incubate at 37°C for 30 min, discard the liquid, blot dry, and wash twice with PBST solution. Add 100 µL of enzyme-labeled secondary antibody to each well, incubate at 37°C for 30 min, discard the liquid, blot dry, and wash four times with PBST solution. Add 100 µL of TMB substrate solution to each well in the dark, incubate at room temperature in the dark for 15 min, and stop the reaction with 50 µL of 2M sulfuric acid per well. Place the ELISA plate on the microplate reader and measure the OD value at 450 nm.

[0035] Table 2 Results of specific screening of human GBM antigen by ELISA

[0036] The results are shown in Table 2. A total of 13 positive antibodies with OD>0.4 were screened from 88 cell samples (columns 1-11, column 12 is the negative control well, cells were not sorted). No non-specific antibodies were found. A total of 13 specific antibodies were obtained.

[0037] V. Antibody Expression and Purification Ten antibodies with ELISA OD values ​​greater than 3 were selected and expressed in moderate batches using 293F cells. The cell density was adjusted to 1.0 × 10⁶ cells at transfection. 6 Cells / mL were transfected with PEI (1 mg / mL) and 80 µg plasmid DNA (light and heavy chains mixed 1:1) at room temperature for 10 min to form a cationic polymer (PEI to DNA mass ratio 3:1). This polymer was slowly added to 293F cells, with each antibody transfecting 80 mL of cells. After 6 days, the cell supernatant was collected for purification. Antibody purification was performed using AT Protein A Diamond (Bestchrom, AA0272). The supernatant was washed with 10 mL of equilibration buffer, followed by antibody elution with 5 mL of 0.1 M glycine. The eluted antibody solution was immediately neutralized with 500 µL of 1 M Tris-HCl and dialyzed against 10 mM PBS at 4 °C. The dialysate was changed every 3 h, and dialyzed 3 times. Antibody purity and concentration were determined by SDS-PAGE and UV absorption.

[0038] VI. Antibody Screening 1. Chemiluminescence method for preliminary screening of antibodies. The prepared antibodies were screened using chemiluminescence immunoassay to identify the antibody with the highest affinity. The specific method is as follows: the coating antigen was a self-produced human GBM antigen, each antibody was used as the analyte, and anti-human IgG secondary antibody was labeled with acridinium ester. Antibody performance was tested on a chemiluminescence platform. The instrument used was an i2910, employing a two-step reaction: first, 50 µL of antigen-coated magnetic bead solution was added, followed by 50 µL of the test antibody (diluted to 10 ng / mL). The reaction was carried out at 37°C for 5 min, followed by washing four times with washing buffer. Then, 50 µL of acridinium ester-labeled anti-human IgG secondary antibody was added, and the reaction was carried out at 37°C for 5 min. After washing four times with washing buffer, the antibody was analyzed. Specific experimental data are shown in Table 3. The antibody with the highest affinity obtained in the initial screening was antibody number 1, i.e., 1F1.

[0039] Table 3 Chemiluminescence screening results

[0040] VII. Analysis of Sequencing Results of Screened Antibody Plasmids Analysis of the 1F1 plasmid sequencing results revealed that the heavy chain variable region includes complementarity-determining regions VH CDR1, VHCDR2, and VH CDR3; the light chain variable region includes complementarity-determining regions VL CDR1, VL CDR2, and VL CDR3. Specifically, the amino acid sequence of VH CDR1 is SDFAWN (SEQ ID NO.1), the amino acid sequence of VH CDR2 is YINNRDTTSNNPSLKS (SEQ ID NO.2), and the amino acid sequence of VH CDR3 is GDYGYGYFDV (SEQ ID NO.3). The amino acid sequence of VL CDR1 is RASESIEYYGTSLMQ (SEQ ID NO.4), the amino acid sequence of VL CDR2 is AASNVES (SEQ ID NO.5), and the amino acid sequence of VL CDR3 is QQSRKVPYT (SEQ ID NO.6).

[0041] In one embodiment, the heavy chain variable region amino acid sequence of the anti-human GBM recombinant monoclonal antibody is shown in SEQ ID NO.7; SEQ ID NO.7: EVQLQESGPGLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYINNRDTTSNNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGDYGYGYFDVWGAGTTVTVSA.

[0042] The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8; SEQ ID NO.8: DVLMTQSPASLAVSLGQRATISCRASESIEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDITMYFCQQSRKVPYTFGGGTKLEIK.

[0043] VIII. Characterization of Monoclonal Antibodies 1. Sensitivity of 1F1 antibody The approximate sensitivity of 1F1 antibody was determined using chemiluminescence immunoassay. The general method is as follows: The coating antigen was purchased human GBM antigen. The 1F1 antibody was serially diluted as the analyte. Acridinium ester was used as a labeling agent to label anti-human IgG secondary antibody. A two-step reaction was performed, and antibody performance was tested on a chemiluminescence platform. First, 50 µL of antigen-coated magnetic bead solution was added, followed by 50 µL of the antibody to be tested. The reaction was carried out at 37°C for 5 min, followed by washing four times with washing buffer. Then, 50 µL of acridinium ester-labeled anti-human IgG secondary antibody was added, and the reaction was carried out at 37°C for 5 min. After washing four times with washing buffer, the sample was analyzed. The detection results for different antibody concentrations are shown in Table 4. Table 4. Results of 1F1 antibody sensitivity detection

[0044] As shown in Table 4, the antibody still had a high signal when diluted to 0.06 ng / mL, indicating that the antibody had high sensitivity and good antigen binding ability.

[0045] 2. Specificity of 1F1 antibody Human GBM antigen and other autoimmune antigens SmD1, SmD2, SmD3, RNPC, JO-1, Sc1-70, RNPA, RNP70, SSA, Ro52, and PCNA were diluted to 1 µg / mL with carbonate coating buffer and added to ELISA plates, 100 µL per well. Incubate overnight at 4°C, and wash once with PBST solution. Block with 5% skim milk, 200 µL per well, incubate overnight at 4°C or 60 min at 37°C, discard the blocking solution, and wash once with PBST solution. Add diluted 1F1 antibody (1 µg / mL) to 100 µL per well, incubate at 37°C for 30 min, discard the liquid, blot dry, and wash twice with PBST solution. Add enzyme-labeled secondary antibody to 100 µL per well, incubate at 37°C for 30 min, discard the liquid, blot dry, and wash four times with PBST solution. Add 100 µL of TMB substrate solution to each well, protected from light, and incubate at room temperature in the dark for 15 min. Terminate the reaction by adding 50 µL of 2 M sulfuric acid to each well. Place the microplate on a microplate reader and measure the OD value at 450 nm. The results are shown in Table 5.

[0046] Table 5 1F1 antibody specificity detection

[0047] Table 5 shows that the human GBM monoclonal antibody 1F1 has good specificity, does not cross-react with other irrelevant autoimmune antigens such as SmD1, and specifically binds to human GBM antigen only.

[0048] 3. Accelerated stability and freeze-thaw stability of 1F1 antibody 1F1 antibodies were diluted to two different concentrations: 1 µg / mL and 0.01 µg / mL, and aliquoted into several 300 µL tubes. Samples for accelerated stability testing were placed in a 37°C incubator and tested using ELISA on days 1, 3, and 7 of accelerated stability. Freeze-thaw stability samples were placed at -80°C. After the antibody was completely frozen, it was removed and thawed at room temperature for 1 hour, then returned to -80°C. This process was repeated once, twice, and three times before ELISA testing. Control antibodies for accelerated and freeze-thaw stability were untreated 1F1 antibodies aliquoted and stored at 2–8°C. For ELISA testing, human GBM antigen was diluted to 1 µg / mL with carbonate coating buffer and added to each well of an ELISA plate at 100 µL. The plate was incubated overnight at 4°C. After removing the antigen coating buffer, the plate was patted dry, and 200 µL of 5% skim milk powder was added. The plate was then blocked at 37°C for 1 hour. Discard the blocking solution and wash the plate three times with 300 µL PBST. Add 100 µL each of the accelerated, freeze-thawed, and untreated 1F1 at concentrations of 1 µg / mL and 0.01 µg / mL to the corresponding microplates and incubate at 37°C for 40 min. Discard the antibody dilution solution and wash the plate twice with PBST. Add HRP-labeled goat anti-human IgG secondary antibody (1:10000 diluted to 5% skim milk powder), incubate at 37°C for 40 min, then wash the plate four times with PBST. Add 100 µL of TMB chromogenic solution to each well and react in the dark for 15 min. Add 50 µL of 2M sulfuric acid stop solution to each well and measure the absorbance at 450 nm using a microplate reader. The results are shown in Table 6. The OD450 signal change of the accelerated and freeze-thawed samples compared to the untreated samples was <15%, which is considered to indicate that the antibody maintained stable functional activity under these conditions.

[0049] Table 6. Results of Antibody Stability Test for 1F1

[0050] The results in Table 6 show that, regardless of whether the concentration (1 µg / mL) or the concentration (0.01 µg / mL) of human GBM antibody 1F1 was accelerated at 37°C for 1 day, 3 days, and 7 days, or after 1, 2, and 3 freeze-thaw cycles, the CV relative to the untreated control sample did not exceed 10%, indicating that the 1F1 antibody has good accelerated stability and freeze-thaw stability.

[0051] In summary, this application integrates single-cell B-cell sorting and genetic engineering techniques. The process involves immunizing mice, isolating lymphocytes, flow cytometry sorting human GBM protein-specific B cells, PCR amplification to obtain the antibody variable region sequence, constructing an antibody expression plasmid, obtaining purified mouse monoclonal antibodies, verifying the antibody's specificity and sensitivity against human GBM protein, constructing a humanized monoclonal antibody expression plasmid, obtaining purified humanized high-affinity and high-specificity anti-human GBM antibodies, and verifying the antibody's specificity and sensitivity against human GBM protein. The use of flow cytometry to sort anti-human GBM memory B cells, combined with single-cell RT-PCR technology to directly obtain the antibody gene, and then humanizing the antibody, reduces the risk of immunogenicity in clinical applications. Compared to traditional hybridoma technology, the preparation cycle is shortened to within two months.

[0052] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0053] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0054] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A recombinant monoclonal antibody against human GBM, characterized in that, It includes variable regions for light chains and variable regions for heavy chains; The heavy chain variable region includes complementarity-determining regions VH CDR1, VH CDR2, and VH CDR3; the light chain variable region includes complementarity-determining regions VL CDR1, VL CDR2, and VL CDR3. The amino acid sequence of VH CDR1 is shown in SEQ ID NO.1, the amino acid sequence of VH CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO.3; The amino acid sequence of VL CDR1 is shown in SEQ ID NO.4, the amino acid sequence of VL CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of VL CDR3 is shown in SEQ ID NO.

6.

2. The anti-human GBM recombinant monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-human GBM recombinant monoclonal antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The anti-human GBM recombinant monoclonal antibody according to claim 1, characterized in that, The anti-human GBM recombinant monoclonal antibody is any one of a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

4. A nucleic acid, characterized in that, The nucleic acid encodes the anti-human GBM recombinant monoclonal antibody according to any one of claims 1-3.

5. The nucleic acid according to claim 4, characterized in that, The nucleotide sequence used to encode the variable region of the heavy chain is shown in SEQ ID NO.9; the nucleotide sequence used to encode the variable region of the light chain is shown in SEQ ID NO.

10.

6. A biomaterial, characterized in that, The biomaterial comprises the nucleic acid as described in claim 4 or 5; the biomaterial is an expression cassette, vector, or transgenic cell.

7. The method for preparing the anti-human GBM recombinant monoclonal antibody according to any one of claims 1-3, characterized in that, Gene expression is performed using the nucleic acid described in claim 4 or 5; or the transgenic cells described in claim 6 are cultured.

8. The use of the anti-human GBM recombinant monoclonal antibody according to any one of claims 1-3 in the preparation of reagents or medical devices for detecting human GBM.

9. A reagent, characterized in that, The reagent is used for the in vitro detection of human GBM, and the reagent includes the anti-human GBM recombinant monoclonal antibody according to any one of claims 1-3, or the nucleic acid according to claim 4 or 5, or the biological material according to claim 6.

10. A reagent kit, characterized in that, The kit comprises the anti-human GBM recombinant monoclonal antibody as described in any one of claims 1-3, or the nucleic acid as described in claim 4 or 5, or the biological material as described in claim 6, or the reagent as described in claim 9.

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