Anti-AAV5 monoclonal antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-AAV5 monoclonal antibody as well as a preparation method and application thereof, and particularly discloses an anti-AAV5 monoclonal antibody 7B4, 7G3 and 7B4 heavy chain variable region amino acid sequence as shown in SEQ ID NO.13, and an anti-AAV5 monoclonal antibody light chain variable region amino acid sequence as shown in SEQ ID NO.15; the amino acid sequence of the 7G3 heavy chain variable region is as shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.19. The invention also discloses a preparation method of the anti-AAV5 monoclonal antibodies 7B4 and 7G3, and an application of the anti-AAV5 monoclonal antibodies 7B4 and 7G3 in preparation of an AAV5 neutralizing antibody detection kit or a kit for quantitative detection of AAV5 viruses. According to the invention, rabbit immunization is adopted, and the obtained monoclonal antibodies 7B4 and 7G3 have higher affinity and stronger neutralizing activity compared with the existing anti-AAV5 antibodies ADK5a and ADK5b.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and in particular to an anti-AAV5 monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Adeno-associated virus (AAV) vectors are widely used in gene therapy. These viruses are considered non-pathogenic and are widely used in gene therapy and vaccine research due to their safety, stability, and ability to infect both dividing and non-dividing cells. AAV5, a serotype, has high delivery efficiency in tissues such as the lungs and liver and is widely used in gene therapy research. Currently, marketed gene therapy drugs based on AAV5 vectors are primarily focused on the treatment of hemophilia. Hemgenix (etranacogene dezaparvovec) received marketing approval from the US FDA in November 2022 for the treatment of adults with hemophilia B. It is administered intravenously and expresses coagulation factor IX in the liver, thereby improving the patient's coagulation function. Roctavian (valoctocogene roxaparvovec) received conditional marketing authorization from the European Union in August 2022 and was approved by the US FDA in June 2023 for the treatment of adults with severe hemophilia A. This gene therapy, administered through a single intravenous dose, enables patients to express their own coagulation factor VIII, reducing the need for long-term reliance on coagulation factor injections.

[0003] During the development of AAV5-based gene therapy drugs, AAV5 antibodies can be used for quantitative analysis of AAV5 drugs. Antibodies with AAV5 neutralizing activity can also serve as positive controls for monitoring and calibrating pre-existing neutralizing antibody levels in clinical trials. Anti-AAV5 antibodies with high affinity and neutralizing activity have broad application in gene therapy drug development and are extremely valuable tool antibodies.

[0004] Currently, the most commonly used anti-AAV5 monoclonal antibodies on the market are ADK5a and ADK5b, which are mouse monoclonal antibodies obtained by immunizing mice with the AAV5 viral vector and then screening them through hybridoma technology (DOI:10.1016 / j.jviromet.2006.10.005). During the discovery and practical application of antibodies, ADK5a and ADK5b are used only as binding antibodies, specifically detecting AAV5 using ELISA. However, ADK5a and ADK5b are used as neutralizing antibodies to characterize their inhibitory effects on AAV5-infected cells, but their neutralization effects are limited. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an anti-AAV5 monoclonal antibody and its preparation method and application. By immunizing rabbits, more diverse antigenic epitopes were screened, and after neutralizing antibody activity characterization screening, neutralizing antibody clones with stronger activity were obtained. Rabbits were immunized with AAV5 virus particles with a complete spatial structure, and specific B cells were isolated from the spleen. Then, the heavy chain variable region and light chain variable region DNA sequences were amplified from these B cells by single-cell PCR technology, and finally expressed in mammalian cells to obtain monoclonal antibodies with biological activity. The obtained monoclonal antibodies have higher affinity than the existing anti-AAV5 antibodies ADK5a and ADK5b. At the same time, the neutralizing antibody screening process was introduced in the screening process. The obtained anti-AAV5 monoclonal antibodies are antibodies with strong neutralizing activity, which solves the problem that the neutralizing effect of anti-AAV5 monoclonal antibodies in the prior art is poor and cannot be used as a good positive control when screening neutralizing antibodies in subjects.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides an anti-AAV5 monoclonal antibody 7B4, comprising a heavy chain and a light chain;

[0008] The heavy chain includes a heavy chain variable region, and the three complementarity determining regions of the heavy chain variable region are:

[0009] CDR1: GFSLNHYA (SEQ ID NO. 61);

[0010] CDR2:LSWVGNT(SEQ ID NO.62);

[0011] CDR3: GRAIWAADSNAIFNL (SEQ ID NO.63);

[0012] The light chain includes a light chain variable region, and the three complementarity determining regions of the light chain variable region are:

[0013] CDR1: QNIYDF (SEQ ID NO.64);

[0014] CDR2:GAS;

[0015] CDR3: QCTYDGGTYVA (SEQ ID NO. 65).

[0016] As some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-AAV5 monoclonal antibody 7B4 is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.15.

[0017] Heavy chain variable region (SEQ ID NO.13):

[0018] QSVEESGGRLVTPGTPLTLTCTVSGFSLNHYAMIWVRQAPGEGLEYIGFLSWVGNTYYATWARGRFTISRTSTTVDLRVTSLTTEDTGTYFCGRAIWAADSNAIFNLWGQGTLVTVSS.

[0019] Light chain variable region (SEQ ID NO.15):

[0020] DVVMTQTPASVEAAVGGTVTIKCQASQNIYDFLAWYQQKPGQPPKLLIYGASTLTSGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCTYDGGTYVAFGGGTEVVVK.

[0021] As some specific embodiments of the present invention, the gene sequence of the heavy chain variable region of the anti-AAV5 monoclonal antibody 7B4 is shown as SEQ ID NO.14, and the gene sequence of the light chain variable region is shown as SEQ ID NO.16.

[0022] In a second aspect, the present invention provides an anti-AAV5 monoclonal antibody 7G3, comprising a heavy chain and a light chain;

[0023] The heavy chain includes a heavy chain variable region, and the three complementarity determining regions of the heavy chain variable region are:

[0024] CDR1: GFSLSSYA (SEQ ID NO.66);

[0025] CDR2: IYAGSGIT (SEQ ID NO.67);

[0026] CDR3: ARAQYANGNIYYGL (SEQ ID NO.68);

[0027] The light chain includes a light chain variable region, and the three complementarity determining regions of the light chain variable region are:

[0028] CDR1:ESISSG(SEQ ID NO.69);

[0029] CDR2: YAS;

[0030] CDR3: QQGFTESNVDNT (SEQ ID NO. 70).

[0031] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-AAV5 monoclonal antibody 7G3 is shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.19.

[0032] Heavy chain variable region (SEQ ID NO.17):

[0033] QSVEESGGRLVTPGTPLTLTCTASGFSLSSYAMGWVRQAPGKGLEWIGIIYAGSGITSYASWANGRFTISKTSTTVDLKITSPTIEDTATYFCARAQYANGNIYYGLWGQGTLVTVSS.

[0034] Light chain variable region (SEQ ID NO.19):

[0035] AYDMTQTPASVEAAVGGTVTIKCQASESISSGLAWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQGFTESNVDNTFGGGTEVVVK.

[0036] As some specific embodiments of the present invention, the gene sequence of the heavy chain variable region of the anti-AAV5 monoclonal antibody 7G3 is shown as SEQ ID NO.18, and the gene sequence of the light chain variable region is shown as SEQ ID NO.20.

[0037] In a third aspect, the present invention provides a method for preparing the anti-AAV5 monoclonal antibody 7B4 or the anti-AAV5 monoclonal antibody 7G3 as described above, comprising the following steps:

[0038] S1. Immunize rabbits with AAV5 virus particles and screen for specific B cells of positive clones;

[0039] S2. Cultivate specific B cells in vitro and screen and confirm positive B cell clones by ELISA;

[0040] S3. Amplify the DNA sequences of the heavy and light chain variable regions from specific B cells of positive clones using single-cell PCR technology and express them in mammalian cells;

[0041] S4. Screen and confirm the neutralizing effect of the expressed positive cloned antibodies through the AAV5 neutralizing antibody method.

[0042] In a fourth aspect, the present invention provides a use of the anti-AAV5 monoclonal antibody 7B4 or the anti-AAV5 monoclonal antibody 7G3 as described above in the preparation of an AAV5 neutralizing antibody detection kit.

[0043] As some specific embodiments of the present invention, the anti-AAV5 monoclonal antibody 7B4 or the anti-AAV5 monoclonal antibody 7G3 is used as a positive control in the AAV5 neutralizing antibody detection kit.

[0044] In a fifth aspect, the present invention provides a use of the anti-AAV5 monoclonal antibody 7B4 or the monoclonal anti-AAV5 antibody 7G3 as described above in the preparation of a kit for quantitative detection of AAV5 virus.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1) The present invention adopts the method of immunizing rabbits to screen monoclonal antibodies that can recognize more diverse antigen epitopes, and through neutralizing antibody activity characterization screening, neutralizing antibody clones with stronger activity are obtained.

[0047] 2) The rabbit monoclonal antibodies obtained by screening immunized rabbits using monoclonal B cloning technology have higher affinity than the existing anti-AAV5 monoclonal antibodies ADK5a and ADK5b, and have better sensitivity when used for AAV5 virus quantification. At the same time, the screening process also introduced a neutralizing antibody screening process. The resulting anti-AAV5 monoclonal antibody has strong neutralizing activity, with a neutralizing effect 28 times stronger than ADK5a and more than 3 times stronger than ADK5b, making it an excellent reference antibody for neutralizing antibody detection.

[0048] 3) Compared with the commercially available products ADK5a and ADK5b, the neutralization activity sensitivity of ADK5a is 1765.2 ng / mL, and the neutralization activity of ADK5b is 197.1 ng / mL, while the neutralization activity of the 7B4 clone of the present invention is 61.7 ng / mL and the neutralization activity of the 7G3 clone is 74.9 ng / mL, showing more significant neutralization activity sensitivity.

[0049] 4) The anti-AAV5 monoclonal antibodies 7B4 and 7G3 of the present invention can be used as key antibody materials for ELISA detection of AAV5 virus particles based on specific binding epitopes, and can be used for quantitative detection of AAV5 virus particles; they can more fully characterize the sensitivity of neutralizing antibody detection methods, can serve as more stable neutralizing antibody positive controls, and can be used as positive control raw materials for companion diagnostic kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0051] Figure 1 This is a flow cytometry sorting diagram of positive single B cells in Example 1. DETAILED DESCRIPTION

[0052] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0053] Example 1. Preparation of anti-AAV5 antibodies

[0054] 1. Immunity

[0055] Three healthy New Zealand white rabbits were immunized with inactivated AAV5 virus [ssAAV.CMV.EGFP.WPRE.SV40pA] (Paigen Biopharm, Catalog No. XH246-6). Immunizations were performed using the immunogen mixed with RTiterFast aqueous adjuvant (Pujian Biopharm, Catalog No. ATO00071). Each immunization used 100 μL of aqueous adjuvant. The amount of antigen was 150 μL AAV5 [ssAAV.CMV.EGFP.WPRE.SV40pA] (1E13 GC / mL) for the first immunization and 100 μL AAV5 [ssAAV.CMV.EGFP.WPRE.SV40pA] (1E13 GC / mL) for the second and third immunizations. Immunizations were injected into the hind leg muscles at two to three sites. Immunizations were administered weekly for a total of four times.

[0056] 2. Antiserum titer detection

[0057] Serum was collected from one of the rabbits to evaluate the immune effect, and the titer of the rabbit serum after three immunizations was detected by ELISA. The specific steps included: coating the ELISA plate with 100 μl / well of 5 μg / mL AAV5 [ssAAV.CMV.EGFP.WPRE.SV40pA] overnight; the next day, the supernatant was discarded, and after washing with PBST, 50 μl / well of 5% skim milk powder was added and incubated at 37°C for 1.5 hours; the supernatant was discarded, and after washing with PBST, the rabbit serum after three immunizations was gradiently diluted with 3% BSA in PBS diluent, starting at 2000 times, and then diluted 2 times to 128,000 times, and 25 μl / well was added to the enzyme labeling plate, incubated at 37°C for 1 hour, the supernatant was discarded, and after washing with PBST, 1:1000 diluted anti-rabbit IgG HRP was added. 100 μl / well was incubated at 37°C for 1 hour. The supernatant was discarded, patted dry, and TMB color development solution was added to terminate the color reaction. The antiserum titer was measured to be >1:128,000. The test results are shown in Table 1.

[0058] Table 1 Results of rabbit serum test after triple immunization with AAV5

[0059]

[0060] The results showed that the rabbit serum after 3 immunizations had a good immune effect and produced stronger anti-AAV5 antibodies.

[0061] 3. Spleen cell isolation and positive B cell screening

[0062] A rabbit with good serum immunization results after 4 immunizations was selected, and PBMC cells were extracted from the blood and spleen. The cells were divided into two groups. Biotin-labeled AAV5 virus was used, diluted with 1×PBS at a ratio of 1:50, incubated at 37°C for 1 hour and centrifuged. Goat anti-rabbit IgG-FITC (1:100, diluted with PBS) and AF647-Streptavindin (1:750, diluted with PBS) were added, incubated at 37°C for 1 hour, and resuspended by centrifugation. Before the flow cytometer was used for detection, 2% 7-AAD was added for staining to eliminate dead cells, and blank tubes and single-stained tubes were used to draw gates to obtain positive cells. Positive single B cells (memory B cells) were obtained by flow cytometry sorting (using membrane-type IgG positivity and antigen positivity for double positive screening). The extracted PBMC sorting diagram is shown in Figure 1 .like Figure 1 As shown, cells in gate R5 with strong positive results in the FITC channel (IgG positive) and the APC channel (AF647-Streptavindin positive) were circled for cell sorting, and the sorted cells were spread on 8 plates and cultured for 7-10 days.

[0063] 4. Detection and screening

[0064] After culture, the cell supernatant was aspirated and screened for antigen-specific positive clones of B cells using ELISA (AAV5 virus coating, similar to the antiserum titer test in step 2). The ELISA results showed 251 positive clones across Plates 1-8.

[0065] 5. Sequencing

[0066] According to the ELISA results, 15 positive clone B cells with stronger ELISA signal values ​​were selected from the 251 positive clones. The clone numbers and signal values ​​of the 15 positive clones are shown in Table 2.

[0067] Table 2 Test results of culture supernatants of 15 positive clones

[0068] serial number Clone number OD value 1 3F6 3.327 2 5A8 3.151 3 5E11 3.167 4 7B4 3.059 5 7G3 3.072 6 7G9 3.013 7 8B5 3.457 8 3B6 3.406 9 4D2 3.043 10 6C8 3.382 11 6E6 3.325 12 7C8 3.38 13 2F11 3.076 14 3G6 3.297 15 7F9 3.436

[0069] The amino acid sequence and gene sequence of the antibody heavy chain variable region were obtained by sequencing. The sequences of the heavy chain variable region and light chain variable region of the 15 positive clones are shown in Table 3.

[0070] Table 3 Antibody heavy chain variable region and light chain variable region sequences of 15 positive clones

[0071]

[0072] 6. Recombinant Expression

[0073] The DNA sequences of the heavy chain variable region and the light chain variable region were synthesized. 15 μg of DNA and 45 μl of PEI transfection reagent were co-transfected into XtenCHO cells in the logarithmic growth phase. The rabbit monoclonal antibody was recombinantly expressed using the mammalian system. The process is as follows:

[0074] (1) One day before transfection, inoculate 27×10 6 XtenCHO cells were cultured in complete medium (without antibiotics and anti-aggregation agents) so that the cells were in the logarithmic growth phase on the day of transfection.

[0075] (2) Preparation of transfection complex

[0076] ① Dilute 15 μg DNA into 1.5 ml complete culture medium and mix gently.

[0077] ② Dilute 45 μl of PEI transfection reagent into 1.5 ml of complete culture medium, mix gently, and incubate at room temperature for 5 minutes.

[0078] ③Add diluted PEI to the diluted DNA to a total volume of 3 ml. Mix gently and incubate at room temperature for 20-30 minutes.

[0079] (3) Transfection: Add 3 ml of transfection mixture to 27 ml of cell suspension, mix gently, and culture at 37°C, 5% CO2, 130 rpm until the cell viability drops below 50%. Collect the sample as the purified sample.

[0080] (4) Purification:

[0081] ① Add the purified rProtein A filler to the gravity column. After the 20% ethanol in the preservation solution has dripped off, add 10 times the column volume of ultrapure water to rinse out the ethanol. Finally, add 10 times the column volume of PBS pH 7.5 to equilibrate the gravity column.

[0082] ② Use a plug to plug the lower end of the gravity column, and use the sample to be purified to evenly suspend the filler, mix with the sample, and place it in a binding shaker. Place the binding shaker in a 4°C refrigerator. The binding time must not be less than 60 minutes.

[0083] ③ Remove the bound sample from the binding shaker and place it in a 4°C refrigerator for 5-10 minutes. Then use a pipette to add the mixture of sample and filler to the empty column and collect the flow-through sample (FT).

[0084] ④ Rinse the column with PBS pH 7.5 to elute non-specifically bound host proteins and collect the sample (W1);

[0085] ⑤ Wash one tube with Citric acid for each column volume, add the amount of neutralization solution tested previously, and gently invert to mix to collect the target recombinant protein.

[0086] The collected target recombinant proteins were tested, and the results showed that 11 out of 15 positive clones expressed recombinant antibody proteins.

[0087] Example 2: Secondary characterization of positive clones

[0088] The positive cloned antibody after recombinant expression and purification in Example 1 was taken, and after protein quantification, the binding strength was determined. The specific method is as follows: AAV5 [ssAAV.CMV.EGFP.WPRE.SV40pA] was diluted with PBS at a ratio of 1:200 and coated on an ELISA plate at 4°C overnight; the next day, 3% BSA-PBS was added, 75 μl / well was blocked at 37°C for 1.5 hours; the plate was washed, and the positive cloned antibody was diluted 2-fold starting from 1 μg / mL, for 7 consecutive points, to 15.63 ng / mL, and incubated at 37°C for 1 hour at 25 μl / well; the plate was washed, and anti-rabbit IgG-HRP enzyme-linked secondary antibody was added, and incubated at 37°C for 0.5 hours at 25 μl / well; the plate was washed, and 50 μl / well of TMB was added for color development. After termination, OD450-OD630 readings were taken. The test results are shown in Table 4.

[0089] Table 4 Binding activity results of purified antibodies from positive clones

[0090]

[0091] The results showed that clones 3F6, 5E11, 7B4, 7G3, 7G9, 6E6, and 7C8 had good binding activity.

[0092] Example 3: Characterization of AAV5 Antibody Neutralizing Activity

[0093] The positive cloned antibody purified after recombinant expression in Example 1 was taken, and the neutralization activity was determined after protein quantification. Cultured 293T cells (manufactured by ATCC, catalog number: CRL-3216) were seeded at 20,000 cells / well in a 96-well white plate (manufactured by Corning, catalog number: 3917), and 100 μl / well of DMEM cell culture medium was used. The plates were cultured in a 37°C 5% CO2 incubator overnight. On the second day, AAV5 [ssAAV.CMV.Fluc.WPRE.SV40pA] (customized by Guangzhou Paizhen Biotechnology Co., Ltd.) was diluted to 1.5E10 vg / ml using DMEM serum-free basal medium; the positive clone sample to be tested and the control sample ADK5a (purchased from Progen, product number: 610148) and ADK5b (purchased from Progen, product number: 610149S) were diluted using DMEM serum-free basal medium, with a starting concentration of 4000 ng / mL, 4-fold gradient dilution, a total of 6 points to 16.46 ng / mL; the AAV5 dilution solution and the serum gradient dilution solution were mixed in a 1:1 ratio and incubated at 37°C for 30 minutes; after incubation, 50 μL / well were added to a 96-well plate, gently mixed with a pipette, and placed in a 37°C 5% CO2 incubator for 24 hours. On the third day, 50 μl Bright-Lumi TM Firefly luciferase assay reagent (manufacturer: Beyotime, catalog number: RG051S) was mixed at 500 rpm for 5 minutes at room temperature and then placed in a chemiluminescence detector for detection. The test results are shown in Table 5.

[0094] Table 5 Neutralization activity results of purified antibodies from positive clones

[0095]

[0096] Using half the negative control signal as the screening threshold to calculate the sensitivity (neutralizing activity) of positive clones, the results in Tables 4 and 5 show that clone 5E11 has strong binding activity but no strong neutralizing activity. 2F11, 3B6, 3G6, and 5A8 have weaker binding activity and also possess weaker neutralizing activity. 3F6, 6E6, 7B4, 7C8, 7G3, and 7G9 have both strong binding activity and strong neutralizing activity. The neutralizing activity of these clones is far superior to that of the ADK5a clone. Furthermore, the neutralizing activities of 7B4 and 7G3 are significantly superior to those of the ADK5a and ADK5b clones.

[0097] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. An anti-AAV5 monoclonal antibody 7B4, characterized in that Includes heavy and light chains; The heavy chain includes a heavy chain variable region, and the three complementarity determining regions of the heavy chain variable region are: CDR1:GFSLNHYA; CDR2:LSWVGNT; CDR3:GRAIWAADSNAIFNL; The light chain includes a light chain variable region, and the three complementarity determining regions of the light chain variable region are: CDR1:QNIYDF; CDR2:GAS; CDR3:QCTYDGGTYVA.

2. The anti-AAV5 monoclonal antibody 7B4 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-AAV5 monoclonal antibody 7B4 is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

15.

3. The anti-AAV5 monoclonal antibody 7B4 according to claim 2, characterized in that The gene sequence of the heavy chain variable region of the anti-AAV5 monoclonal antibody 7B4 is shown in SEQ ID NO.14, and the gene sequence of the light chain variable region is shown in SEQ ID NO.

16.

4. An anti-AAV5 monoclonal antibody 7G3, characterized in that Includes heavy and light chains; The heavy chain includes a heavy chain variable region, and the three complementarity determining regions of the heavy chain variable region are: CDR1:GFSLSSYA; CDR2: IYAGSGIT; CDR3:ARAQYANGNIYYGL; The light chain includes a light chain variable region, and the three complementarity determining regions of the light chain variable region are: CDR1:ESISSG; CDR2: YAS; CDR3:QQGFTESNVDNT.

5. The anti-AAV5 monoclonal antibody 7G3 according to claim 4, characterized in that The amino acid sequence of the heavy chain variable region of the anti-AAV5 monoclonal antibody 7G3 is shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

19.

6. The anti-AAV5 monoclonal antibody 7G3 according to claim 5, characterized in that The gene sequence of the heavy chain variable region of the anti-AAV5 monoclonal antibody 7G3 is shown in SEQ ID NO.18, and the gene sequence of the light chain variable region is shown in SEQ ID NO.

20.

7. A method for preparing the anti-AAV5 monoclonal antibody 7B4 according to any one of claims 1 to 3 or the anti-AAV5 monoclonal antibody 7G3 according to any one of claims 4 to 6, characterized in that: The steps include: S1. Immunize rabbits with AAV5 virus particles and screen for specific B cells of positive clones; S2. Cultivate specific B cells in vitro and screen and confirm positive B cell clones by ELISA; S3. Amplify the DNA sequences of the heavy and light chain variable regions from specific B cells of positive clones using single-cell PCR technology and express them in mammalian cells; S4. Screen and confirm the neutralizing effect of the expressed positive cloned antibodies through the AAV5 neutralizing antibody method.

8. Use of the anti-AAV5 monoclonal antibody 7B4 according to any one of claims 1 to 3 or the anti-AAV5 monoclonal antibody 7G3 according to any one of claims 4 to 6 in preparing an AAV5 neutralizing antibody detection kit.

9. The use according to claim 8, characterized in that The anti-AAV5 monoclonal antibody 7B4 or the anti-AAV5 monoclonal antibody 7G3 is used as a positive control in the AAV5 neutralizing antibody detection kit.

10. Use of the anti-AAV5 monoclonal antibody 7B4 according to any one of claims 1 to 3 or the anti-AAV5 monoclonal antibody 7G3 according to any one of claims 4 to 6 in preparing a kit for quantitative detection of AAV5 virus.

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