Anti-AAV6 antibody and application thereof in AAV6 titer determination

Abstract

The invention belongs to the technical field of biology, and relates to an anti-AAV6 antibody and application thereof in AAV6 titer determination. The anti-AAV6 monoclonal antibody 3G8 with high specificity is obtained through mouse immunization and antibody screening, and the antibody can specifically recognize and combine with complete AAV6 virus particles or carrier particles. The mouse Anti-AAV6 monoclonal antibody 3G8 has high serotype specificity, is only combined with AAV6 complete virus particles, and does not react with virus particles of other serotypes such as AAV2, AAV5, AAV8, AAV9 and the like. Meanwhile, conformational epitope dependency is achieved, only the native conformational epitope of the AAV6 capsid is recognized, and VP1 subunits, VP2 subunits and VP3 subunits in a depolymerization state are not combined.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an anti-AAV6 antibody and its application in the determination of AAV6 titer. Background Technology

[0002] Adeno-associated virus (AAV) has become a core delivery vector for gene therapy due to its advantages such as low pathogenicity, long-term gene expression, and tissue-specific targeting. The highly symmetrical capsid structure of AAV is a core component that completes all key steps in its viral life cycle, playing an irreplaceable role from attaching to host cells, entering the cell, releasing the genome, to immune evasion. It not only protects the viral genome from degradation but also guides the virus to recognize cells, cross the cell barrier, and initiate the infection process. Therefore, the AAV capsid is not only valuable for research in virology but also the core of AAV as a therapeutic gene delivery platform.

[0003] The AAV capsid is assembled from 60 VP protein subunits into an icosahedral structure with a diameter of approximately 26 nm, where the ratio of VP1:VP2:VP3 is 1:1:10. The N-terminal sequences of VP1 / 2 / 3 are different, and VP1 and VP2 extend additional functional domains based on the VP3 sequence. The β-sheet barrel-shaped core on the capsid surface is composed of multiple symmetry axes (pentaplex, triple, and biplex), and its connecting loops form a prominent spike domain around the triple symmetry axis. This region is a key target for host receptor binding and antibody recognition.

[0004] Differences in the capsid sequence of different AAV serotypes lead to varying specificities in their interaction with host receptors, resulting in significant tissue tropism. Currently, more than ten serotypes are in clinical trials, primarily targeting the eye, liver, muscle, and central nervous system. Due to its highly efficient transduction capabilities into respiratory epithelial cells, muscle tissue, and cardiomyocytes, AAV6 shows great potential in gene therapy for muscle-related diseases. For example, in some studies of muscular dystrophy, AAV6 vectors have been used to deliver therapeutic genes, aiming to improve muscle function. AAV6 also exhibits a certain affinity in lung tissue, making it a potential gene delivery vector in gene therapy explorations targeting certain genetic lung diseases or lung conditions.

[0005] In the current field of gene therapy, accurate titer determination is a crucial step in drug quality control for therapeutic AAV6 vectors. Similar to AAV2, the AAV6 vector capsid titer is commonly determined using ELISA, requiring antibodies that specifically bind to the AAV6 vector as capture and detection antibodies. However, like other AAV vectors used in gene therapy, AAV6 exhibits low immunogenicity, posing significant challenges to mouse immunization and the development of highly effective antibodies. Currently, there are no commercially available AAV6 quantitative detection kits in China. Developing highly specific AAV6 antibodies and reliable quantitative methods is essential for advancing the research, quality control, and clinical application of AAV6-based gene therapy drugs. This not only helps improve the efficacy of gene therapy but also better ensures patient safety and treatment effectiveness. Research on AAV6 antibodies and the development of related technologies have significant practical implications and application value for the field of gene therapy. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention obtains a highly specific anti-AAV6 monoclonal antibody 3G8 through mouse immunization and antibody screening. This antibody can specifically recognize and bind to intact AAV6 virus particles or vector particles.

[0007] The first aspect of this invention discloses an Anti-AAV6 antibody, wherein the variable region of the antibody light chain includes CDR-L1, CDR-L2, and CDR-L3; and the variable region of the antibody heavy chain includes CDR-H1, CDR-H2, and CDR-H3. According to the official standards of the International Immunogenetic Information System (IMGT), the amino acid sequence position of the complementarity-determining region (CDR) (CDR-IMGT) is as follows: Antibody light chain: CDR-L1: QSVDYDGDNY (SEQ ID NO: 1); CDR-L2::VAS (SEQ ID NO: 2); CDR-L3: SQSYEDPPT (SEQ ID NO: 3); Antibody heavy chain: CDR-H1: GYSFTDYS (SEQ ID NO: 4); CDR-H2:INTETGEP (SEQ ID NO: 5); CDR-H3: TPRIYFAFTH (SEQ ID NO: 6).

[0008] The amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO: 7: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDNYMNWYQQKPGQPPKLLIYVASTLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYFCSQSYEDPPTFGGGTKLEIE (SEQ ID NO: 7); The amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO: 8: QIHLVQSGPELKKPGETVKISCRASGYSFTDYSMHWVEQAPGKGLKWMGWINTETGEPTYADDFTGRFAFSLETSASTAYLQISNLKNEDTATYFCTPRIYFAFTHWGQGTLVTVSA (SEQ ID NO: 8).

[0009] The amino acid sequence of the constant region of the antibody light chain is shown in SEQ ID NO: 9: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 9); The amino acid sequence of the constant region of the antibody heavy chain is shown in SEQ ID NO: 10: ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEV HTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPGK (SEQ ID NO: 10).

[0010] The amino acid sequence of the antibody light chain is shown in SEQ ID NO: 11: METDTILLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDNYMNWYQQKPGQPPKLLIYVASTLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYFCSQSYEDPPT FGGGTKLEIERADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 11); The amino acid sequence of the antibody heavy chain is shown in SEQ ID NO: 12: MAWVWTLLFLMAAAQSIQTQIHLVQSGPELKKPGETVKISCRASGYSFTDYSMHWVEQAPGKGLKWMGWINTETGEPTYADDFTGRFAFSLETSASTAYLQISNLKNEDTATYFCTP RIYFAFTHWGQGTLVTVSAATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEP RIPKPSTPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIE RTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPGK (SEQ ID NO: 12).

[0011] The amino acid sequence of the signal peptide on the antibody light chain is shown in SEQ ID NO: 13: METDTILLWVLLLWVPGSTG (SEQ ID NO: 13); The amino acid sequence of the signal peptide on the antibody heavy chain is shown in SEQ ID NO: 14: MAWVWTLLFLLMAAAQSIQT (SEQ ID NO: 14).

[0012] Among them, the nucleotide sequence of the antibody light chain is shown as SEQ ID NO: 15: ATGGAAACCGACACAATATTGCTTTGGGTACTCCTGCTCTGGGTCCCCGGGAGTACAGGCGACATTGTCCTTACACAGAGTCCTGCTAGTTTGGCCGTTTCACTCGGACAAAGGGCGACCATCTCTTGCAAGGCTAGCCAAAGTGTGGACTACGACGGCGACAATTATATGAATTGGTATCAACAGAAGCCCGGTCAGCCTCCCAAACTCCTGATCTATGTCGCTTCCACCCTTGAGAGTGGAATCCCTGCAAGGTTTTCTGGATCAGGCTCTGGGACCGACTTCACCCTGAATATTCACCCTGTGGAGGAGGAAGATGCAGCAACATACTTCTGCTCCCAGTCCTACGAAGATCCCCCTACTTTCGGAGGAGGAACCAAGCTGGAAATTGAACGCGCCGATGCCGCTCCTACAGTGAGCATCTTTCCTCCTTCCTCCGAGCAGCTGACAAGCGGCGGCGCCAGCGTGGTGTGTTTCCTGAACAACTTCTATCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACTCCTGGACCGACCAGGATTCCAAGGACTCCACCTACTCCATGTCCTCCACACTGACCCTGACCAAGGATGAGTACGAGAGGCACAACAGCTACACATGCGAGGCCACACACAAGACCTCCACCAGCCCTATCGTGAAGAGCTTCAATAGAAACGAGTGC (SEQ ID NO: 15); The nucleotide sequence of the antibody heavy chain is shown as SEQ ID NO: 16.

[0013]

[0014] The second aspect of this invention discloses a titer determination ELISA kit, which uses 3G8 antibody as the capture and detection antibody to develop an AAV6 titer determination ELISA kit.

[0015] This kit uses the principle of double-antibody sandwich ELISA: Capture: The monoclonal antibody 3G8 is coated onto an ELISA plate to specifically capture intact AAV6 particles in the sample. Unassembled or depolymerized capsid proteins lack the conformational epitopes recognized by this antibody and are therefore not captured, thus significantly reducing the risk of false positives.

[0016] Detection: Biotin-labeled monoclonal antibody 3G8 was added as the detection antibody. The intact AAV6 particle surface contains multiple identical conformational epitopes, therefore biotinylated 3G8 can bind to the captured AAV6 particles.

[0017] Signal amplification and color development: Streptavidin-horseradish peroxidase (SA-HRP) was added, which binds to the detection antibody via biotin. HRP substrate was then added for the colorimetric reaction.

[0018] Quantitative analysis: The color intensity is directly proportional to the number of AAV6 virus particles captured and bound, thereby enabling accurate determination of AAV6 vector titers.

[0019] Detection Principle: This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the AAV6 capsid content in samples. AAV6 monoclonal antibodies are pre-coated into 96-well reaction plates and packaged to ensure their activity. After adding calibrators or test samples, the AAV6 capsid in the standards or samples specifically binds to the reaction plate. Then, detection antibodies and HRP markers are added, forming an antibody-antigen-[detection antibody]-[HRP marker] complex. Excess detection antibody and HRP markers are removed by washing. After adding the chromogenic solution, HRP catalyzes the color development; the color intensity is directly proportional to the AAV6 capsid titer in the sample. After stopping the reaction with a stop solution, the absorbance at 450 nm is read on a microplate reader to calculate the AAV6 capsid titer in the sample.

[0020] A third aspect of this invention discloses the application of the aforementioned Anti-AAV6 antibody or titer assay ELISA kit in the quality control of AAV gene therapy drugs. The Anti-AAV6 antibody or titer assay ELISA kit is used to determine the dosage of AAV6 viral particles in AAV gene therapy drugs.

[0021] Compared with the prior art, the present invention has the following advantages: 1. The mouse Anti-AAV6 monoclonal antibody 3G8 exhibits high serotype specificity, binding only to intact AAV6 viral particles and not reacting with viral particles from other serotypes such as AAV2, AAV5, AAV8, and AAV9. It also exhibits conformational epitope dependence, recognizing only the native conformational epitopes of the AAV6 capsid and not binding to the depolymerized VP1, VP2, and VP3 subunits.

[0022] 2. This AAV6 viral titer quantitative detection kit has significant performance advantages. The kit exhibits strict serotype specificity, generating a specific signal only for intact AAV6 viral particles. It does not cross-react with viral particles from other serotypes such as AAV2, AAV5, AAV8, and AAV9. The AAV6 ELISA kit showed a linear range of 1.00E+08 capsids / mL–6.40E+09 capsids / mL, a sensitivity of 5E+07 capsids / mL, and intra-assay and inter-assay CV% of 5.9% and 7.9%, respectively, both within the allowable error range (intra-assay CV% < 10%, inter-assay CV% < 10%). The results demonstrate high precision and good repeatability of the kit, avoiding false positives that could occur in practical applications.

[0023] 3. This invention addresses the lack of commercially available AAV6 quantitative detection kits in China, providing a crucial quality control tool for the development of AAV6-based gene therapy drugs. By specifically detecting intact viral particles (rather than total protein), it more accurately reflects the viral titer of infectious agents, providing better assurance for the safety and efficacy of gene therapy products. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or existing methods and experiments, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is a graph showing the specificity of the 3G8 antibody.

[0026] Figure 2 The image shows the detection of binding of 3G8 antibody to undenatured and denatured depolymerized AAV6 capsid.

[0027] Figure 3 This is a linear range diagram of the AAV6 ELISA quantitative kit based on the 3G8 antibody. Detailed Implementation

[0028] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this does not limit the present invention to the scope of the described embodiments. Experimental methods and techniques in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions in the relevant art or according to the conditions recommended by the manufacturer.

[0029] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0030] Experimental materials: 1. AAV2, 5, 6, 8, 9-GFP: Vigen Biotech, Belief Biotech, Progen; 2. Varioskan™ Lux (Multifunctional Microplate Analyzer): ThermoFisher Scientific; 3. Microplates, plate washer, 37°C constant temperature shaking incubator: Thermo Fisher Scientific; 4. Immunized mice: Shanghai Shengchang Biotechnology Co., Ltd.

[0031] Example 1: AAV6 antigen immunization and antibody screening 1. Mouse immunization: 1 μL of AAV6-GFP (1E+13 capsids / mL) was diluted 100-fold with PBS and mixed with an equal volume of aluminum hydroxide adjuvant. This mixture was then administered subcutaneously at multiple sites to immunize female Balb / C mice aged 6–8 weeks at a dose of 200 μL per mouse. Booster immunizations were administered two weeks apart with the same dose and method. After four immunizations, serum titer was measured, reaching a titer of 1:800,000. A booster immunization was then administered intraperitoneally with 100 μL of diluted AAV6-GFP (without adjuvant). Three days later, SP2 / 0 fusion of myeloma cells was performed.

[0032] 2. Screening of hybridoma cell lines: Three days after booster immunization of mice, spleen cells were fused with logarithmic growth phase myeloma cells SP2 / 0 using PEG1500 to prepare hybridoma cells. Ten days later, cells coated with AAV6-GFP and AAV2 / 5 / 8 / 9-GFP were screened using indirect ELISA to identify cell lines that reacted only with AAV6-GFP and not with other serotypes. These cell lines were then subjected to limiting dilution to a monoclonal state, expanded culture, and cryopreserved. The cell supernatant from the expanded culture was then subjected to indirect ELISA to obtain the highly active, specific anti-AAV6 antibody cell line 3G8.

[0033] 3. Hybridoma monoclonal antibody sequencing and recombinant expression and purification: The selected 3G8 hybridoma cell line was expanded to a density of 1E+07 cells / mL and sent for hybridoma monoclonal antibody sequencing (Baiying Biotechnology Co., Ltd.). The variable region sequences of the 3G8 heavy and light chain antibodies were fused with the constant region sequences (mouse IgG3 / mouse Kappa) respectively. Lactogenic codon optimization gene synthesis was performed, and the resulting genes were constructed into lactogenic cell expression vectors. The pTT5 expression vector was used (any commercially available lactogenic cell expression vector such as pCDNA3.1 can also be used). Expi293 lactogenic cells were transfected with the plasmid to secrete and express the 3G8 antibody. The cell expression supernatant was purified by protein A affinity and molecular sieve purification to obtain the AAV6-specific recombinant antibody 3G8. This antibody was used for antibody characterization, antibody biotin labeling, and kit development.

[0034] Example 2: 3G8 antibody specificity AAV2 / 5 / 6 / 8 / 9-GFP was coated in microplates at a concentration of 1E+09 capsids / mL. Recombinant monoclonal 3G8 antibody was initially added at a concentration of 10 μg / mL, serially diluted 3-fold, with 100 μL added to each well. Goat anti-mouse Fc-HRP antibody at 0.5 μg / mL was used as the secondary antibody. Figure 1 The results showed that the 3G8 mouse monoclonal antibody only reacted with AAV6, but not with AAV2 / 5 / 8 / 9, which are commonly used AAV serotypes for gene delivery, indicating that the recombinant monoclonal 3G8 antibody has good specificity.

[0035] Example 3: 3G8 antibody binds to AAV6 particles but not to the denatured VP subunit. Sandwich ELISA: 3G8 antibody was coated onto an ELISA plate at a concentration of 2 μg / mL. AAV6-GFP (7E+9 capsids / mL) was serially diluted 3-fold with PBST. A portion of the diluted sample was heated at 95°C for 5 minutes to depolymerize and denature. Undenatured AAV6-GFP particles and denatured AAV6 (VP1 / VP2 / VP3) samples were added separately to the 3G8 antibody-coated ELISA plate for binding. Detection was then performed using 1 μg / mL of biotinylated 3G8 antibody. Figure 2 The 3G8 antibody showed that it only binds to the undepolymerized AAV6-GFP capsid and not to the denatured and depolymerized AAV6 VP1, 2, 3 subunits.

[0036] Example 4: Linear range of AAV6 ELISA quantitative kit based on 3G8 antibody 1. Preparation of reagents for AAV6 ELISA quantitative kit: (1) Prepare 1× washing solution: 1XPBS, 0.05%, Tween 20; (2) Prepare 1× analysis buffer: 1XPBS, 0.05%, Tween 20, 3% BSA; (3) Preparation of detection antibody: Use activated biotin (NHS-biotin) at a biotin to 3G8 antibody mass ratio of 1:10, mix well, label at 37 degrees for half an hour, and dialyze overnight with 1X PBS. Dilute the dialyzed detection antibody with 1X analytical buffer to 0.5ug / mL for later use; (4) Preparation of the stop solution: Dilute 98% concentrated sulfuric acid with ultrapure water to 1 M / L; (5) Standards: Dilute AAV6 (1E+13 capsids / mL) with 1X analytical buffer to 2.50E+09 capsids / mL; (6) Prepare SA-HRP working solution: Dilute SA-HRP at a concentration of 1 mg / mL with 1X analytical buffer by 10,000 times.

[0037] 2. AAV6 ELISA Quantitative Reagent Kit Operation Procedure: (1) Before the experiment, take the kit out of the refrigerator and place it at room temperature to equilibrate to room temperature.

[0038] (2) Prepare 1× buffer: Dilute 20× buffer with ddH2O to 1× for later use.

[0039] (3) Prepare 1× detection antibody: Dilute the detection antibody 20 times with 1× buffer solution for later use.

[0040] (4) Preparation of standard: Take out the standard, add 700uL ddH2O, dissolve at room temperature for 10-20 min, mix well and avoid vortexing, take 250 μL of standard and dilute the 2.5E+09 capsids / mL standard with 1× buffer at a ratio of 2 to obtain 7 titers: 1.25E+09 capsids / mL, 6.25E+08 capsids / mL, 3.13E+08 capsids / mL, 1.56E+08 capsids / mL, 7.81E+07, and 3.91E+07 capsids / mL. Use 1× buffer as the zero-point standard. Each titer standard should be replicated.

[0041] (5) Pre-wash: Take out the 96-well reaction plate according to the required test volume, wash the plate once, add 300 μL of 1× buffer to each well, pat dry after washing, and put the unused strips into a sealed bag as soon as possible and store at 2-8℃.

[0042] (6) Adding samples: Add 100 μL of standard and sample to each well of the 96-well reaction plate and incubate at 37°C for 60 min.

[0043] (7) Add 1× detection antibody: Wash the 96-well plate after the previous reaction three times with 1× buffer, add 300 μL of 1× buffer to each well, pat dry, and immediately add 100 μL of 1× detection antibody to each well. Incubate at 37℃ for 60 min.

[0044] (8) Add HRP label: Wash the 96-well plate three times after the previous reaction, add 300 μL of 1× buffer to each well, pat dry, add 100 μL of HRP label to each well, and incubate at 37℃ for 60 min.

[0045] (9) Colorimetric reading: Wash the 96-well plate three times after the previous reaction. Add 300 μL of 1× buffer to each well, pat dry, and then add 100 μL of colorimetric solution to each well. Incubate at 37°C in the dark for 10 min. Add 50 μL of stop solution to each well and mix gently. Immediately read the OD value of each well at 450 nm using a microplate reader. (It is recommended to preheat the colorimetric solution at 37°C, and complete the reaction from termination to OD value reading within 5 min).

[0046] like Figure 3 As shown, the linear range of the AAV6 Elisa kit is 1.00E+08 capsids / mL–6.40E+09 capsids / mL, and the sensitivity is 5E+07 capsids / mL.

[0047] Example 5: Precision of the ELISA quantitative reagent kit AAV6 capsid particles were diluted with 1X dilution buffer to three titers: 1.6E+09 capsids / mL, 8E+08 capsids / mL, and 4E+08 capsids / mL (high, medium, and low titers). The AAV6 ELISA kit was used to continuously analyze the high, medium, and low titers of AAV6 particles diluted within the linear range 20 times, once daily for 20 consecutive days. The results showed that the intra-assay and inter-assay CV% were 5.9% and 7.9%, respectively, both within the allowable error range (intra-assay CV% < 10%, inter-assay CV% < 10%). These results indicate that the kit has high precision and good reproducibility.

[0048] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall also fall within the protection scope of the present invention.

Claims

1. An Anti-AAV6 antibody, characterized in that, The antibody light chain variable region includes CDR-L1, CDR-L2, and CDR-L3; the antibody heavy chain variable region includes CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of the CDR-L1 is shown in SEQ ID NO: 1; The amino acid sequence of the CDR-L2 is shown in SEQ ID NO: 2; The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 3; The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 4; The amino acid sequence of the CDR-H2 is shown in SEQ ID NO: 5; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO:

6.

2. The Anti-AAV6 antibody according to claim 1, characterized in that, The amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO: 7; the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO:

8.

3. The Anti-AAV6 antibody according to claim 1, characterized in that, The amino acid sequence of the constant region of the antibody light chain is shown in SEQ ID NO: 9, and the amino acid sequence of the constant region of the antibody heavy chain is shown in SEQ ID NO:

10.

4. The Anti-AAV6 antibody according to claim 1, characterized in that, The amino acid sequence of the antibody light chain is shown in SEQ ID NO: 11; the amino acid sequence of the antibody heavy chain is shown in SEQ ID NO:

12.

5. The Anti-AAV6 antibody according to claim 1, characterized in that, The amino acid sequence of the signal peptide on the antibody light chain is shown in SEQ ID NO: 13; the amino acid sequence of the signal peptide on the antibody heavy chain is shown in SEQ ID NO:

14.

6. The Anti-AAV6 antibody according to claim 1, characterized in that, The antibody light chain nucleotide sequence is shown in SEQ ID NO: 15; the antibody heavy chain nucleotide sequence is shown in SEQ ID NO:

16.

7. A titer determination ELISA kit, characterized in that, The kit includes the Anti-AAV6 antibody as described in any one of claims 1-6.

8. The titer determination ELISA kit according to claim 7, characterized in that, Based on the antibody sandwich ELISA method, Anti-AAV6 antibodies that specifically recognize AAV6 capsid conformational epitopes are pre-coated on a kit plate to capture AAV6 virus particles from the sample.

9. The use of the Anti-AAV6 antibody as described in any one of claims 1-6 or the titer assay ELISA kit as described in any one of claims 7-8 in the quality control of AAV gene therapy drugs.

10. The application according to claim 9, characterized in that, The dosage of AAV virus particles in AAV gene therapy drugs can be determined using the Anti-AAV6 antibody or titer assay ELISA kit.

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