AQP4 antibody and its application

Abstract

The present invention provides an AQP4 antibody and its application. Specifically, the present invention provides an AQP4 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6; or the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:12, and HCDR3 as shown in SEQ ID NO:13; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO:14, LCDR2 as shown in SEQ ID NO:15, and LCDR3 as shown in SEQ ID NO:16. After verification by CBA and TBA methods, the AQP4 monoclonal antibodies of the present invention can successfully and specifically recognize the extracellular region of the AQP4 antigen.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to AQP4 antibodies and applications thereof. Background Art

[0002] Inflammatory demyelinating diseases of the central nervous system are autoimmune diseases associated with a variety of autoantibodies, including anti-aquaporin 4 (AQP4) antibodies, anti-myelin oligodendrocyte glycoprotein (MOG) antibodies, glial fibrillary acid protein (GFAP) antibodies, anti-myelin basic protein (MBP) antibodies, and anti-myelin associated glycoprotein antibodies. Among them, AQP4 antibodies are the most common, and AQP4-positive patients account for more than 75% of this type of disease.

[0003] The commercial monoclonal antibodies for AQP4 are as follows: Thermo MA5-35079 and Sigma AMAB90537 are mouse monoclonal antibodies, and they both recognize the C-terminal (amino acids 256-323) antigen of AQP4; CST 59678 is a rabbit monoclonal antibody, which also uses an unknown C-terminal antigen; and abcam's mouse monoclonal antibody ab9512, which uses the 301-318aa peptide segment of the C-terminus of AQP4. Therefore, the antigen epitopes recognized by these AQP4 monoclonal antibodies are concentrated at the carboxyl end of AQP4 and are located in the intracellular region. Therefore, when performing an immune response, cells or tissues need to be permeabilized so that the antibody can enter the cell and react with the C-terminus of the AQP4 antigen. Summary of the invention

[0004] In view of the shortcomings of the current AQP4 monoclonal antibodies, that is, they all recognize the intracellular region and are of animal origin, the present invention provides a humanized AQP4 antibody or an antigen-binding fragment thereof. After verification by CBA and TBA methods, the two AQP4 monoclonal antibodies obtained by the present invention can successfully and specifically recognize the extracellular region of the AQP4 antigen.

[0005] Therefore, in one aspect, the present invention provides an AQP4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6;

[0006] Or the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:12, and HCDR3 as shown in SEQ ID NO:13; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO:14, LCDR2 as shown in SEQ ID NO:15, and LCDR3 as shown in SEQ ID NO:16.

[0007] In some embodiments, the heavy chain variable region of the AQP4 antibody or antigen-binding fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:7; and the light chain variable region of the AQP4 antibody or antigen-binding fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:8.

[0008] In some embodiments, the heavy chain variable region of the AQP4 antibody or antigen-binding fragment thereof may comprise an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 17; and the light chain variable region of the AQP4 antibody or antigen-binding fragment thereof may comprise an amino acid sequence as shown in SEQ ID NO: 18, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 18.

[0009] In some embodiments, the AQP4 antibody or antigen-binding fragment thereof may comprise a heavy chain and a light chain, the heavy chain may comprise an amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO:9; and the light chain may comprise an amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO:10.

[0010] In some embodiments, the AQP4 antibody or antigen-binding fragment thereof may comprise a heavy chain and a light chain, the heavy chain may comprise an amino acid sequence as shown in SEQ ID NO: 19, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 19; and the light chain may comprise an amino acid sequence as shown in SEQ ID NO: 20, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 20.

[0011] In some embodiments, the AQP4 antibody is a monoclonal antibody.

[0012] In some embodiments, the antigen binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (diabody), disulfide bond stabilized V region (dsFv) and an antigen binding fragment of a peptide comprising CDR.

[0013] In another aspect, the present invention provides a nucleic acid molecule comprising a polynucleotide encoding the above-mentioned AQP4 antibody or antigen-binding fragment thereof.

[0014] In yet another aspect, the present invention provides a recombinant vector, in particular a recombinant expression vector, comprising the above-mentioned nucleic acid molecule.

[0015] In some embodiments, the vector can be a prokaryotic expression vector or a eukaryotic expression vector.

[0016] In some embodiments, the vector can be a plasmid.

[0017] In yet another aspect, the present invention provides a host cell, which is introduced into or contains the above-mentioned recombinant vector.

[0018] In some embodiments, the cell is selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells. In some embodiments, examples of the cell include, but are not limited to, HEK293T cells, Hela cells, Hep2 cells, and the like.

[0019] In another aspect, the present invention provides a method for preparing the above-mentioned AQP4 antibody or antigen-binding fragment thereof, the method comprising the step of culturing the above-mentioned host cell in a culture medium to obtain the AQP4 antibody or antigen-binding fragment thereof.

[0020] In some embodiments, the method further comprises the step of recovering the AQP4 antibody or antigen-binding fragment thereof from the culture.

[0021] In another aspect, the present invention provides a use of the AQP4 antibody or antigen-binding fragment thereof.

[0022] In some embodiments, the present invention provides the use of the AQP4 antibody or antigen-binding fragment thereof in the preparation of a preparation (especially a positive control) for detecting AQP4 antibodies.

[0023] In some embodiments, the present invention provides the use of the AQP4 antibody or antigen-binding fragment thereof in the preparation of a product for quality inspection of AQP4 antigen-transfected cells.

[0024] In some embodiments, the present invention provides use of the AQP4 antibody or antigen-binding fragment thereof in the study of antigenic determinants of the AQP4 antigen.

[0025] In yet another aspect, the present invention provides an AQP4 antibody detection kit, the kit comprising the AQP4 antibody or an antigen-binding fragment thereof as a positive standard.

[0026] The present invention separates B cells or plasma cells from the body fluids of patients with AQP4 antibody-positive cerebrospinal fluid, and obtains hundreds of paired antibody sequences through 10X single-cell immune repertoire sequencing. Some antibody sequences are selected and completed, constructed into eukaryotic expression plasmids, transfected with Expi293, and expressed and purified full antibodies. After screening and verification, two recombinantly expressed human AQP4 monoclonal antibodies were selected. Performance analysis shows that these two antibodies can be directly used for immunofluorescence detection without permeabilization of cells or tissues because they recognize the extracellular region of the AQP4 antigen.

[0027] Therefore, the present invention has the following technical effects:

[0028] (1) The human AQP4 monoclonal antibody of the present invention can be directly used for immunofluorescence detection of AQP4 antigen in cell or tissue samples without prior permeabilization treatment;

[0029] (2) The human AQP4 monoclonal antibody of the present invention can tolerate various cell permeabilization reagents such as Triton and Saponin, and can be stably used for immunofluorescence detection;

[0030] (3) The human AQP4 monoclonal antibody of the present invention can be used as a positive standard product of an AQP4 antibody detection kit, using the same set of anti-human fluorescent secondary antibodies, thereby simplifying the components of the kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the staining image of the purified human AQP4 monoclonal antibody (BCR1, BCR2) in Example 3.

[0032] Figure 2This is a diagram showing that the human AQP4 monoclonal antibody (BCR1, BCR2) in Example 4 can recognize different AQP4 protein subtypes.

[0033] Figure 3 The immunofluorescence reaction diagrams of the human AQP4 monoclonal antibody and different extracellular loop region mutants of AQP4 in Example 5 are shown. A is a schematic diagram of the structure of the AQP4 protein. AQP4 has three extracellular loop regions: loop A, loop C and loop E; B is the result of the reaction between cells transfected with loop A, loop C and loop E mutants of AQP4 and AQP4 BCR1 / 2 antibodies.

[0034] Figure 4 This is a diagram showing that the immunofluorescence experiment can be performed without cell permeabilization in Example 6, compared with the human AQP4 monoclonal antibody and the tag antibody.

[0035] Figure 5 The immunofluorescence images show the reaction between the human AQP4 monoclonal antibody in Example 6 and the AQP4 detection material stored for different time periods.

[0036] Figure 6 This is the immunofluorescence image of the human AQP4 monoclonal antibody and mouse brain tissue in Example 7. Wherein: Cerebellum represents cerebellum; Hippo represents hippocampus. DETAILED DESCRIPTION

[0037] Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Materials and instruments

[0041] PCR enzyme: Max DNA Polymerase (Takara, catalog number: R045A), KOD DNA Polymerase (brand: Toyobo, catalog number: KOD-201);

[0042] Cell line: HEK293T cells, FreeStyle TM 293-F cells were purchased from Thermofisher, catalog number: R79007; Expi293 TMExpression Medium was purchased from Thermofisher, product number: A1435101;

[0043] Gel Extraction Kit: Omega Gel Extraction Kit;

[0044] Plasmids: pCDNA3.4 was purchased from Newpro Biotechnology, catalog number: V001453#; pEGFP-N1 was purchased from Addgene, 54767; AQP4 M1 / M23 expression plasmids were purchased from Shandong Weizhen, catalog numbers CH895708 / CH811833 respectively, and the expression vector was pEnter, and the inserted expression protein C-terminus would have a His and Flag double tag;

[0045] Plasmid extraction kit: Omega plasmid mini kit;

[0046] Homologous recombination kit: HiFi DNA Assembly Master Mix, Cat. No. E2621L;

[0047] Water Purifier: Millipore 5UV Water Purification System;

[0048] Pipette: eppendorf;

[0049] PCR instrument: Bio-Rad T100;

[0050] Anti-His mouse monoclonal antibody: purchased from Thermofisher, catalog number R930-25;

[0051] Gene synthesis was performed by General Biotech;

[0052] Valproic Acid (VPA) was purchased from Sigma, catalog number P4543;

[0053] Fluorescent secondary antibody: Alexa Fluor 488 Anti-human IgG purchased from Thermofisher, catalog number: A11013;

[0054] Chromium Single Cell 5' Library Kit: Chromium Next GEM Single Cell 5'Kit v2, 4rxns100265;

[0055] V(D)J amplification kit: Chromium Single Cell Human BCR Amplification Kit, 16rxns 1000253.

[0056] Example 1: BCR sequencing to obtain paired human antibody sequences

[0057] 1. Patient inclusion: Patients with antibody-positive clinical diagnosis of neuromyelitis optica spectrum disorder (NMOSD) were included, and their body fluids were obtained with clinical knowledge;

[0058] 2. Flow cytometry analysis: cells were separated from body fluids for flow staining, and anti-human CD45, CD3, CD19, CD27, IgD, CD38, and CD138 surface markers were used to stain the cells. The FACSARIA III (BD) instrument was used to detect naive B cells, memory B cell subsets, and plasma cells / plasmablasts.

[0059] 3.10X Genomics transcriptome sequencing, VDJ 5′RACE amplification sequencing and data analysis:

[0060] The cell sample after removing dead cells was encapsulated in droplets using a 10X single cell chip (Chromium Next GEM SingleCell 3'GEM, Library&Gel Bead Kit v3.1, 16rxns PN-1000121). Single cells and gel beads were co-encapsulated in droplets. VDJ library construction was performed using the Chromium Single Cell 5' Library Kit and the Chromium Single Cell VDJ Enrichment Kit, respectively. The reverse transcribed cDNA encapsulated in the droplets was divided into three parts for 5' single cell transcriptome and B cell library construction, respectively. Sequencing was performed on the Illumina NovaSeq platform. The pooled 5' single cell transcriptome was sequenced on the Illumina NextSeq 500. The obtained FASTQ file was input into the Linux-based Cellranger. The GEX sequencing library was aligned with the ref data-Cellranger-GRCh38 reference genome, and the VDJ sequencing library was aligned with the VDJ GRCh38 alts ensembl reference. And generate preliminary VDJ genotype and clonal type identification.

[0061] Example 2: Small-scale expression and initial screening of human AQP4 antibodies

[0062] Small-scale expression of human AQP4 antibody:

[0063] Based on the results of single-cell sequencing, for the selected 10 paired BCR heavy and light chains, human IgG1kappa / lammda were complemented, and the synthetic genes were inserted into the eukaryotic expression vector pcDNA3.4 (synthesized and subcloned by General Bio). HEK293T in a six-well plate was used to produce antibodies. The specific steps are as follows:

[0064] 1) 293T cells were cultured at 1x 10 6 Cells / well were seeded into 6-well plates, 2 mL of DMEM high-glucose medium was added, mixed evenly, and cultured overnight in a CO2 incubator at 37°C;

[0065] 2) When the cell density is about 80%, transfection is performed: 2 μg of paired antibody light and heavy chain expression plasmids (light and heavy chain plasmid ratio 1:1) and 6 μL of PEI MAX 40K (1 mg / mL) are added to 250 μL OptiMEM, mixed, and allowed to stand at room temperature for 10-15 minutes;

[0066] 3) Replace the medium of each well of cells by adding 1 mL of OptiMEM, and then add the transfection reagent in step 2), mix gently, and culture in a CO2 incubator at 37°C for 4 hours, then replace the medium with OptiMEM containing 3% FBS and continue to culture. Change the medium every 3 days, and take 2 times of culture medium for concentration to obtain a crude product of AQP4 antibody.

[0067] Initial screening of human AQP4 antibodies:

[0068] The cell immunofluorescence assay was used to screen the possible human AQP4 antibodies that had been expressed. The main steps are as follows:

[0069] 1.293T / Hep2 cells were cultured in 10% FBS+DMEM high glucose medium at 37°C in a 5% CO2 incubator;

[0070] 2. When the cell density reaches 40%-50%, the AQP4 M23 recombinant plasmid vector pEnter-AQP4 M23 is transfected into the cells using PEI, and fresh culture medium is replaced 6 hours after transfection;

[0071] 3. Cell fixation: 24-48 hours after transfection, when the cell density reaches 80%-90%, fix the cells with 4% paraformaldehyde at room temperature for 5-15 minutes, and wash three times with 200 μL of PBS / TBS washing solution;

[0072] 4. Cell Immunofluorescence Experiment

[0073] Step 1: Dilute the concentrated AQP4 candidate antibody to 200 μL and add it to the detection well;

[0074] Step 2: Incubate at 37°C for 1 hour in the dark;

[0075] Step 3: washing 5 times with a rinse solution, wherein the rinse solution is PBS or TBS (containing 0.2% Tween 20);

[0076] Step 4: Add the secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG and incubate for 1 hour;

[0077] Step 5: Wash 5 times with a rinse solution, wherein the rinse solution is PBS or TBS (containing 0.2% Tween 20);

[0078] Step 6: Observe the green fluorescence signal under a 20X fluorescence microscope.

[0079] After testing, 10 antibodies were expressed, 8 of which had no green fluorescence signal after reaction with AQP4 transfected cell slides, which was a negative result, but 2 of them produced obvious green fluorescence signals after reaction with AQP4 transfected cell slides. Therefore, these two antibodies are specific AQP4 antibodies, named AQP4 BCR1 and AQP4 BCR2 respectively. The heavy chain of AQP4 BCR1 is AQP4 BCR1 H: SEQ ID NO: 9; the light chain of AQP4 BCR1 is AQP4 BCR1 L: SEQ ID NO: 10; the heavy chain of AQP4 BCR2 is AQP4 BCR2 H: SEQ ID NO: 19, and the light chain of AQP4 BCR2 is AQP4BCR2 L: SEQ ID NO: 20.

[0080] The sequencing results were analyzed by bioinformatics. After blast comparison, the heavy chain variable region of AQP4 BCR1 (AQP4 BCR1 VH) was QITLKESGPTLVKPTETLTLTCTFSGFSLSTSGVGVGWVRQPPGKALEWLALLYWDGDKRYSPSLKSRLTITKDTSKNLVVLTVTNMDPVDTATYYCARRLMGSIWANWGQGTLVTVSS (SEQ ID NO:7), and the light chain variable region of AQP4 BCR1 (AQP4 BCR1 VL) was QSVLTQPPSVSAAPGQKVTISCSGSTSNIGNNFVSWYQQLPGTAPKLLIYDNNKRASGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDISLSAGVFGGGTKLTVL (SEQ ID NO:8); the heavy chain variable region of AQP4 BCR2 (AQP4 BCR2 VH) is EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVRQAPGKGLEWVANIKQEGSEKYYVDAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVHRDFWSGYFYPDYWGQGTLVTVSS (SEQ ID NO: 17), the light chain variable region of AQP4 BCR2 (AQP4 BCR2 VL) is DIQMTQSPSTLSASSVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIK (SEQ ID NO: 18).

[0081] According to Antibody CDR annotation analysis, the heavy chain variable region of AQP4 BCR1 has HCDR1 as shown in TSGVGVG (SEQ ID NO: 1), HCDR2 as shown in LLYWDGDKRYSPSLKS (SEQ ID NO: 2), and HCDR3 as shown in RLMGSIWAN (SEQ ID NO: 3), and the light chain variable region of AQP4 BCR1 has LCDR1 as shown in SGSTSNIGNNFVS (SEQ ID NO: 4), LCDR2 as shown in DNNKRAS (SEQ ID NO: 5), and LCDR3 as shown in GTWDISLSAGV (SEQ ID NO: 6); the heavy chain variable region of AQP4 BCR2 has HCDR1 as shown in SYWMT (SEQ ID NO: 11), HCDR2 as shown in NIKQEGSEKYYVDAVKG (SEQ ID NO: 12), HCDR3 as shown in VHRDFWSGYFYPDY (SEQ ID NO: 13), and AQP4 The light chain variable region of BCR2 has LCDR1 as shown in RASQSISSWLA (SEQ ID NO: 14), LCDR2 as shown in KASSLES (SEQ ID NO: 15), and LCDR3 as shown in QQYNSYSWT (SEQ ID NO: 16).

[0082] Example 3. Expression and purification of human AQP4 antibodies

[0083] Construction of expression plasmid for human AQP4 antibody:

[0084] The human AQP4 antibody (BCR1, BCR2) genes were synthesized by General Motors and subcloned into the expression vector pCDNA3.4. The light and heavy chain expression plasmids of AQP4 BCR1 were pCDNA3.4-AQP4-BCR1-H and pCDNA3.4-AQP4-BCR1-L, and the light and heavy chain expression plasmids of AQP4 BCR2 were pCDNA3.4-AQP4-BCR2-H and pCDNA3.4-AQP4-BCR2-L.

[0085] Expression of human AQP4 antibody:

[0086] 1. Culture Expi293 cells to a density of 2.5-3.0 x 10 6 cells / mL, ready for transfection;

[0087] 2. Add 30 μg of human AQP4 monoclonal antibody expression plasmid pCDNA3.4-AQP4-BCR1-H / L or pCDNA3.4-AQP4-BCR2-H / L to 3 mL OptiMEM at a light chain to heavy chain plasmid ratio of 1:2 and mix well.

[0088] 3. Take 120 μL of PEI MAX 40K (1 mg / mL) and add it to OptiMEM, mix well, and let it stand at room temperature for 10-15 minutes;

[0089] 4. Add the mixed DNA and PEI mixture to 27 mL of Expi293 cells and culture on a shaker at 37°C.

[0090] 5. 24 hours after transfection, add 300 μL of VPA and 270 μL of glucose solution to a final VPA concentration of 1 mM;

[0091] 6. Continue shaking culture for 4 days and then collect the culture medium.

[0092] Purification of human AQP4 antibody:

[0093] 1. Centrifuge the collected Expi293 cell culture medium at 15,000 rpm and 4°C for 10 minutes to remove dead cells and cell debris;

[0094] 2. Add 1 mg of Protein A beads to every 200 mL of culture supernatant, rotate the culture supernatant and protein A beads, and incubate them together;

[0095] 3. After incubation for 1 hour, rinse the Protein A beads with Wash buffer for 3-5 times to remove impurities that are weakly bound to the beads or antibodies;

[0096] 4. Then use a strong acidic buffer (pH 3.0-3.5) to elute the antibody bound to Protein A, and then use an alkaline buffer (pH about 12.0) to neutralize;

[0097] 5. After the first step of affinity chromatography, the eluate was concentrated to about 0.5 mL, and the second step of molecular sieve chromatography was performed using Superdex200Increase to collect the flow-through of different protein peaks, and the flow-through of the intact monoclonal antibody position was concentrated to obtain human AQP4 antibodies BCR1 and BCR2 ( Figure 1 ).

[0098] Example 4: Recombinant human AQP4 monoclonal antibody recognizes different subtypes of AQP4 protein

[0099] There are two main isoforms of AQP4: M1 and M23. These two isoforms differ by 22 amino acids in the N-terminal cytoplasmic region. M1 isoform: full length 323 amino acids, translation start point at Met-1, Met-23 isoform: shorter, contains 301 amino acids, translation start site is Met-23. Although the 22 amino acids of N are not the target of AQP4 antibodies, they affect the quaternary structure of cell surface proteins. Both isoforms can be assembled into homotetrameric AQP4, but only the M23 isoform is regularly arranged in clusters on the cell membrane, called orthogonal tetramers (OAPs).

[0100] In this example, two human AQP4 monoclonal antibodies and two AQP4 isoforms of the present invention were subjected to cell immunofluorescence detection: recombinant human AQP4 monoclonal antibodies BCR1 and BCR2 were used as positive standards for testing different AQP4 isoforms. The detection steps are as follows:

[0101] 1.293T / Hep2 cells were cultured in 10% FBS+DMEM high glucose medium at 37°C in a 5% CO2 incubator;

[0102] 2. When the cell density reaches 40%-50%, the two recombinant plasmid vectors AQP4 M1 and M23 are transfected into the cells using PEI, and fresh culture medium is replaced 6 hours after transfection;

[0103] 3. Cell fixation: 24-48 hours after transfection, when the cell density reaches 80%-90%, fix the cells with 4% paraformaldehyde at room temperature for 5-15 minutes, and wash three times with 200 μL of PBS / TBS washing solution;

[0104] 4. Cell Immunofluorescence Experiment

[0105] Step 1: Dilute the purified AQP4 antibodies BCR1 and BCR2 to 0.1-1 μg / μL and add to the detection wells of transfected M1 and M23 cells;

[0106] Step 2: Incubate at 37°C for 1 hour in the dark;

[0107] Step 3: washing 5 times with a rinse solution, wherein the rinse solution is PBS or TBS (containing 0.2% Tween 20);

[0108] Step 4: Add the secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG and incubate for 1 hour;

[0109] Step 5: Wash 5 times with a rinse solution, wherein the rinse solution is PBS or TBS (containing 0.2% Tween 20);

[0110] Step 6: Observe and take fluorescence photos under a 20X fluorescence microscope.

[0111] Test results such as Figure 2 As shown, the purified BCR1 and BCR2 antibodies can produce green fluorescence signals when reacting with cells transfected with AQP4 M1 and M23, while there is no green fluorescence when transfected with the control plasmid. Therefore, these two human AQP4 monoclonal antibodies can recognize M1 and M23. The difference is that cells transfected with AQP4 M23 can produce stronger and more aggregated signals, which may be related to the fact that AQP4 M23 isoforms can form orthogonal array (OAP) structures.

[0112] Example 5: Spatial structure of AQP4 antigen recognized by recombinant human AQP4 antibody

[0113] The present invention found that the recombinant human AQP4 monoclonal antibody can recognize the extracellular region of AQP4, and the extracellular region structure of AQP4 has only three short loop regions ( Figure 3 In Figure A), in order to verify which extracellular loop region is recognized by the recombinant human AQP4 monoclonal antibody, a series of AQP4 mutant proteins were constructed in the present invention, the extracellular region of the homologous protein AQP1 of the AQP4 family was replaced with the corresponding extracellular region of AQP4, and three AQP mutant expression plasmids were obtained. The final immunofluorescence experiment proved that loop A and loop C of the extracellular region of AQP4 are the main antigen recognition regions, while loop E does not affect the recognition of AQP4 antigen and recombinant human AQP4 monoclonal antibody.

[0114] Construction of AQP4 mutants: Using the overlap extension PCR vector construction method, loop A of AQP1 (YPVGNNQTAVQD, SEQ ID NO:21) was replaced with loop A of AQP4 (NWGGTEKPLPVD, SEQ ID NO:22); loop C of AQP1 (TSSLTGNSLGRNDLADGVNSG, SEQ ID NO:23) was replaced with loop C of AQP4 (TPPSVVGGLGVTMVHGNLT, SEQ ID NO:24); loop E of AQP1 (VITHNFS, SEQ ID NO:25) was replaced with loop E of AQP4 (GNWENHW, SEQ ID NO:26); three AQP4 mutant plasmids were obtained.

[0115] CBA cell preparation

[0116] Step 1: 293T / Hep2 cells were cultured in 10% FBS+DMEM high glucose medium at 37°C in a 5% CO2 incubator;

[0117] Step 2: When the cell density reaches 40%-50%, the three AQP4 mutants and AQP1 plasmid are transfected into the cells using PEI, and fresh culture medium is replaced 6 hours after transfection;

[0118] Step 3: Cell fixation: 24-48 hours after transfection, when the cell density reaches 80%-90%, fix the cells with 4% paraformaldehyde at room temperature for 5-15 minutes, and wash three times with 200 μL of PBS / TBS washing solution;

[0119] Cell immunofluorescence assay

[0120] Step 1: Dilute the purified AQP4 antibodies BCR1 and BCR2 to 0.1-1 μg / μL, and add Anti-His mouse monoclonal antibody (Thermo, R930-25) to the detection wells according to the instructions;

[0121] Step 2: Incubate at 37°C for 1 hour in the dark;

[0122] Step 3: washing 5 times with a rinse solution, wherein the rinse solution is PBS or TBS (containing 0.2% Tween 20);

[0123] Step 4: Add secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG or Alexa Fluor 488 Anti-mouse IgG and mix well and incubate for 1 hour;

[0124] Step 5: Wash 5 times with a rinse solution, wherein the rinse solution is PBS or TBS (containing 0.2% Tween 20);

[0125] Step 6: Observe and take fluorescence photos under a 20X fluorescence microscope.

[0126] Test results such as Figure 3 As shown in B, the three AQP4 mutants and the AQP1 plasmid itself carry a His tag, and the transfected cells are stained with an Anti-AQP4 antibody. The results show that although the three mutant proteins have changed different loop regions, they do not affect the expression and membrane localization of the protein. The staining results of the human AQP4 monoclonal antibodies BCR1 and BCR2 of the present invention show that loop A and loop C are very important for the recognition of AQP4 antibodies BCR1 and BCR2 and AQP4 antigens, and neither of them can be missing. A change in a loop will cause the antibody and antigen to be unable to recognize, while loop E is relatively unimportant, and an obvious green fluorescence signal can still be detected after replacement. This also shows that the recognition of AQP4 antibodies BCR1 and BCR2 and AQP4 antigens depends on the spatial conformation of the AQP4 antigen, especially the spatial structure formed by loop A and loop C, which are indispensable.

[0127] Example 6: Advantages of using recombinant human AQP4 monoclonal antibody for quality control of cell-beased assay (CBA)

[0128] There are currently two methods for detecting AQP4 antibodies: cell-based indirect immunofluorescence (CBA) or ELISA. The CBA method is still recognized as the gold standard because the antigen protein expressed by the cell can still maintain its natural spatial structure after fixation and has better antigenicity. The AQP4 CBA detection kits currently circulating on the market do not contain positive controls. When a negative result is obtained, it cannot be ruled out that it is due to problems with cell expression, such as transfection operation errors, which cannot make users completely assured. Many laboratories rely on screening positive samples from patient populations as positive controls, but individual samples are relatively limited. After use, they can only look for new positive samples and cannot maintain good consistency. In addition, although there are commercial AQP4 antibodies on the market, they are expensive and most of them are mouse or rabbit-derived, requiring corresponding secondary antibodies, which are different from the anti-human secondary antibodies used to detect human autoantibodies, adding additional reagents. In addition, due to the Figure 3 As shown in A, the N-terminus and C-terminus of AQP4 are both located intracellularly, and the usual labels are added to the N-terminus or C-terminus of the protein, so when using the label antibody (such as His), the cells need to be permeabilized in advance, and AQP4 antibody detection does not require permeabilization, because AQP4 autoimmune antibodies mainly recognize the extracellular region, so there is an urgent need to develop a simpler AQP4 antibody as a positive standard for CBA detection. The inventors found that human AQP4 BCR antibodies have multiple advantages over other forms of AQP4 positive antibody standards: First, human AQP4 BCR antibodies do not need to be permeabilized and punched, and can be used for immunofluorescence reactions. And the punching process will not affect its immunofluorescence reaction; second, human AQP4 BCR antibodies have obvious advantages over ordinary label antibodies due to their recognition of the spatial structure of the AQP4 antigen.

[0129] AQP4 antibody detection cell preparation:

[0130] Step 1: 293T / Hep2 cells were cultured in 10% FBS+DMEM high glucose medium at 37°C in a 5% CO2 incubator;

[0131] Step 2: When the cell density reaches 40%-50%, the AQP4 M23 plasmid is transfected into the cells using PEI, and fresh culture medium is replaced 6 hours after transfection;

[0132] Step 3: Cell fixation: 24-48 hours after transfection, when the cell density reaches 80%-90%, fix the cells with 4% paraformaldehyde at room temperature for 5-15 minutes, wash three times with 200 μL of PBS / TBS washing solution, and store in a sterile environment for later use;

[0133] Human AQP4 monoclonal antibody does not require permeabilization for immunofluorescence experiments:

[0134] The prepared 48-well cells were subjected to immunofluorescence experiments using AQP4 antibody BCR1 / 2 and His antibody (Thermo, R930-25).

[0135] Step 1: The AQP4-expressing cell detection wells were treated with no perforation, Saponin perforation, and Triton X-100 perforation for 10 minutes, and then rinsed once;

[0136] Step 2: Dilute the purified AQP4 antibodies BCR1 and BCR2 to 0.1-1 μg / μL, and add His mouse monoclonal antibody (Thermo, R930-25) to the punched or unpunched detection wells according to the instructions;

[0137] Step 3: Incubate at 37°C for 1 hour in the dark;

[0138] Step 4: Wash 5 times with a rinse solution, wherein the rinse solution is PBS or TBS (containing 0.2% Tween 20);

[0139] Step 5: Add secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG or Alexa Fluor 488 Anti-mouse IgG and mix well and incubate for 1 hour;

[0140] Step 6: Wash 5 times with a rinse solution, wherein the rinse solution is PBS or TBS (containing 0.2% Tween 20);

[0141] Step 7: Observe and take fluorescence photos under a 20X fluorescence microscope.

[0142] Test results such as Figure 4As shown, with or without perforation treatment, human AQP4 monoclonal antibody BCR1 and BCR2 can react with AQP4 expressing cells to produce specific green fluorescence signals. However, His mouse monoclonal antibody (Thermo, R930-25) can only dye green fluorescence signals when using Saponin or Triton X-100. If perforation and permeation treatment is not performed, the His monoclonal antibody cannot enter the cells and react with the His tag and cannot react to produce fluorescence signals. Therefore, the experiment also shows that the human AQP4 antibody monoclonal antibody of the present invention can tolerate a variety of cell permeation reagents such as Triton and Saponin, and can be stably used for immunofluorescence detection.

[0143] In addition, the storage time of the detection reagent is also an important consideration for the stability of the detection reagent. The present invention finds that the AQP4 antibody that recognizes the spatial conformation is more suitable as a positive standard to detect the cell matrix used for the CBA method to detect the AQP4 autoimmune antibody, because the use of commonly used label antibodies can detect the presence of the small label at the end of the antigen, but it cannot reflect the conformational changes of the antigen protein. Based on the above speculation, the present invention uses human AQP4 antibodies and label antibodies (AQP4 antibodies) to simultaneously detect AQP4 antigen-expressing cell matrices stored for different times.

[0144] Immunofluorescence experiment of AQP4 antibody to detect cell matrix:

[0145] The prepared AQP4 antigen-expressing cells were placed at room temperature for 0, 1 and 2 weeks, and then immunofluorescence experiments were performed using AQP4 antibody BCR1 / 2 and His antibody.

[0146] Step 1: Dilute the purified AQP4 antibodies BCR1 and BCR2 to 0.1-1 μg / μL, and add His mouse monoclonal antibody (Thermo, R930-25) to the detection wells in a sterile environment at room temperature for different periods of time according to the instructions;

[0147] Step 2: Incubate at 37°C for 1 hour in the dark;

[0148] Step 3: washing 5 times with a rinse solution, wherein the rinse solution is PBS or TBS (containing 0.2% Tween 20);

[0149] Step 4: Add secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG or Alexa Fluor 488 Anti-mouse IgG, mix well and incubate for 1 hour;

[0150] Step 5: Wash 5 times with a rinse solution, wherein the rinse solution is PBS or TBS (containing 0.2% Tween 20);

[0151] Step 6: Observe and take fluorescence photos under a 20X fluorescence microscope.

[0152] Test results such as Figure 5 As shown in the figure, the staining results of human AQP4 monoclonal antibodies BCR1 and BCR2 show that after the cells were placed for 1 week, the positive signal generated by the cells transfected with AQP4 M23 antigen was significantly weakened, and after 2 weeks, almost no obvious green fluorescence signal was seen, which is consistent with the change in the positive signal of known AQP4 positive serum (clinical samples from hospitals). The AQP4 antibody only slightly weakened the signal at 1 week and 2 weeks, which may be caused by slight terminal degradation of the AQP4 protein.

[0153] Therefore, human AQP4 BCR monoclonal antibody can more sensitively reflect the spatial conformational changes of AQP4 antigen, which has obvious advantages over ordinary tag antibodies.

[0154] Example 7: Use of recombinant human AQP4 monoclonal antibody in tissue immunofluorescence experiments

[0155] Human AQP4 BCR antibody can effectively recognize AQP4 expressed in plasmid-transfected cells. The present invention further uses mouse brain tissue to detect whether human AQP4 BCR antibody can recognize mouse natural AQP4 protein. The specific experimental steps are as follows:

[0156] 1. Take out the frozen OCT-embedded mouse brain tissue samples from the -80℃ refrigerator for later use;

[0157] 2. Slice: Mouse brain tissue samples were sliced ​​10 μm thick and placed directly in PBS at room temperature;

[0158] 3. Block peroxisomes: 3% H2O2, (1mL 30% H2O2 + 9mL methanol);

[0159] 4. PBS 5 minutes × 3 times;

[0160] 5. High temperature antigen repair: 95℃ water bath, citrate (PH=6.0) repair solution, 6 minutes. After completion, let it stand for 20-30 minutes to cool down;

[0161] 6. PBS 5 minutes x 3 times;

[0162] 7. Permeabilize the membrane with 0.2% Triton at room temperature for 20 minutes;

[0163] 8. Blocking: 10% goat serum, 1h;

[0164] 9. Incubate the purified BCR1 and BCR2 antibodies at 4°C overnight;

[0165] 10. PBS 5 minutes × 3 times;

[0166] 11. Incubate with anti-human fluorescent secondary antibody Alexa Fluor 488 Anti-human IgG at room temperature for 1 hour;

[0167] 12. PBS 5 minutes × 3 times;

[0168] 13. Add DAPI and incubate in dark for 15 minutes;

[0169] 14. Use PBS for 5 minutes x 2 times, and wipe off the PBS outside the specimen with filter paper;

[0170] 15. Seal the slides with anti-fluorescence quencher and dry them at 37°C;

[0171] 16. Take photos to record the results.

[0172] Test results such as Figure 6 As shown, human AQP4 monoclonal antibodies BCR1 and BCR2 can detect obvious AQP4 positive signals in mouse brain tissue sections, and the positive signals are mainly in the hippocampus and cerebellum of mice.

[0173] After verification by CBA and TBA, the human AQP4 BCR antibody screened by the present invention can effectively identify AQP4, and therefore can be used as a positive standard for the AQP4 antibody detection kit. The human AQP4 monoclonal antibody has two advantages as a positive standard: first, it can share the anti-human secondary antibody with the human sample for detection, without the need to add additional reagent components; second, it can more accurately reflect the preservation state of the transfected AQP4 antigen cell matrix, whether it has lost its spatial conformation and cannot be used for the detection of AQP4 antibodies, so that users can truly rest assured. Therefore, the two human AQP4 monoclonal antibodies screened by the present invention can be used as positive standards for the AQP4 immunofluorescence detection kit, laying a solid foundation for establishing a stable AQP4 antibody detection system.

[0174] Sequence Listing

[0175] AQP4 BCR1 HCDR1(SEQ ID NO:1):TSGVGVG

[0176] AQP4 BCR1 HCDR2(SEQ ID NO:2):LLYWDGDKRYSPSLKS

[0177] AQP4 BCR1 HCDR3(SEQ ID NO:3):RLMGSIWAN

[0178] AQP4 BCR1 LCDR1(SEQ ID NO:4): SGSTSNIGNNFVS

[0179] AQP4 BCR1 LCDR2 (SEQ ID NO:5): DNNKRAS

[0180] AQP4 BCR1 LCDR3 (SEQ ID NO:6): GTWDISLSAGV

[0181] AQP4 BCR1 VH (SEQ ID NO:7):

[0182] QITLKESGPTLVKPTETLTLTCTFSGFSLSTSGVGVGWVRQPPGKALEWLALLYWDGDKRYSPSLKSRLTITKDTSKNLVVLTVTNMDPVDTATYYCARRLMGSIWANWGQGTLVTVSSAQP4 BCR1 VL (SEQ ID NO:8):

[0183] QSVLTQPPSVSAAPGQKVTISCSGSTSNIGNNFVSWYQQLPGTAPKLLIYDNNKRASGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDISLSAGVFGGGTKLTVLAQP4 BCR1 heavy chain (SEQ ID NO:9):

[0184] QITLKESGPTLVKPTETLTLTCTFSGFSLSTSGVGVGWVRQPPGKALEWLALLYWDGDKRYSPSLKSRLTITKDTSKNLVVLTVTNMDPVDTATYYCARRLMGSIWANWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0185] AQP4 BCR1 light chain (SEQ ID NO:10):

[0186] QSVLTQPPSVSAAPGQKVTISCSGSTSNIGNNFVSWYQQLPGTAPKLLIYDNNKRASGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDISLSAGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0187] AQP4 BCR2 HCDR1 (SEQ ID NO:11): SYWMT

[0188] AQP4 BCR2 HCDR2 (SEQ ID NO:12): NIKQEGSEKYYVDAVKG

[0189] AQP4 BCR2 HCDR3 (SEQ ID NO:13): VHRDFWSGYFYPDY

[0190] AQP4 BCR2 LCDR1 (SEQ ID NO:14): RASQSISSWLA

[0191] AQP4 BCR2 LCDR2 (SEQ ID NO:15): KASSLES

[0192] AQP4 BCR2 LCDR3 (SEQ ID NO:16): QQYNSYSWT

[0193] AQP4 BCR2 VH (SEQ ID NO:17):

[0194] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVRQAPGKGLEWVANIKQEGSEKYYVDAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVHRDFWSGYFYPDYWGQGTLVTVSS

[0195] AQP4 BCR2 VL (SEQ ID NO:18):

[0196] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIK

[0197] AQP4 BCR2 heavy chain (SEQ ID NO:19):

[0198] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVRQAPGKGLEWVANIKQEGSEKYYVDAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVHRDFWSGYFYPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0199] AQP4 BCR2 light chain (SEQ ID NO:20):

[0200] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0201] AQP1 Loop A (SEQ ID NO:21): YPVGNNQTAVQD

[0202] AQP4 Loop A(SEQ ID NO:22):NWGGTEKPLPVD

[0203] AQP1 Loop C(SEQ ID NO:23):TSSLTGNSLGRNDLADGVNSG

[0204] AQP4 Loop C(SEQ ID NO:24):TPPSVVGGLGVTMVHGNLT

[0205] AQP1 Loop E(SEQ ID NO:25):VITHNFS

[0206] AQP4 Loop E(SEQ ID NO:26):GNWENHW

Claims

1. An AQP4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; Or the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:12, and HCDR3 as shown in SEQ ID NO:13; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO:14, LCDR2 as shown in SEQ ID NO:15, and LCDR3 as shown in SEQ ID NO:

16.

2. The AQP4 antibody or antigen-binding fragment thereof according to claim 1, wherein: The AQP4 antibody is a monoclonal antibody.

3. The AQP4 antibody or antigen-binding fragment thereof according to claim 1, wherein: The antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single chain antibody, dimerized V region and disulfide bond stabilized V region.

4. The AQP4 antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region of the AQP4 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence that is at least 70% identical to SEQ ID NO:7; and the light chain variable region of the AQP4 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence that is at least 70% identical to SEQ ID NO:8, or The heavy chain variable region of the AQP4 antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 17; and the light chain variable region of the AQP4 antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence that is at least 70% identical to SEQ ID NO:

18.

5. The AQP4 antibody or antigen-binding fragment thereof according to claim 1, wherein: The AQP4 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 70% identity with SEQ ID NO:9; and the light chain comprises an amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 70% identity with SEQ ID NO:10, or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence that is at least 70% identical to SEQ ID NO:19; and the light chain comprises the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence that is at least 70% identical to SEQ ID NO:

20.

6. A nucleic acid molecule encoding the AQP4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. A recombinant vector comprising the nucleic acid molecule according to claim 6 .

8. The recombinant vector according to claim 7, wherein The recombinant vector is a recombinant expression vector.

9. A host cell introduced with or containing the recombinant vector according to claim 8.

10. A method for preparing an AQP4 antibody or an antigen-binding fragment thereof, the method comprising the step of culturing the host cell according to claim 9 in a culture to obtain the AQP4 antibody or the antigen-binding fragment thereof.

11. The method according to claim 10, wherein: The method further comprises the step of recovering the AQP4 antibody or antigen-binding fragment thereof from the culture.

12. Use of the AQP4 antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein the use is selected from: use of the AQP4 antibody or its antigen-binding fragment in preparing a product for detecting AQP4 antibodies; use of the AQP4 antibody or its antigen-binding fragment in preparing a product for quality inspection of AQP4 antigen-transfected cells; use of the AQP4 antibody or its antigen-binding fragment in research on antigenic determinants of AQP4 antigens.

13. An AQP4 antibody detection kit, comprising the AQP4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 as a positive standard.

Images