Anti-GAD65 antibodies and their applications

Abstract

The present application provides an anti-GAD65 antibody and its application, wherein the antibody 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: 2, HCDR2 as shown in SEQ ID NO: 3, and HCDR3 as shown in SEQ ID NO: 4; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 6, LCDR2 as shown in SEQ ID NO: 7, and LCDR3 as shown in SEQ ID NO: 8. After verification by cell immunofluorescence, tissue immunofluorescence and protein immunoblotting methods, the anti-GAD65 monoclonal recombinant antibody obtained by the present invention can specifically recognize different forms of GAD65 antigens and can be used for a variety of experimental purposes.

Description

Technical Field

[0001] The present application belongs to the field of proteins and biological detection. Specifically, the present application provides an anti-GAD65 antibody and its application. Background Art

[0002] Glutamate decarboxylase 65 (GAD65) is an enzyme that plays a key role in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) from glutamate. The enzyme is responsible for converting glutamate into gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the brain. GAD65 and its isoform GAD67 are responsible for regulating GABA levels in the brain and are essential for inhibitory signaling in the central nervous system (CNS). GAD65 is primarily found in GABAergic neurons of the CNS and, unlike GAD67, which is more evenly distributed throughout neurons, is highly concentrated in synaptic terminal vesicles and is primarily membrane-bound, anchored to the synaptic vesicle membrane via its palmitoylated (fatty acid-modified) N-terminus. This positioning enables it to regulate GABA synthesis in response to neuronal activity.

[0003] In recent years, anti-GAD65 antibodies (anti-GAD65 or glutamic acid decarboxylase 65 antibodies) have been found in different patients. These antibodies are closely related to a variety of neurological autoimmune diseases, especially those affecting the central nervous system (CNS). They include: stiff neck syndrome (SPS), cerebellar ataxia and autoimmune encephalitis. Since GAD65 is also expressed in pancreatic β cells, the presence of GAD65 in the pancreas makes it a target for autoantibodies in patients with type 1 diabetes. Anti-GAD65 antibodies are also associated with autoimmune diseases such as type 1 diabetes.

[0004] The presence of anti-GAD65 antibodies is often used as a diagnostic marker for the above diseases, and its detection often relies on laboratory testing. Currently, the commonly used techniques for GAD65 antibody detection are roughly the following: cell-based immunofluorescence assay (CBA), enzyme-linked immunosorbent assay (ELISA) and line immunoassay (LIA).

[0005] Positive standards are essential components of IVD diagnostic kits. At present, there is no standard positive standard in the diagnostic kit for anti-GAD65 antibodies. Even if you buy commercial GAD65 antibodies yourself, they are all rabbit or mouse-derived, and cannot be directly used as positive standards for human autoantibody detection kits because different secondary antibodies are required. Therefore, we obtained a positive antibody for GAD65 through single-cell immune repertoire sequencing. And tested its function and application, we found that the antibody has a wide range of applications and can be used in cell immunofluorescence (IF), tissue immunofluorescence (IHC), immunoblotting (LIA) and other experiments, indicating that GAD65 antibodies can recognize linear epitopes of proteins. Therefore, this antibody can be used as a positive standard for anti-GAD65 antibody CBA, ElISA and LIA detection kits, and has broad commercial value. Summary of the invention

[0006] The present invention provides a humanized anti-GAD65 antibody or an antigen-binding fragment thereof. After verification by cell immunofluorescence (IF), tissue immunofluorescence (IHC) and protein immunoblotting (Line immunoassay, LIA) methods, the anti-GAD65 monoclonal recombinant antibody obtained by the present invention can specifically recognize different forms of GAD65 antigens, and can be used as a positive standard in an anti-GAD65 antibody detection kit to improve the accuracy of detection.

[0007] On the one hand, the present application provides an anti-GAD65 antibody or an antigen-binding fragment thereof, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4; and the light chain variable region comprising LCDR1 as shown in SEQ ID NO:6, LCDR2 as shown in SEQ ID NO:7, and LCDR3 as shown in SEQ ID NO:8.

[0008] Furthermore, the antibody is a monoclonal antibody.

[0009] Furthermore, the antibody is a human monoclonal antibody.

[0010] Further, the heavy chain variable region amino acid sequence of the antibody is SEQ ID NO:1, or an amino acid sequence that is at least 75%, such as 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:1; and the light chain variable region amino acid sequence of the antibody is SEQ ID NO:5, or an amino acid sequence that is at least 75%, such as 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:5.

[0011] Furthermore, the heavy chain amino acid sequence of the antibody is SEQ ID NO:9, or an amino acid sequence that is at least 75%, such as 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:9; and the light chain amino acid sequence is SEQ ID NO:10, or an amino acid sequence that is at least 75%, such as 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:10.

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

[0013] On the other hand, the present application provides a nucleic acid molecule comprising a polynucleotide encoding the above-mentioned anti-GAD65 antibody or its antigen-binding fragment.

[0014] On the other hand, the present application provides a recombinant vector, in particular a recombinant expression vector, which comprises the above-mentioned nucleic acid molecule.

[0015] Furthermore, the vector may be a prokaryotic expression vector or a eukaryotic expression vector.

[0016] Furthermore, the vector may be a plasmid.

[0017] On the other hand, the present application provides a host cell into which the above-mentioned recombinant vector is introduced or contains.

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

[0019] On the other hand, the present application provides a method for preparing the above-mentioned anti-GAD65 antibody or its antigen-binding fragment, which comprises the step of culturing the above-mentioned host cell in a culture to obtain the anti-GAD65 antibody or its antigen-binding fragment.

[0020] Furthermore, the method further comprises the step of recovering the anti-GAD65 antibody or antigen-binding fragment thereof from the culture.

[0021] On the other hand, the present application provides the use of the anti-GAD65 antibody or its antigen-binding fragment in the preparation of a membrane strip method product or a kit (especially a positive control) for detecting anti-GAD65 antibodies.

[0022] On the other hand, the present application provides the use of the anti-GAD65 antibody or its antigen-binding fragment in the preparation of a product for quality inspection of GAD65 antigen-transfected cells.

[0023] On the other hand, the present application provides an anti-GAD65 antibody detection kit, which contains the anti-GAD65 antibody or its antigen-binding fragment as a positive control.

[0024] On the other hand, the present application provides the use of the anti-GAD65 antibody or its antigen-binding fragment in the preparation of a kit for detecting GAD65.

[0025] Furthermore, the kit is a cell immunofluorescence, tissue immunofluorescence and protein immunoblotting detection kit.

[0026] On the other hand, the present application provides a kit for detecting GAD65, wherein the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-6.

[0027] The present invention uses 10X Genomics' single-cell immune repertoire and transcriptome sequencing from the cerebrospinal fluid of patients with positive anti-GAD65 antibodies, selects plasma cells by gene expression patterns, and obtains dozens of heavy chain and light chain pairing sequences. The antibody sequence is completed, constructed into a eukaryotic expression plasmid, transfected with FreeStyle™ CHO-S cells, and the cell supernatant is collected to purify the antibody. After screening and verification, a human anti-GAD65 antibody was selected. Performance analysis shows that this antibody can be directly used for cell immunofluorescence (IF), tissue immunofluorescence (IHC) and protein immunoblotting (LIA) and other detections because it recognizes human / rat GAD65 antigens.

[0028] The present invention has the following technical effects:

[0029] (1) The humanized anti-GAD65 antibody or antigen-binding fragment thereof of the present invention can be used for immunofluorescence detection of GAD65 antigen in cell or tissue samples;

[0030] (2) The humanized anti-GAD65 antibody or antigen-binding fragment thereof of the present invention can be used for GAD65 protein immunoblotting (LIA) analysis;

[0031] (3) The humanized anti-GAD65 antibody or antigen-binding fragment thereof of the present invention can be used as a positive standard in an anti-GAD65 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

[0032] Figure 1 The screening results of anti-GAD65 positive samples;

[0033] Figure 2 The SDS-PAGE Coomassie blue staining image of 12 human B cell sequencing antibodies;

[0034] Figure 3 For immunofluorescence validation of anti-GAD65 B cell antibodies;

[0035] Figure 4 The molecular screening image and Coomassie brilliant blue staining image of the expressed and purified human anti-GAD65 antibody (L7-21-1);

[0036] Figure 5 This is an immunofluorescence image of the reaction between human anti-GAD65 antibody (L7-21-1) and transfected GAD65 cells;

[0037] Figure 6 This is the immunofluorescence image of rat brain tissue with human anti-GAD65 antibody (L7-21-1);

[0038] Figure 7 The results of the reaction of human anti-GAD65 antibody (L7-21-1) with paraneoplastic membrane products. DETAILED DESCRIPTION

[0039] 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.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] Reagents:

[0043] Cell lines: HEK293T cells were purchased from ATCC, catalog number: crl-3216; FreeStyle™ CHO-S cells were purchased from ThermoFisher, catalog number: R80007; Hela cells were purchased from ATCC, catalog number: CCL-2;

[0044] Plasmid: GAD65 expression plasmid was synthesized by General Biotechnology;

[0045] Anti-His mouse monoclonal antibody: purchased from Thermofisher, catalog number R930-25;

[0046] Alexa Fluor 488 Anti-human IgG was purchased from ThermoFisher, catalog number: A11013;

[0047] Alexa Fluor 488 Anti-mouse IgG was purchased from ThermoFisher, catalog number: A-11001;

[0048] Bovine albumin (BSA) was purchased from Vetec, catalog number: V900933;

[0049] Horseradish enzyme-labeled goat anti-mouse IgG (H+L) was purchased from Zhongshan Jinqiao, catalog number: ZB-5305;

[0050] Horseradish enzyme-labeled goat anti-human IgG (H+L) was purchased from Beyotime, catalog number: A0201;

[0051] ECL luminescent liquid Immobilon ECL Ultra Western HRP Substrate was purchased from Millipore, catalog number: WBULS0500;

[0052] PBS was purchased from VivaCell, catalog number: C3580-0500;

[0053] 10% FBS + DMEM cell culture medium was purchased from procell, catalog number: PM150210B;

[0054] FreeStyle™ CHO-S cell culture medium was purchased from ThermoFisher, catalog number: 12651014;

[0055] Triton was purchased from Sigma, catalog number: X100-500mL;

[0056] Opti-MEM was purchased from Gibco, catalog number: 31985-070;

[0057] Transfection reagent PEI MAX 40K, purchased from Polysciences, catalog number: 24765-100;

[0058] Valproic Acid (VPA) was purchased from Sigma, catalog number: P4543;

[0059] SurePAGE™ Bis-Tris SDS-PAGE electrophoresis gel was purchased from GenScript, catalog number: M00656;

[0060] 10X Genomics single cell array: Chromium Next GEM Single Cell 3' GEM, Library&Gel Bead Kit v3.1, 16 rxns PN-1000121;

[0061] V(D)J amplification kit: Chromium Single Cell Human BCR Amplification Kit, 16rxns, catalog number: 1000253;

[0062] Protein A beads were purchased from ThermoFisher, catalog number: 10001D;

[0063] Amicon Ultra 4 mL ultrafiltration tubes were purchased from Millipore, catalog number: UFC803096;

[0064] Wash buffer: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2.0 mM KH2PO4, 0.1% Tween-20, pH 7.2~7.4;

[0065] Acidic glycine buffer (pH 3.0-3.5): 100 mM Gly, 0.1% Tween-20, pH 3.0-3.5;

[0066] SDS-loading buffer was purchased from Beyotime, catalog number: P0015;

[0067] Neutralization Tris-HCl: 1.0 M Tris-HCl, pH 9.0;

[0068] 0.2% Triton: add 20 μl Triton X-100 to 10 ml PBS and dissolve in a 37°C water bath;

[0069] 10% BSA: weigh 5 g BSA and dissolve it in 50 ml PBS buffer;

[0070] DAPI staining solution was purchased from Beyotime, catalog number: C1005;

[0071] Strong RIPA lysis buffer was purchased from Beyotime, catalog number: P0013B;

[0072] PVDF membrane was purchased from Merck, catalog number: IPFL00010;

[0073] TBS was purchased from Beyotime, catalog number: ST665-1L;

[0074] The anti-fluorescence quenching sealing solution was purchased from Beyotime, item number: P0126-5ml.

[0075] instrument:

[0076] Water Purifier: Millipore Direct-Q ® 5 UV Water Purification System;

[0077] Pipette: Eppendorf;

[0078] PCR instrument: Bio-Rad T100;

[0079] Chemical exposure instrument: Bio-Rad ChemiDoc XRS+.

[0080] Film spraying machine: Biodot-AD 3220

[0081] Example 1 Screening of human anti-GAD65 antibody positive samples

[0082] Patient inclusion: Patients with encephalitis who were positive for anti-GAD65 antibodies in cerebrospinal fluid were enrolled to obtain cerebrospinal fluid cells. The patients had signed the clinical informed consent.

[0083] The patient's cerebrospinal fluid first undergoes an immune reaction with antigens and antibodies to verify that it does contain anti-GAD65 antibodies. Currently, the detection of anti-GAD65 antibodies is mainly based on cellular immunofluorescence (IF).

[0084] Preparation of cell matrix for anti-GAD65 antibody detection:

[0085] HEK293T cells were plated in 48-well plates and cultured in 10% FBS+DMEM cell culture medium at 37°C in a 5% CO2 incubator;

[0086] When the cell density reaches 40%-50%, co-transfect the GAD65 plasmid with PEI MAX 40K. Replace the culture medium with fresh medium 6 hours after transfection to remove the transfection reagent. Transfect for 24-48 hours until the cell density reaches 80%-90%;

[0087] Cell fixation: Fix the cells with 1-4% paraformaldehyde at room temperature, wash 2-3 times with 200µL PBS, and store in a sterile environment until use.

[0088] Immunofluorescence experiments:

[0089] About 150-200 μl of the patient's cerebrospinal fluid was added to a 48-well plate transfected with GAD65 antigen expression and incubated at 37°C for 1 hour in the dark;

[0090] Wash five times with PBS;

[0091] Add secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG and incubate for 1 hour;

[0092] Wash five times with PBS;

[0093] Observe and take fluorescence photos under a 20X fluorescence microscope.

[0094] The patient's cerebrospinal fluid contained anti-GAD65 antibodies, which produced a clear green fluorescent signal after transfection of GAD65 cells ( Figure 1 ).

[0095] Example 2 Small-scale expression of human anti-single cell sequencing antibodies:

[0096] The anti-GAD65 antibody positive samples obtained by the test were sent to the company for single cell (B cell) sequencing, and a certain number of light and heavy chain paired antibody sequences were obtained. According to the characteristics of the antibody amino acid sequence and the distribution of B cells, different types of antibodies such as IgG1, IgG2 or IgG3 were selected for further expression and verification. 12 groups of paired heavy and light chains were selected to complement human IgG1 κ / λ, and the synthetic genes were inserted into the eukaryotic expression vector pTT5 (synthesized and subcloned by General Biotech) after codon optimization to obtain light and heavy chain expression plasmids. HEK293T was transfected in a six-well plate to produce antibodies.

[0097] The specific steps are as follows:

[0098] HEK293T cells were cultured at 1 x 10 6 Cells / well were seeded into 6-well plates, 2 mL of DMEM cell culture medium was added, mixed evenly, and cultured overnight at 37 °C in a CO2 incubator;

[0099] 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 1:2) 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;

[0100] Replace the medium for each well of cells by adding 1 mL of OptiMEM, then add the transfection reagent, mix gently, and culture in a CO2 incubator at 37°C for 4 hours. Then replace the medium with Opti-MEM containing 3% FBS and continue to culture. Change the medium every 3 days, take 2 culture media and use Pall 30K centrifugal concentrators to concentrate by ultracentrifugation to obtain the crude product of B cell sequencing antibody.

[0101] Take 2-5 μg of B cell sequencing antibody and add SDS-loading buffer containing β-mercaptoethanol (β-ME) to prepare samples. Use SurePAGE™ Bis-Tris SDS-PAGE 4-12% precast gel at 100V constant voltage for 1 hour. Remove the gel plate and stain with Coomassie Brilliant Blue staining solution for 20 minutes. Then you can see the protein bands of B cell sequencing antibody (such as Figure 2 ).

[0102] Example 3 Cellular Immunofluorescence Screening of Human B Cell Sequencing Antibodies Screening:

[0103] As in Example 2, all 12 B cell sequencing antibodies were expressed, and then it was verified whether these 12 B cell sequencing antibodies could recognize GAD65, and an immunofluorescence experiment was performed on a small amount of recombinant antibodies expressed in a 6-well plate.

[0104] The specific steps are as follows:

[0105] HEK293T cells were seeded into 6-well plates at 1 x 106 cells / well, 2 mL of DMEM cell culture medium was added, mixed evenly, and cultured overnight in a CO2 incubator at 37°C;

[0106] When the cell density is about 60-70%, transfection is performed: 2 µg of paired antibody light and heavy chain expression plasmids (light and heavy chain plasmid ratio 1:2) 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;

[0107] Replace the medium for each well of cells by adding 1 mL of OptiMEM, then add the transfection reagent, mix gently, and culture in a CO2 incubator at 37°C for 4 hours. Then replace the medium with Opti-MEM containing 3% FBS and continue to culture. Change the medium every 3 days, take 2 culture media and use Pall 30K centrifugal concentrators to concentrate by ultracentrifugation to obtain the crude product of B cell sequencing antibody.

[0108] Take about 150-200 μl of crude product of B cell sequencing antibody, add it to the 48-well plate transfected with GAD65 antigen expression, and incubate at 37°C for 1 hour in the dark;

[0109] Wash five times with PBS;

[0110] Add secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG and incubate for 1 hour;

[0111] Wash five times with PBS;

[0112] Observe and take fluorescence photos under a 20X fluorescence microscope.

[0113] If the crude product contains specific anti-GAD65 antibodies, it will produce obvious green fluorescence signals after reacting with transfected GAD65 cells, such as B cell sequencing antibody L7-21-1 ( Figure 3 ).

[0114] One of the 12 B cell sequencing antibodies (L7-21-1) produced an obvious green fluorescence signal after reacting with transfected GAD65 cells.

[0115] L7-21-1 is a humanized anti-GAD65 antibody of the present invention. After IMGT IgBlast comparison analysis, the heavy chain variable region (L7-21-1-VH) of L7-21-1 is: SEQ ID NO: 1, HCDR1 is SEQ ID NO: 2, HCDR2 is SEQ ID NO: 3, HCDR3 is SEQ ID NO: 4, the light chain variable region (L7-21-1-VL) of L7-21-1 is: SEQ ID NO: 5, LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ ID NO: 7, LCDR3 is SEQ ID NO: 8, the complete heavy chain of L7-21-1 is L7-21-1-H: SEQ ID NO: 9; the complete light chain of L7-21-1 is L7-21-1-L: SEQ ID NO: 10. The details are as follows:

[0116] L7-21-1-VH (SEQ ID NO:1):

[0117] QLQLQESGPGLVKPSETLSLTCTVSGGSFSSSTHYWVWIRQPPGKGLDWIGSIYYGGSTYYNPSLKSRVAISGDTSNSQFSLRLRSVTAADTAVYHCARGSGSGRFDFWGQGILVTVSS

[0118] L7-21-1 HCDR1 (SEQ ID NO:2):

[0119] SSTHYW

[0120] L7-21-1 HCDR2 (SEQ ID NO:3):

[0121] SIYYGGSTYYNPSLKS

[0122] L7-21-1 HCDR3 (SEQ ID NO:4):

[0123] GSGSGRFDF

[0124] L7-21-1 - VL (SEQ ID NO:5):

[0125] DIQMSQSPSSLSASVGDRVTIPCRASQSIATYVNWYQHRPGKAPRLLIYGASSLQSGVPSRFSGGGSGTDFTLTINSLQPEDFATYYCQQSFSMPFTFGQGTKLEIK

[0126] L7-21-1 LCDR1 (SEQ ID NO:6):

[0127] RASQSIATYVN

[0128] L7-21-1LCDR2 (SEQ ID NO:7):

[0129] GASSLQS

[0130] L7-21-1LCDR3 (SEQ ID NO:8):

[0131] QQSFSMPFT

[0132] L7-21-1 heavy chain (SEQ ID NO:9):

[0133] MHSSALLCCLVLLTGVRAQLQLQESGPGLVKPSETLSLTCTVSGGSFSSSTHYWVWIRQPPGKGLDWIGSIYYGGSTYYNPSLKSRVAISGDTSNSQFSLRLRSVTAADTAVYHCA RGSGSGRFDFWGQGILVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0134] L7-21-1 light chain (SEQ ID NO: 10):

[0135] MHSSALLCCLVLLTGVRADIQMSQSPSSSLSASVGDRVTIPCRASQSIATYVNWYQHRPGKAPRRLLIYGASSLQSGVPSRFSGGGSGTDFTLTINSLQPEDFATYYCQQSFSMPFTF GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0136] Purification of human anti-GAD65 antibody:

[0137] The harvested FreeStyle™ CHO-S cell culture was centrifuged at 15000 rpm, 4°C for 10 minutes, and the supernatant was collected to remove dead cells and cell debris;

[0138] Add the corresponding volume of Protein A beads to the supernatant according to the instruction manual of Protein A beads, and rotate the supernatant and Protein A beads for binding and incubation;

[0139] 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;

[0140] Then, the antibody bound to the Protein A beads was eluted with acidic glycine buffer (pH 3.0-3.5), and the eluate was neutralized with 1 / 10 volume of acidic glycine buffer 1 M Tris-HCl pH 9.0;

[0141] After the protein A affinity chromatography was completed, the eluate was concentrated to a volume of about 0.5 mL using an Amicon Ultra 4 mL ultrafiltration tube, and the second step of molecular sieve chromatography was performed using a BioCore SEC-300 chromatography column. The protein peak flow-through was collected and ultrafiltered and concentrated to obtain about 1 ml of human GAD65 antibody;

[0142] Take 2 μg of human GAD65 antibody and add SDS-loading buffer with / without beta-mercaptoethanol (β-ME) to prepare samples. Use SurePAGE™ Bis-Tris SDS-PAGE 4-12% precast gel and run electrophoresis at 100V for 1 hour. Remove the gel plate and stain with Coomassie Brilliant Blue staining solution for 20 minutes to see the protein bands of human GAD65 antibody.

[0143] Figure 4 The molecular screening image and Coomassie brilliant blue staining image of the expressed and purified human anti-GAD65 antibody. Figure 4 Part A is the molecular sieve diagram, showing that the obtained recombinant antibody is a single peak with no aggregation. Figure 4 Part B is the antibody Coomassie Brilliant Blue staining image, M represents protein marker, 1 is the human GAD65 antibody under reducing conditions, and 2 is the human GAD65 antibody under non-reducing conditions.

[0144] The protein concentration of about 1 ml of human GAD65 antibody obtained was measured using nanodrop A280 and was 1.53 mg / mL. Based on this, it can be estimated that about 1.53 mg of antibody can be expressed by 30 ml of FreeStyle™ CHO-S cells. Through the above measurement and calculation, it can be known that the expression level of human GAD65 antibody in FreeStyle™ CHO-S cells is about 51 mg / L.

[0145] Example 5 Recombinant human anti-GAD65 antibody for cell-based immunofluorescence detection (CBA)

[0146] At present, the detection of anti-GAD65 antibodies appears in the antibody spectrum detection of autoimmune encephalitis and paraneoplastic syndrome. Since the main detection method for autoimmune encephalitis is cell-based immunofluorescence assay (CBA), we used the L7-21-1 antibody to recognize the titer of GAD65 protein for preliminary analysis.

[0147] Preparation of cell matrix for anti-GAD65 antibody detection:

[0148] Hela cells were cultured in 10% FBS+DMEM cell culture medium at 37°C in a 5% CO2 incubator;

[0149] When the cell density reaches 40%-50%, transfect the cells with GAD65-His plasmid using PEI MAX 40K. Replace the culture medium with fresh medium 6 hours after transfection to remove the transfection reagent. Transfect for 24-48 hours until the cell density reaches 80%-90%;

[0150] Cell fixation: Fix the cells with 4% paraformaldehyde at room temperature, wash 2-3 times with 200µL PBS, and store in a sterile environment until use.

[0151] Using a 48-well plate, the cells prepared above were subjected to immunofluorescence experiments using the 11 anti-GAD65 antibodies and Flag antibodies expressed in Example 1 as follows:

[0152] For GAD65-His-expressing cells fixed with 4% paraformaldehyde, they were treated with Triton X-100 for 10 min and rinsed once with PBS before use;

[0153] The purified anti-GAD65 antibody was diluted to 1 μg / mL, and the His mouse monoclonal antibody was diluted 1:1000, added to a 48-well plate, and incubated with cells expressing the GAD65 antigen protein at 37°C in the dark for 1 hour;

[0154] Wash five times with PBS;

[0155] Add secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG (for anit-GAD65 antibody) or conjugated with Alexa Fluor 488 Anti-mouse IgG (for Anti-His) and incubate for 1 hour;

[0156] Wash five times with PBS;

[0157] Observe and take fluorescence photos under a 20X fluorescence microscope.

[0158] L7-21-1 antibody (gradient dilution: 1:1000-10000), His antibody and cells transfected with GAD65-his produced obvious green fluorescence signals ( Figure 5 ). And the positive signal of L7-21-1 antibody gradually weakened with the increase of dilution ratio, and the positive signal can still be seen at a dilution of 1:10000. The above results show that the human GAD65 antibody of the present invention can be used for immunofluorescence experiments of transfected cells.

[0159] Example 6 Recombinant human anti-GAD65 antibody for IHC

[0160] Human GAD65 antibodies can effectively recognize GAD65 overexpressed in plasmid-transfected cells. The present invention further uses rat brain tissue to detect whether human GAD65 antibodies can recognize GAD65 protein in rat brain tissue.

[0161] The specific experimental steps are as follows:

[0162] Frozen rat brain tissue sections were taken out of the -80°C freezer and rewarmed;

[0163] The rat brain tissue sections were washed with PBS for 5 min;

[0164] Rat brain tissue sections were permeabilized in 0.2% Triton for 20 min;

[0165] Rat brain tissue sections were blocked in 10% BSA for 1 h;

[0166] Rat brain tissue sections were incubated with 100 μl of anti-GAD65 antibody dilution (antibody concentration 1 μg / ml, diluted in PBS containing 2% BSA) at 4°C overnight;

[0167] The rat brain tissue sections were washed three times with PBS;

[0168] Add anti-human fluorescent secondary antibody Alexa Fluor 488 Anti-human IgG and incubate at room temperature for 1 h;

[0169] The rat brain tissue sections were washed three times with PBS;

[0170] Add DAPI and incubate in the dark for 15 minutes;

[0171] Seal the slides with anti-fluorescence quenching sealing solution and store them in a dry oven at 37°C.

[0172] The rat brain tissue sections were observed under a 10X objective lens of a fluorescence microscope and fluorescent photographs were taken.

[0173] Test results such as Figure 6 shown. Figure 6 To show that the human anti-GAD65 antibody according to the present invention can identify the immunofluorescence image of the synapses of rat hippocampal neurons, the staining results of the rat hippocampal neurons cells incubated with the human anti-GAD65 antibody primary antibody and using the anti-human fluorescent secondary antibody. As shown in the figure, the human anti-GAD65 antibody according to the present invention can detect obvious positive signals on rat brain tissue sections, and the positive signals appear in the synaptic bulb layer area of ​​the rat hippocampus. This is consistent with the known synaptic localization of the GAD65 protein.

[0174] Figure 6 The results show that the humanized anti-GAD65 antibody of the present invention can identify the GAD65 protein of neuronal cells in the rat hippocampus on rat brain tissue sections.

[0175] Example 7 Recombinant human anti-GAD65 antibody for immunostrip detection

[0176] This example tests and further verifies whether the recombinant human anti-GAD65 antibody can recognize the GAD65 protein in the membrane strip product.

[0177] Preparation of GAD65 membrane strip test products

[0178] The GAD65 protein comes from the previous patented technology: an expression vector for detecting neurological paraneoplastic syndrome and its preparation method (application number: 202310428950.3). The protein is then prepared into an immune membrane strip product for testing.

[0179] The specific experimental steps are as follows:

[0180] Dilute GAD65 protein to working concentration using antigen diluent;

[0181] Add samples to the 96-well plate in the order of spraying;

[0182] Place the 96-well plate into the sample loading area of ​​the film sprayer and execute the film spraying procedure;

[0183] The nitrocellulose membrane after spraying is dried at room temperature;

[0184] The dried nitrocellulose membrane is placed in the blocking solution for blocking;

[0185] The blocked nitrocellulose membrane was taken out and placed in a 37°C oven for drying.

[0186] Use a membrane cutter to cut the nitrocellulose membrane and set aside.

[0187] GAD65 membrane strip test product testing

[0188] The membrane strip of the present invention is used to perform membrane strip method detection, and the specific steps are as follows:

[0189] Add 1 ml of dilution buffer to the incubation tank to ensure that the GAD65 membrane strips are completely covered;

[0190] Add 5 µl of positive control to each reaction tank;

[0191] Mix carefully, then incubate on a shaker at room temperature (18-25°C) for 30 min;

[0192] Wash with washing buffer: Use a pipette to carefully aspirate or gently pour out the liquid in the incubation tank, then add 1 ml of washing buffer and shake for 30 seconds. Repeat the above steps 5 times;

[0193] Add 1 ml of enzyme conjugate to each incubation tank;

[0194] Incubate on a shaker at room temperature for 30 min.

[0195] Wash with washing buffer: Use a pipette to carefully aspirate or gently pour out the liquid in the incubation tank, then add 1 ml of washing buffer, shake for 30 seconds, and repeat the above steps 5 times;

[0196] Add 1 ml of substrate solution to each incubation tank;

[0197] At room temperature, incubate for 15-20 min until the bands on the membrane strip are clearly visible, using the positive quality control membrane strip as a reference;

[0198] Terminate the reaction with deionized water: Use a pipette to carefully aspirate or gently pour out the liquid in the incubation tank, then add 1 ml of deionized water to each incubation tank and shake for 30 seconds. Repeat the above steps 5 times;

[0199] Place the membrane strip on absorbent paper and wait for it to dry completely, then read the result. Figure 7 shown.

[0200] Figure 7 The top band in the test membrane strips in Part A is the front mark of the membrane made with a ballpoint pen, the middle band is the quality control line, and the lower band is the band detected using the GAD65 antibody (L7-21-1), such as Figure 7 On the left side of part A is a schematic diagram of all the bands. The order of the paraneoplastic protein bands, from top to bottom, is: Ma1, PKCγ, Amphiphysin, CV2, GAD65, Hu, Ma2, Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, Zic4. The 5th band from top to bottom is the GAD65 antigen. The experimental results show that the GAD65 antibody (L7-21-1) can recognize the expressed human GAD65 recombinant protein and produce a strong purple detection band.

[0201] The antibodies of the present invention were also validated using membrane strips from a commercial kit (Ravo line 14 assay). Figure 7 As shown in part B, the 4th protein band was detected using the GAD65 antibody (L7-21-1). According to the order of the protein bands in the Ravo membrane strip, from top to bottom they are: Titin, Recoverin, PKCγ, GAD65, Zic4, Tr (DNER), SOX1, Ma2, Ma1, Amphiphysin, CV2, Ri, Yo, Hu. The 4th band from top to bottom is the GAD65 antigen. The GAD65 antibody (L7-21-1) of the present invention can also react immunologically with the GAD65 protein in the commercial product membrane strip to produce a purple detection band.

[0202] The human GAD65 antibody screened by the present invention can effectively identify GAD65. Through the above-mentioned ICC, IHC, LIA and other experiments, it is shown that the human GAD65 antibody of the present invention recognizes the linear epitope of the GAD65 protein. This linear epitope can exist in both natural GAD65 protein and denatured GAD65 protein. Therefore, the GAD65 antibody of the present invention has a wide range of applications. The GAD65 antibody of the present invention can be used as a positive standard for autoimmune encephalitis and paraneoplastic syndrome antibody detection kits. The human anti-GAD65 antibody has several advantages as a positive standard: it can widely identify human GAD65 proteins after different treatments; it shares the anti-human secondary antibody with the patient sample for detection, and no additional reagent components are required; it is a fully human sequence, which simulates the binding of antibodies and antigens in the human body to the greatest extent, and has better representativeness.

Claims

1. An anti-GAD65 antibody, characterized in that The antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4; and the light chain variable region comprising LCDR1 as shown in SEQ ID NO:6, LCDR2 as shown in SEQ ID NO:7, and LCDR3 as shown in SEQ ID NO:

8. The antibody according to claim 1 , which is a monoclonal antibody. The antibody according to claim 2 , which is a human monoclonal antibody.

4. The antibody according to any one of claims 1 to 3, wherein the heavy chain variable region amino acid sequence of the antibody is SEQ ID NO: 1 or an amino acid sequence that is at least 75% identical to SEQ ID NO: 1; and the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 5 or an amino acid sequence that is at least 75% identical to SEQ ID NO:

5.

5. The antibody according to claim 4, wherein the heavy chain amino acid sequence of the antibody is SEQ ID NO: 9 or an amino acid sequence that is at least 75% identical to SEQ ID NO: 9; and the light chain amino acid sequence is SEQ ID NO: 10 or an amino acid sequence that is at least 75% identical to SEQ ID NO:

10.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a polynucleotide encoding the antibody according to any one of claims 1-5.

7. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule according to claim 6. The recombinant vector according to claim 7 , which is a plasmid.

9. A host cell, characterized in that The host cell is introduced with or contains the recombinant vector according to claim 7 or 8. The host cell according to claim 9 , which is a eukaryotic cell.

11. A method for preparing the antibody according to any one of claims 1 to 5, characterized in that: The method comprises the step of culturing the host cell according to claim 9 or 10 in a culture medium to obtain the antibody.

12. Use of the antibody according to any one of claims 1 to 5 in the preparation of a membrane strip product or a membrane strip kit for detecting anti-GAD65 antibodies.

13. The use according to claim 12, wherein the antibody according to any one of claims 1 to 5 is a positive control in a membrane strip product or a membrane strip kit.

14. An anti-GAD65 antibody detection kit, characterized in that: The kit comprises the antibody according to any one of claims 1 to 5 as a positive control.

15. Use of the antibody according to any one of claims 1 to 5 in preparing a kit for detecting GAD65.

16. The use according to claim 15, wherein the kit is a cell immunofluorescence, tissue immunofluorescence and protein immunoblotting detection kit.

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