Monoclonal antibody 5F11 capable of identifying a plurality of antigens of AAV in a broad spectrum and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody 5F11 capable of recognizing a plurality of antigens of AAV in a broad spectrum and application thereof. The monoclonal antibody 5F11 comprises three complementarity determining regions in the heavy chain and light chain variable regions respectively, corresponding to SEQ ID NO. 1-3 and SEQ ID NO. 4-6. The monoclonal antibody 5F11 can specifically recognize AAV2, AAV8 and AAV9 antigens, and has high sensitivity and broad spectrum. A double antibody sandwich detection method based on the antibody can simultaneously detect three kinds of AAV antigens, and significantly reduces the detection cost. The clear variable region sequence lays a foundation for humanization modification, recombinant expression and application in an in vitro detection platform such as an ELISA kit, an immunochromatography test strip and the like.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a monoclonal antibody 5F11 that can broadly recognize multiple antigens of AAV and its applications. Background Technology

[0002] Virus-based gene vectors, which can deliver target genes into cultured cells or animals, are common molecular biology tools widely used in scientific experiments and clinical research. Currently, commonly used viral vectors include lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors.

[0003] Adeno-associated virus (AAV) is the simplest, non-enveloped, single-stranded DNA-deficient virus discovered to date, and a member of the parvovirus family. AAV has an icosahedral structure, a diameter of approximately 22 nm, and a viral genome length of only about 4.7 kb. Currently, more than 200 AAV genotypes have been discovered, and they can be classified into different serotypes based on differences in the capsid protein (Cap). In humans, 12 serotypes have been identified, including AAV1 to AAV11 and DJ. Different serotypes of AAV exhibit varying infectivity in different tissues and cells. AAV is non-pathogenic in humans and can be integrated and modified at specific sites, demonstrating effective transfection in various tissues and cells. It is currently widely used in basic research and clinical trials, and AAV vectors have become one of the most commonly used gene therapy vectors in the world. For example, Zolgensma (using the AAV9 vector) for treating spinal muscular atrophy and Luxturna (using the AAV2 vector) for treating hereditary blindness, among other AAV therapeutics, have been approved for marketing, bringing hope for the treatment of intractable diseases.

[0004] As the number of trials using AAV vectors as gene therapy vectors steadily increases, the demand for AAV vector production, purification, titer, and purity identification is also steadily increasing. However, AAV has multiple serotypes, and traditionally, different antibodies are needed to identify different serotypes, which undoubtedly increases testing costs significantly. Therefore, developing broad-spectrum antibodies against different serotypes of AAV virus has significant application value. Summary of the Invention

[0005] Given that existing detection methods for AAV2, AAV8, and AAV9 largely rely on serotype-specific antibodies, requiring the use of different antibodies to detect different serotypes, resulting in complex operations, high detection costs, and difficulties in standardization, this invention provides a monoclonal antibody 5F11 that can broadly recognize multiple AAV antigens and its applications. This solves the problems of insufficient specificity, low sensitivity, and cumbersome multi-serotype detection procedures in existing detection methods, providing a rapid, accurate, and standardized detection tool for virus identification, titer determination, and clinical research of AAV2, AAV8, and AAV9.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] In a first aspect, the present invention provides a monoclonal antibody 5F11 capable of broadly recognizing multiple AAV antigens. The heavy chain variable region of the monoclonal antibody 5F11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 5F11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0008] In some embodiments, the AAV multiple antigens include AAV2 antigen, AAV8 antigen, and AAV9 antigen.

[0009] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 5F11 is shown in SEQ ID NO.7.

[0010] In some embodiments, the amino acid sequence of the variable region of the light chain of monoclonal antibody 5F11 is shown in SEQ ID NO.8.

[0011] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5F11 is shown in SEQ ID NO. 9.

[0012] In some embodiments, the nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 5F11 is shown in SEQ ID NO.10.

[0013] Secondly, the present invention provides the application of the above-mentioned monoclonal antibody 5F11 in the preparation of tools for detecting multiple AAV antigens, including AAV2 antigen, AAV8 antigen and AAV9 antigen.

[0014] In some embodiments, the tools include reagents, kits, test strips, and antibody chips.

[0015] In some embodiments, the kit includes a double-antibody sandwich ELISA kit.

[0016] In some embodiments, the kit is coated and labeled with the monoclonal antibody 5F11.

[0017] In some embodiments, the test strip uses monoclonal antibody 5F11 as the capture antibody and monoclonal antibody 5F11 as the labeling antibody.

[0018] Beneficial effects:

[0019] The monoclonal antibody 5F11 provided by this invention comprises three complementarity-determining regions (CDRs) in both its heavy and light chain variable regions. The amino acid sequences of these CDRs are shown in SEQ ID NO. 1 to SEQ ID NO. 3 (heavy chain) and SEQ ID NO. 4 to SEQ ID NO. 6 (light chain). Monoclonal antibody 5F11 can sensitively and specifically bind to AAV2, AAV8, and AAV9 serotype antigens, enabling its use in the field of AAV immunoassay. This allows for sensitive and efficient detection of the titers of the three AAV viruses, significantly reducing production costs and demonstrating significant market potential in large-scale AAV vector production and gene therapy. The clearly defined CDR sequence information provides a foundation for the humanization, affinity optimization, recombinant expression, and development of multi-platform detection reagents (such as ELISA kits and immunochromatographic test strips) of this antibody. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Image showing the results of hybridoma cell screening;

[0022] Figure 2 This is a graph showing the results of the monoclonal antibody binding affinity test.

[0023] Figure 3 The results are for the sensitivity and specificity tests of the double-antibody sandwich.

[0024] Figure 4 The graph shows the detection results of the binding activity of monoclonal antibody 5F11 to AAV2 antigen.

[0025] Figure 5 The graph shows the detection results of the binding activity of monoclonal antibody 5F11 to AAV8 antigen.

[0026] Figure 6The image shows the results of detecting the binding activity of monoclonal antibody 5F11 against the AAV9 antigen. Detailed Implementation

[0027] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0028] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated in these embodiments should be interpreted as merely exemplary and not as limiting.

[0029] The monoclonal antibody 5F11, which can broadly recognize AAV2 / 8 / 9 antigens, indicates that it can simultaneously recognize and bind to common antigenic epitopes on the protein capsids of the three serotypes of AAV2, AAV8, and AAV9, and therefore can be used to detect any one or more of these three viruses.

[0030] Unless otherwise specified, all reagents and materials used in this application are conventional commercially available products in this field. The recombinant adeno-associated viruses AAV1 / 2 / 3 / 5 / 6 / 8 / 9 were packaged by Shandong Weizhen Biotechnology Co., Ltd., and their titers were all between 1×10⁻⁶. 12 above vg / ml.

[0031] This application provides a monoclonal antibody 5F11 that can broadly recognize multiple AAV antigens. The heavy chain variable region of monoclonal antibody 5F11 includes three complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2 and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.3.

[0032] The light chain variable region of monoclonal antibody 5F11 includes three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.6.

[0033] In this application, the kit includes a double-antibody sandwich ELISA kit for in vitro detection of AAV2 / 8 / 9 antigens.

[0034] A dual-antibody sandwich assay based on the monoclonal antibody 5F11 can simultaneously detect AAV2, AAV8, and AAV9 antigens, significantly reducing testing costs and simplifying the procedure. This method is not intended for disease diagnosis.

[0035] Example 1:

[0036] 1. Mouse immunization

[0037] Freund's complete adjuvant and an equal volume of AAV9-EGFP (stock solution 5.2 × 10⁻⁶) 12 After emulsifying with a mixture of vg / ml (diluted 10-fold with PBS), 200 μl / mouse was administered subcutaneously at multiple sites to immunize female Balb / C mice aged 6-8 weeks. Two weeks after the initial immunization, Freund's incomplete adjuvant was mixed with an equal volume of AAV9-EGFP (5.2 × 10⁻⁶ vg / ml) to induce subcutaneous immunization at multiple sites. 12 Subcutaneous immunization was performed after emulsification with a concentration of vg / ml (diluted 10-fold with PBS). A booster immunization was administered two weeks later using the same dose and method. Serum titer was measured, and cell fusion was initiated after the titer reached 10,000. Three days before fusion, 200 μl of AAV9-EGFP (stock solution 5.2 × 10⁻⁶) was injected. 12 (vg / ml, diluted 10 times with PBS, without adjuvant) One intraperitoneal booster immunization.

[0038] Although only AAV9 was used as the antigen during immunization, the high amino acid sequence similarity of the capsid proteins of AAV2, AAV8, and AAV9 means that some antibodies produced after mouse immunization may recognize these common epitopes. Through subsequent parallel screening of multiple AAV serotypes, a broad-spectrum monoclonal antibody, 5F11, specifically binding to AAV2, AAV8, and AAV9 was successfully obtained, demonstrating that immunization with only a single serotype and subsequent screening for broad-spectrum antibodies is entirely feasible.

[0039] 2. Screening of hybridoma cell lines

[0040] Three days after booster immunization of mice, mouse spleen cells were fused with myeloma cells SP2 / 0 using PEG1450 to prepare hybridoma cells. Ten days later, cells were coated with AAV9 and an irrelevant antigen (adenovirus AdV5) and detected by indirect ELISA. Cell lines that reacted with AAV9 antigen but not with other irrelevant antigens (adenovirus AdV5) were selected. After limiting dilution to monoclonal state, these cells were expanded and cryopreserved. The preparation method of the adenovirus AdV5 antigen is described in our company's patents CN120271698B or CN120309720B, and will not be repeated here.

[0041] The indirect ELISA method is as follows:

[0042] First, AAV9 antigen (stock solution 5.2 × 10⁻⁶) was coated in microplates. 12 Vg / ml, 500-fold dilution, coating buffer (carbonate buffer: sodium carbonate 1.59g, sodium bicarbonate 2.93g, diluted to 1L pure water, pH 9.6) and irrelevant antigen AdV5 (500-fold dilution), 50μl per well, incubated overnight at 4℃; 3% BSA, 2% sucrose blocking, 150μl per well, incubated at 37℃ for 2 hours, washed once with washing buffer, patted dry; 50μl of cell culture supernatant from each well was added to the antigen-coated microplate and reacted at 37℃ for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μl of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS, v / v) to each well. Incubate at 37°C for 30 min, wash four more times, blot dry, and add 50 μl of TMB chromogenic buffer per well. Incubate at room temperature for 10 min. Finally, add 50 μl of 0.5 M sulfuric acid to terminate the reaction. Measure the OD using a microplate reader. 450 Values ​​and screening results can be found in [link / reference]. Figure 1 .

[0043] 3. Preparation of monoclonal antibody ascites

[0044] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were pretreated with Freund's incomplete adjuvant. After about 10 days, when the mice's abdomens were significantly swollen, ascites was collected using a sterile syringe needle.

[0045] 4. Purification of monoclonal antibodies

[0046] Centrifuge the ascites fluid at 12000 r / min for 10 min to remove the upper layer of oil and the lower layer of precipitate, and collect the supernatant. Dilute the ascites fluid supernatant with binding buffer (0.02M PB, 150mM NaCl, pH 7.4) at a volume ratio of 1:10, filter through a 0.22 μm filter membrane, and then bind it to a Protein G column (equilibrate with binding buffer first). Wash with binding buffer for approximately 5 column volumes, elute with elution buffer (0.1M glycine, pH 2.7), and collect the elution peak. Then quickly neutralize with 1M Tris-HCl solution (pH 9.0) to a neutral range. Dialyze the purified antibody to 0.01M PBS, determine the concentration, and aliquot and store.

[0047] 5. Binding affinity test of purified monoclonal antibody to AAV9 antigen

[0048] AAV9 antigen was coated onto microplates, and the purified monoclonal antibodies were serially diluted (50 μl per well) using the indirect ELISA method described above. The binding affinity of each monoclonal antibody was then assessed. Results are as follows: Figure 2 As shown, 5F11 has the best detection titer.

[0049] The binding affinity test only measures AAV9 because it is the initial screening method, and the goal is to select the candidate antibody that binds most strongly to the immunogen; while whether it can recognize AAV2 and AAV8 is systematically verified in downstream specialized specific identification experiments.

[0050] 6. Specificity identification of purified monoclonal antibodies

[0051] AAV1 / 2 / 3 / 5 / 6 / 8 / 9 and the irrelevant antigen adenovirus AdV5 were coated onto microplates, with AAV1 / 2 / 3 / 5 / 6 / 8 diluted to the same concentration as AAV9 before coating. Monoclonal antibodies diluted to 1 μg / ml were added to each plate using the indirect ELISA method described above. The results are shown in Table 1. 1G1, 1H3, 4G3, 5F11, and 5A12 reacted with AAV2 / 8 / 9 antigens simultaneously but not with other AAV antigens or the irrelevant AdV5. Five broad-spectrum AAV2 / 8 / 9 antibodies were screened for double-antibody sandwich pairing testing.

[0052] Table 1: Results of monoclonal antibody specificity identification.

[0053]

[0054] 7. Double-antibody sandwich ELISA method for identifying paired antibodies

[0055] HRP labeling of antibodies:

[0056] The purified monoclonal antibody was labeled with horseradish peroxidase (HRP), specifically as follows:

[0057] (1) Dissolve 2 mg HRP in 0.5 ml of pure water, add 0.5 ml of freshly prepared 0.06 M NaIO4 solution (10 ml of pure water + 128 mg NaIO4), mix well and let stand at 4 °C for 30 minutes.

[0058] (2) Add 0.5 ml of 0.16 M ethylene glycol aqueous solution (10 ml of pure water + 0.09 ml of ethylene glycol) to the mixture, mix well and keep at room temperature in the dark for 30 minutes.

[0059] (3) Mix 2 mg of antibody with 1 ml of the above mixture and dialyze overnight with 0.05 M pH 9.6 carbonate buffer (sodium carbonate 1.59 g, sodium bicarbonate 2.93 g to 1 L).

[0060] (4) Add 0.2 ml of NaBH4 solution (5 mg / ml), mix well, and let stand at room temperature in the dark for 2 hours. Then dialyze with 0.01 M PBS at 4 °C to change the solution.

[0061] (5) Finally, add an equal volume of glycerol and store at -20℃.

[0062] Screening for paired monoclonal antibodies:

[0063] The purified monoclonal antibody was diluted to a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) and added to the microplate at 50 μL / well. The plate was coated overnight at 4 °C. The coating buffer was discarded the next day, and the plate was washed once with washing buffer (PBST, PBS containing 0.05% Tween-20). The plate was patted dry and blocked with 3% BSA and 2% sucrose at 150 μL / well. The plate was incubated at 37 °C for 2 h, the blocking buffer was discarded, and the plate was patted dry. AAV2 / 8 / 9 and irrelevant antigen AdV5 were diluted 1000-fold and added to ELISA plates at 50 μL / well. The plates were incubated at 37°C for 35 min. After washing four times with PBST buffer, the plates were blotted dry. Then, 50 μL / well of enzyme-labeled monoclonal antibody diluted 1000-fold with PBS was added and the plates were incubated at 37°C for 35 min. After washing four more times and blotting dry, 50 μL / well of TMB chromogenic buffer was added and the plates were incubated at room temperature for 10 min. Finally, 50 μL / well of 0.5 M sulfuric acid was added to terminate the reaction. The OD values ​​were measured using an ELISA reader. 450 nm value. The condition pairing with the most significant difference between positive and negative results was selected as the optimal combination for further optimization. The results are shown in Table 2. Monoclonal antibody 5F11 as the coating antibody and monoclonal antibody 5F11 as the labeling antibody had the highest P / N value, and this combination showed the best detection effect for AAV2 / 8 / 9.

[0064] Table 2: Screening results of double antibody sandwich pairing.

[0065]

[0066] 8. Optimization of double antibody sandwich

[0067] The purified monoclonal antibody 5F11 was added to each well at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, respectively, and coated with 50 μL / well of coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6). The mixture was incubated overnight at 4°C. The coating buffer was discarded the next day, and the plates were blocked with 1% BSA (150 μL / well). The plates were incubated at 37°C for 2 hours, and the blocking buffer was discarded. The test antigen and control antigen were diluted with PBS at 100 ng / mL and added to each well (50 μL / well). The plates were incubated at 37°C for 35 minutes. The plates were washed four times with PBST wash buffer, and then coated with PBS. HRP-labeled monoclonal antibody 5F11 diluted 1000, 2000, and 3000 times, 50 μL / well, was incubated at 37°C for 35 min. After washing the plate four times and patting it dry, 50 μL / well of TMB chromogenic buffer was added, and the plate was incubated at room temperature for 10 min. Finally, 50 μL / well of 0.5 M sulfuric acid was added to terminate the reaction, and the OD was measured using a microplate reader. 450 nm value. The results are shown in Table 3. The pairing condition with the highest P / N value was selected for sensitivity and specificity testing. The results show that the best detection results were obtained by double antibody sandwich ELISA when the HRP-labeled monoclonal antibody 5F11 was diluted 2000 times with 1ug / ml as the coating antibody.

[0068] Table 3: Optimization results of double-antibody sandwich ELISA.

[0069]

[0070] The optimal detection results for double-antibody sandwich ELISA were obtained when the coating antibody concentration was 1 μg / ml and the HRP-labeled antibody was diluted 2000 times.

[0071] 9. Sensitivity and specificity test of double-antibody sandwich

[0072] Optimal coating concentration (1 μg / ml), HRP-labeled monoclonal antibody dilution (2000-fold), and other reaction conditions were determined. Following the above detection steps, AAV2 / 8 / 9 were first serially diluted with PBS buffer (the original solution had an AAV9 concentration of 5.2 × 10⁻⁶). 12 vg / ml, AAV2 was 4.8×10 12 vg / ml, AAV8 was 3.9×10 12 All vg / ml were diluted to 2×10⁻⁶. 12(The concentration was initially set at vg / ml and then serially diluted.) The resulting concentrations were 100, 1000, 10000, 100000, and 1000000 times. Simultaneously, AAV1 / 3 / 6 (all diluted to the same concentration as AAV2 / 8 / 9), AdV5, and AdV7 were used as irrelevant controls. 50 μL of each well was added at the same concentration for detection to determine the sensitivity and specificity of the assay system for the antigen. Results are shown below. Figure 3 , Figure 3 The horizontal axis represents the dilution factor. The double-antibody sandwich assay consisting of 5F11 coating and labeling did not detect adenovirus AdV5, AdV7 antigens, or adeno-associated virus AAV1 / 3 / 6, demonstrating good specificity and showing similarity to AAV2 / 8 / 9 antigens within 2×10⁻⁶. 12 The presence of a positive reaction even when diluted 100,000 times from a concentration of vg / ml indicates that the method has good sensitivity and specificity.

[0073] 10. Identification of binding activity of paired monoclonal antibodies

[0074] Referring to the aforementioned indirect ELISA method, AAV2 / 8 / 9 antigen was coated, and monoclonal antibody 5F11 was serially diluted at 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 0.1 ng / ml. AdV5 monoclonal antibody 4A6 (see our company's patents CN120309720B or CN120271698B) was used as a negative control. The binding activity of monoclonal antibody 5F11 against AAV2 / 8 / 9 antigen was measured. Results are shown below. Figure 4-6 5F11 is the monoclonal antibody 5F11, and Ctrl is the AdV5 monoclonal antibody 4A6. The monoclonal antibody 5F11 showed a positive reaction with AAV2 / 8 / 9 at a dilution of 10 ng / ml, indicating a high binding affinity.

[0075] 11. Determination of the variable region gene of monoclonal antibodies

[0076] Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0077] Monoclonal antibody 5F11 variable region gene sequence:

[0078] The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 5F11 is shown in SEQ ID NO.10:

[0079] GACATTTGTGATGTCACAGTCTCCATCCAGTCTGTTTGCATTCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTAAACTGGTACCAGCAGAAACCAGGAAATATTCCTAAAGTATTGATCTATGAGGCTTCCAACTTGCAC ACAGGGCTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACACTAATCATCAGGAACCTGCAGCCTGAAGACATTGCCACTTACTACTGTCAACACGGTCAAAGTTATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTG.

[0080] The amino acid sequence of the variable region of the light chain of monoclonal antibody 5F11 is shown in SEQ ID NO. 8:

[0081] DIVMSQSPSSLFAFLGDTITITCHASQNINVWLNWYQQKPGNIPKVLIYEASNLHTGVPSRFSGSGSGTGFTLIIRNLQPEDIATYYCQHGQSYPRTFGGGTKLEIKRTV.

[0082] Light chain CDR area annotation:

[0083] The complementarity-determining region (CDR-L1) sequence of the light chain variable region of monoclonal antibody 5F11 is shown in SEQ ID NO.4: HASQNINVWLN;

[0084] The complementarity-determining region (CDR-L2) sequence of the light chain variable region of monoclonal antibody 5F11 is shown in SEQ ID NO.5: EASNLHT;

[0085] The CDR-L3 sequence of the light chain variable region of monoclonal antibody 5F11 is shown in SEQ ID NO.6: QHGQSYPRT.

[0086] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5F11 is shown in SEQ ID NO.9:

[0087] CAGATGCAGCTTCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATTTCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACTACATGCACTGGGTGAACATAGCCATGTAAAGAGTCTTGAGTGGATTGGACGTATTAATCCGTACAGTGGTACTACTACCTACAG CCAGAGTTTCAAGGACAAGGCCAGGTTGACTGTAGATAGGTCCTCCAACACAGTCTACATGGAGCTCCACAGCCTGACATCTGAAGACTCAGCAGTCTATTACTGTGCAAGAGGGGCCTACGGTGATAACCACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACTGTCTCTGCA.

[0088] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5F11 is shown in SEQ ID NO.7:

[0089] QMQLQESGPELVKPGASVKISCKASGYSFTGYYMHWVKHSHVKSLEWIGRINPYSGTTTYSQSFKDKARLTVDRSSNTVYMELHSLTSEDSAVYYCARGAYGDNHWYFDVWGAGTTVTVSA.

[0090] Heavy chain CDR region annotation:

[0091] The complementarity-determining region (CDR-H1) sequence of the heavy chain variable region of monoclonal antibody 5F11 is shown in SEQ ID NO.1: GYYMH;

[0092] The heavy chain variable region complementarity-determining region (CDR-H2) sequence of monoclonal antibody 5F11 is shown in SEQ ID NO.2: RINPYSGTTTYSQSFKD;

[0093] The heavy chain variable region complementarity-determining region (CDR-H3) sequence of monoclonal antibody 5F11 is shown in SEQ ID NO.3: GAYGDNHWYFDV.

[0094] The embodiments of this application have now been described in detail. To avoid obscuring the concept of this application, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0095] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application.

Claims

1. A monoclonal antibody 5F11 capable of broadly recognizing multiple AAV antigens, characterized in that, The heavy chain variable region of the monoclonal antibody 5F11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 5F11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

2. The monoclonal antibody 5F11, which can broadly recognize multiple AAV antigens according to claim 1, is characterized in that, The AAV antigens include AAV2 antigen, AAV8 antigen, and AAV9 antigen.

3. The monoclonal antibody 5F11, which can broadly recognize multiple AAV antigens according to claim 1, is characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5F11 is shown in SEQ ID NO.

7.

4. The monoclonal antibody 5F11, which can broadly recognize multiple AAV antigens according to claim 3, is characterized in that, The amino acid sequence of the variable region of the light chain of the monoclonal antibody 5F11 is shown in SEQ ID NO.

8.

5. The monoclonal antibody 5F11, which can broadly recognize multiple AAV antigens according to claim 4, is characterized in that... The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5F11 is shown in SEQ ID NO.

9.

6. The monoclonal antibody 5F11, which can broadly recognize multiple AAV antigens according to claim 5, is characterized in that, The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 5F11 is shown in SEQ ID NO.

10.

7. The use of the monoclonal antibody 5F11 according to claim 1 in the preparation of a tool for detecting multiple AAV antigens.

8. The application according to claim 7, characterized in that, The tools include reagents, kits, test strips, and antibody chips.

9. The application according to claim 8, characterized in that, The kit includes a double-antibody sandwich ELISA kit.

10. The application according to claim 9, characterized in that, The kit is coated and labeled using the monoclonal antibody 5F11; The test strip uses monoclonal antibody 5F11 as the capture antibody and monoclonal antibody 5F11 as the labeling antibody.

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