A monoclonal antibody 1E12 for specific detection of AAV6 and its application

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to a monoclonal antibody 1E12 for specific detection of AAV6 and its application. The monoclonal antibody 1E12 provided by the present invention comprises three complementarity determining regions (CDRs) in its heavy and light chain variable regions, corresponding to SEQ ID NOs. 1-3 and 4-6, respectively. These CDRs confer high specificity and affinity to the antibody for AAV6, enabling accurate identification of the AAV6 virus without cross-reaction with other serotypes, with a detection sensitivity of 4×10 6 vg / ml level. This antibody is suitable for the development of various detection tools, including double-antibody sandwich ELISA kits, test strips, and antibody chips. It performs particularly well in the biotin-avidin amplification system and can be used for AAV6 virus identification, titer determination, and clinical dose assessment, providing an effective means for standardized, high-throughput detection.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a monoclonal antibody 1E12 for specific detection of AAV6 and its application. Background Art

[0002] Adeno-associated virus (AAV) is a member of the Parvoviridae family. Currently, over 200 AAV genotypes have been identified, classified into different serotypes based on differences in their capsid protein, Cap. Thirteen AAV serotypes have been isolated from human and non-human primate tissues, and different AAV serotypes have varying infection efficiencies in different tissues and cells.

[0003] Adeno-associated virus (AAV) is a viral vector widely used in gene therapy and vaccine development. Its low immunogenicity, long-term expression, high infection capacity, and tissue specificity make it a crucial tool in gene therapy. AAV6, one of its serotypes, exhibits high transduction efficiency in skeletal muscle, lung, and heart tissues and is commonly used in gene therapy for conditions such as Duchenne muscular dystrophy (targeting muscle), cystic fibrosis (targeting lung), and other ophthalmic diseases. As an important gene therapy vector, quantitative detection and quality control of AAVs, including AAV6, are critical steps in gene therapy product development. Identification of capsid protein serotypes is also crucial for improving the specificity and efficiency of gene therapy. Therefore, the National Medical Products Administration recommends AAV serotype identification and requires thorough evaluation of all human gene therapy products. Currently, commonly used AAV6 detection methods primarily include nucleic acid and protein assays, such as qPCR, Western blot, and mass spectrometry. However, these methods suffer from complex procedures, insufficient sensitivity, and low specificity. Therefore, the development of a highly sensitive, specific, and user-friendly AAV6 detection method is of great importance. Summary of the Invention

[0004] In view of the problems of complex operation, low sensitivity and poor specificity in existing AAV6 detection methods, the present invention provides a highly specific monoclonal antibody 1E12 for AAV6 and its application, which solves the problems of poor specificity, low sensitivity and cumbersome operation of existing detection methods, and provides a fast, accurate and standardized detection tool for AAV6 virus identification, titer determination and clinical research.

[0005] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0006] In a first aspect, the present invention provides a monoclonal antibody 1E12 for specific detection of AAV6, wherein the heavy chain variable region of the monoclonal antibody 1E12 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively;

[0007] The light chain variable region of monoclonal antibody 1E12 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 4 to SEQ ID NO. 6, respectively.

[0008] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1E12 is shown in SEQ ID NO.7.

[0009] In some embodiments, the amino acid sequence of the light chain variable region of monoclonal antibody 1E12 is shown in SEQ ID NO.8.

[0010] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1E12 is shown in SEQ ID NO.9.

[0011] In some embodiments, the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1E12 is shown in SEQ ID NO.10.

[0012] The second aspect is the use of the above-mentioned monoclonal antibody 1E12 in the preparation of a tool for specific detection of AAV6.

[0013] In some embodiments, the tools include reagents, kits, test strips, and antibody chips.

[0014] In some embodiments, the kit comprises a double antibody sandwich ELISA kit.

[0015] In some embodiments, the kit includes a double antibody sandwich ELISA kit with a biotin-avidin amplification system.

[0016] In some embodiments, the kit is coated and labeled with monoclonal antibody 1E12.

[0017] Beneficial effects:

[0018] The monoclonal antibody 1E12 of the present invention has three complementarity-determining regions (CDRs) in its heavy and light chain variable regions, respectively. The amino acid sequences of these CDRs are shown in SEQ ID NOs. 1 to 3 (heavy chain) and 4 to 6 (light chain). These CDR regions determine the antibody's high specificity and affinity for AAV6. Through this structural design, the monoclonal antibody 1E12 provided by the present invention can accurately recognize the AAV6 virus without cross-reacting with other AAV serotypes, thereby achieving highly specific detection of AAV6. At the same time, the antibody also has high sensitivity.

[0019] The monoclonal antibody 1E12 of the present invention can be used in the development of a variety of detection tools, including but not limited to double-antibody sandwich ELISA kits, rapid test strips, and antibody chips, greatly expanding its application range. In particular, in double-antibody sandwich ELISA kits constructed with a biotin-avidin amplification system, this antibody is used as both a coating and a labeling antibody, fully leveraging its high specificity and sensitivity. It can be applied to AAV6 virus identification, titer determination, and dosage determination in clinical research, providing strong support for the establishment of a standardized, high-throughput AAV6 detection platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0021] Figure 1 Figure 1 is the identification result of the purified monoclonal antibody;

[0022] Figure 2 Initially establish the result graph for the biotin-avidin amplification system;

[0023] Figure 3 This is a screening graph for coating monoclonal antibody concentration;

[0024] Figure 4 This is a dilution screening chart for biotinylated monoclonal antibodies;

[0025] Figure 5 is the screening graph of HRP-streptavidin dilution;

[0026] Figure 6 This is the sensitivity detection diagram of biotin-avidin amplified ELISA. DETAILED DESCRIPTION

[0027] The following detailed description of the embodiments of the present application is provided in conjunction with the accompanying drawings and examples. The detailed description of the following examples and the accompanying drawings are intended to illustrate the principles of the present application, but are not intended to limit the scope of the present application. The present application may be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but rather includes all technical solutions within the scope of the claims.

[0028] The present application provides these embodiments to make this application thorough and complete, and to fully express the scope of this application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangement of parts and steps, the composition of materials, numerical expressions and numerical values ​​set forth in these embodiments should be interpreted as merely exemplary, and not as limiting.

[0029] Unless otherwise specified, the reagents and materials used in this application are conventional commercial products in the field. Among them, the recombinant adeno-associated viruses AAV1 / 2 / 3 / 5 / 6 / 8 / 9 were packaged by Shandong Weizhen Biotechnology Co., Ltd., and the titers were all 1×10 12 vg / ml or above.

[0030] Example 1

[0031] 1. Mouse immunization

[0032] Freund's complete adjuvant and an equal volume of AAV6-CAG-GFP (stock solution 4 × 10 12 vg / ml, diluted 10-fold with PBS) and then emulsified, 200 μl / mouse was used for subcutaneous multi-site immunization of 6-8 week old female Balb / C mice. Two weeks after the first immunization, an equal volume of AAV6-CAG-GFP (stock solution 4×10 12 vg / ml, diluted 10-fold with PBS) and emulsified for subcutaneous immunization. Two weeks later, the same dose and method were used for booster immunization. Serum titer was tested. Cell fusion was performed after the titer reached 10,000. Three days before fusion, 200 μl AAV6-CAG-GFP (stock solution 4×10 12 vg / ml, diluted 10-fold with PBS, without adjuvant) and intraperitoneal booster immunization once.

[0033] 2. Screening of hybridoma cell lines

[0034] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and cultured in HAT medium for selection. When the fused cells reached half the bottom of the well, they were tested using indirect ELISA using AAV6-CAG-GFP and AAV1 / 2 / 3 / 5 / 8 / 9-coated plates. Cell lines that reacted exclusively with AAV6-CAG-GFP and not with other serotypes were identified (Table 1). After limiting dilution to monoclonal status, they were expanded and cryopreserved.

[0035] Among them, the indirect ELISA method is specifically as follows:

[0036] First, AAV6-CAG-GFP (stock solution 4×10 12 vg / ml, 500-fold dilution, coating buffer (phosphate buffer: 8.5g sodium chloride, 0.26g sodium dihydrogen phosphate, 2.88g disodium hydrogen phosphate, fixed to 1L pure water) and AAV1 / 2 / 3 / 5 / 8 / 9 (all converted to the same concentration as AAV6), 50μl per well, 4℃ overnight; 3% BSA, 2% sucrose blocking, 150μl per well, 37℃ 2 hours, wash the plate once with washing solution, pat dry; take 50μl of the supernatant from the cell culture wells and add it to the antigen-coated microplate, react at 37℃ for 30min. The liquid in the wells was shaken off, and the plate was washed 4 times with PBST solution. After patting dry, HRP-labeled goat anti-mouse secondary antibody (diluted with PBS at a volume ratio of 1:5000) was added to each well, 50 μl, and the reaction was carried out at 37°C for 30 min. The plate was washed again 4 times, and after patting dry, 50 μl / well of TMB color development solution was added. The color was developed at room temperature for 10 min. Finally, 50 μl of 2M sulfuric acid was added to stop the reaction, and the OD was measured with a microplate reader. 450 value.

[0037] Table 1: Clonal reaction intensity (OD) corresponding to different AAV serotypes 450 value).

[0038]

[0039] 3. Preparation of Monoclonal Antibody Ascites

[0040] After the screened hybridoma cell lines were expanded and cultured, 0.2 ml (containing 2.5×10 6 Female BALB / c mice (100 cells) were pretreated with incomplete Freund's adjuvant. Approximately 10 days later, when the abdomen became noticeably swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000 rpm for 10 minutes, and the mid-layer was collected.

[0041] 4. Purification of Monoclonal Antibodies

[0042] Centrifuge the ascites at 12000r / min for 5 minutes, take 5ml of the supernatant, add 20ml of acetate buffer (68mmol / L, pH 4.5), mix well, slowly add 50μl of octanoic acid, stirring while adding, continue stirring for 30 minutes after adding, centrifuge at 12000r / min for 30 minutes at 2~8℃, and take the supernatant. Filter the supernatant with absorbent cotton, add saturated ammonium sulfate at a ratio of 50% (V / V) of the final volume, stirring while adding, continue stirring for 30 minutes after adding, let it precipitate at 2~8℃ for 4~5 hours, centrifuge at 12000r / min for 30 minutes at 2~8℃, and take the precipitate. Use 3ml Tris-HCl

[0043] (10mmol / L, pH 9.0), resuspend, put into dialysis bag (MW: 8000~14000), and place in 2L Tris-HCl at 2~8℃.

[0044] (10mmol / L, pH 9.0) solution and dialyzed for 14 hours. The liquid in the dialysis bag was transferred to a centrifuge tube.

[0045] Centrifuge at 12,000 rpm for 5 minutes. The supernatant is the purified monoclonal antibody. Store at -20°C to avoid repeated freezing and thawing.

[0046] 5. Specificity identification of purified monoclonal antibodies

[0047] AAV1 / 2 / 3 / 5 / 6 / 8 / 9 were coated on microplates and reacted with monoclonal antibodies diluted to 1 μg / ml using the indirect ELISA method described above. The results are shown in Table 2. 1B4, 1H4, 1E12, 1B11, 1E2, 1H11, and 1H6 reacted only with AAV6 and not with other serotypes, demonstrating the good specificity of the monoclonal antibodies. The identification results are shown in Table 2.

[0048] Table 2: Specificity identification results of monoclonal antibodies.

[0049]

[0050] 6. Binding analysis of purified monoclonal antibodies

[0051] AAV6-CAG-GFP was coated on a microplate. According to the above indirect ELISA method, the purified monoclonal antibody was diluted to a concentration gradient of 1000ng / ml, 100ng / ml, 10ng / ml, and 1ng / ml, with 50μl per well, and the binding activity of each monoclonal antibody was determined. The results are shown in Figure 2. Figure 1 As shown, 1H6 and 1E12 showed higher affinity.

[0052] 7. Double antibody sandwich ELISA method to determine paired antibodies

[0053] HRP labeling of antibodies:

[0054] The purified monoclonal antibody was labeled with horseradish peroxidase (HRP) as follows:

[0055] (1) Dissolve 2 mg of 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 of NaIO4), mix well and let stand at 4°C for 30 minutes.

[0056] (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 incubate at room temperature in the dark for 30 minutes.

[0057] (3) 2 mg of antibody (1 ml) was mixed with the above mixture and dialyzed overnight against 0.05 M pH 9.6 carbonate buffer (1.59 g sodium carbonate and 2.93 g sodium bicarbonate to 1 L).

[0058] (4) Add 0.2 ml of NaBH4 solution (5 mg / ml) and mix well. Place the mixture in the dark at room temperature for 2 hours, and dialyze with 0.01 M PBS at 4°C to replace the solution.

[0059] (5) Finally, add an equal volume of glycerol and store at -20°C.

[0060] Screening of paired monoclonal antibodies:

[0061] The purified monoclonal antibodies were 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 pure water, pH 9.6) and added to the ELISA plate at 50 μL / well for overnight coating at 4°C. The coating solution was discarded the next day, and the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), 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 solution was discarded, and the plate was patted dry. AAV6 antigen and irrelevant antigen AAV2 were diluted to 1000 times and added to the enzyme-labeled plate, 50 μL / well, incubated at 37°C for 35 minutes, washed 4 times with PBST, patted dry, and enzyme-labeled monoclonal antibody diluted to 1000 times with PBS was added, 50 μL / well, incubated at 37°C for 35 minutes, washed 4 times, patted dry, and TMB color development solution was added 50 μL / well, developed at room temperature for 10 minutes, and finally 0.5 M sulfuric acid was added, 50 μL / well, to terminate the reaction, and the OD was measured with a microplate reader. 450 nm value. The combination with the largest P / N value was selected for subsequent optimization. The results are shown in Table 3.

[0062] Table 3: Screening results of double antibody sandwich pairings.

[0063]

[0064] The results showed that the P / N value was the largest when monoclonal antibody 1E12 was used as the coating antibody and monoclonal antibody 1E12 was used as the labeling antibody. This combination was selected for biotin system optimization.

[0065] 8. Biotin-antibody coupling

[0066] The biotin (Thermo, EZ-Link NHS Biotin, 20217) was conjugated to the antibody at a molar ratio of 20:1. First, 2.0 mg of biotin was dissolved in 360 μL of ultrapure water to prepare a 10 mM biotin solution. Then, 2 mg of monoclonal antibody 1E12 was reacted with 26.6 μL of 10 mM biotin under shaking at room temperature for 3 hours (the reaction volume was kept around 2 mL). The biotin-antibody mixture was then dialyzed against 0.01 M PBS to remove excess biotin. After dialysis, the antibody concentration was determined, and an equal volume of glycerol was added and stored at -20°C. The final labeled antibody concentration was approximately 0.5 mg / mL.

[0067] 9. Establishment of biotin-streptavidin amplified ELISA system

[0068] The monoclonal antibody 1E12 screened above was used for coating, and the reaction with AAV6 was identified by ELISA. First, the microplate was coated with monoclonal antibody 1E12 (coating buffer: carbonate buffer: 1.59g sodium carbonate, 2.93g sodium bicarbonate, diluted to 1L of pure water) at a coating concentration of 1μg / mL, 50μl / well, and incubated at 4°C overnight. The plate was washed once with washing buffer (PBST, PBS containing 0.05% Tween-20, pH 7.4), patted dry, and blocked with 3% sucrose and 2% BSA, 150μl per well, incubated at 37°C for 2 hours, patted dry, and stored dry. AAV6 (stock solution 4×10 12vg / ml) was diluted with PBS to 1000-fold and 10,000-fold concentrations, and 50 μL was added to the microplate coated with the monoclonal antibody. AAV2 / 8 was also diluted to 1000-fold and 10,000-fold as a negative control. The plates were incubated at 37°C for 30 min. The liquid in the wells was discarded, the plates were washed four times with PBST, and patted dry. Biotin-conjugated monoclonal antibody 1E12 (0.5 mg / ml) was diluted 2000-fold with PBS, and 50 μL / well was added to the microplate. The plates were incubated at 37°C for 30 min. The liquid in the wells was shaken out, and the plate was washed 4 times with PBST solution. After patting dry, 50 μL / well of HRP-labeled streptavidin (BIOSYNTH, 65R-S105PHRP, diluted 1:10000 with PBS) was added. The plate was reacted at 37°C for 30 min. The plate was washed 4 times again. After patting dry, 50 μL / well of TMB color development solution was added and color was developed at room temperature for 10 min. Finally, the reaction was terminated by adding stop solution and the OD was measured with a microplate reader. 450 nm value, see the results Figure 2 , Figure 2 The numbers 1000 and 10000 indicate the dilution factor. The biotin-avidin amplified ELISA system can effectively detect AAV6 adeno-associated virus.

[0069] 10. Optimization of biotin-streptavidin amplification system

[0070] Optimization of monoclonal antibody coating concentration: 0.25ug / ml, 0.5ug / ml, and 1ug / ml coating concentrations were selected, and 100μl / well was used for coating. Biotinylated monoclonal antibody 1E12 was diluted 2000 times, HRP-streptavidin was diluted 10000 times, and AAV2 / 6 / 8 (stock solution 4×10 12 vg / ml) antigen was diluted 100, 1000, 10000, 100000, and 1000000 times. Based on the test results, the AAV6 monoclonal antibody was selected with no non-specific binding to other serotypes of AAV and a higher coating volume with a positive reaction value. Figure 3 As shown, AAV2 / 6 / 8 was used as the test sample, the horizontal axis is the dilution multiple, and the vertical axis represents OD 450 The nm value reflects the intensity of the positive reaction. Under the premise of ensuring the detection specificity and sensitivity, a smaller amount of antibody was selected based on comprehensive considerations, and the coating concentration of monoclonal antibody 1E12 was determined to be 0.5ug / ml.

[0071] Optimization of biotinylated monoclonal antibody dilution: After determining the optimal monoclonal antibody coating concentration, biotinylated monoclonal antibody 1E12 was diluted 2000, 4000, and 6000 times with PBS, HRP-streptavidin was diluted 10,000 times, and AAV2 / 6 / 8 antigens were diluted 100, 1000, 100,000, 100000, and 1000000 times. Based on the test results, the dilution with no nonspecific binding to other serotypes of AAV and a higher positive reaction value was selected. Figure 4 As shown, AAV2 / 6 / 8 was used as the test sample with a coating concentration of 0.5ug / ml. The horizontal axis is the dilution multiple and the vertical axis represents the OD 450 The nm value reflects the intensity of the positive reaction. The biotin-labeled monoclonal antibody 1E12 was diluted. Considering the specificity and sensitivity, the optimal dilution of the biotin-labeled monoclonal antibody 1E12 was determined to be 1:4000.

[0072] Optimization of HRP-streptavidin dilution: The optimal monoclonal antibody coating concentration was determined to be 0.5ug / ml, the biotin-labeled monoclonal antibody 1E12 dilution was 4000 times, HRP-streptavidin was diluted with PBS at 10,000, 20,000, and 40,000 times, and AAV2 / 6 / 8 antigens were diluted at 100, 1,000, 10,000, 100,000, and 1,000,000 times. Based on the test results, the dilution with no nonspecific binding to AAV6 monoclonal antibody and a higher positive reaction value was selected. The results are as follows Figure 5 As shown, a coating concentration of 0.5ug / ml, a biotinylated monoclonal antibody diluted 4000 times, and AAV2 / 6 / 8 were used as test samples. The horizontal axis is the dilution multiple and the vertical axis represents the OD 450 The nm value reflects the intensity of the positive reaction. Taking into account the specificity and sensitivity, the optimal dilution of HRP-streptavidin was determined to be 1:20000.

[0073] 11. Sensitivity test of biotin-avidin amplified ELISA

[0074] Determine the optimal coating concentration, biotinylated monoclonal antibody dilution, HRP-streptavidin dilution and other reaction conditions, refer to the above detection steps, and perform the assay on AAV1 / 2 / 3 / 5 / 6 / 8 / 9 (stock solution 4×10 12 vg / ml) for gradient dilution, the dilution multiples were 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7, 100 μL was added to each well at the same concentration for detection to determine the detection sensitivity of the detection system for AAV6.

[0075] The results are as follows Figure 6 , Figure 6 The middle horizontal axis represents the dilution factor, and the AAV6 antigen is diluted to 10 6 The detection limit of AAV6 is 4×10 6 vg / ml or so.

[0076] 12. Antibody variable region gene cloning and sequencing

[0077] Total RNA from hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Universal primers for the mouse antibody variable regions were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the third-round PCR primers. The PCR products were gel-cleaved and purified, then ligated into the pUC19 vector and transformed into the TOP10 strain. After incubation at 37°C for 14 hours, single colonies were picked and sequenced to obtain the gene sequences of the monoclonal antibody light and heavy chains.

[0078] Monoclonal antibody variable region gene sequence:

[0079] Monoclonal antibody 1E12 heavy chain:

[0080] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1E12 is shown in SEQ ID NO. 9: GAGGTGCAGCTGCAGCAGAGCGTGGCCGAGCTGGTGAGGCCCGGCACCAGCGTGAAGCTGAGCTGCGTGGAGAGCGGCTACACCTTCACCGGCTACTGGATCAACTGGGTGAAGCAGAGGAGGGGCCAGGGCCTGGAGTGGATCGGCAACATCTACCCCGCCGACAGCGACAGCTTCGGCGACTGCCTGTTCAAGGACAGGGCCACCCTGACCGTGGACAAGAGCGCCAGCACCGCCTACATGCAGCTGAGCAGCCCCACCAGCGAGGACAGCGCCGTGTACTACTGCACCAGGAGCAGGAGGGAGCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCGCC.

[0081] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1E12 is shown in SEQ ID NO. 7: EVQLQQSVAELVRPGTSVKLSCVESGYTFTGYWINWVKQRRGQGLEWIGNIYPADSDSFGDCLFKDRATLTVDKSASTAYMQLSSPTSEDSAVYYCTRSRRELDYWGQGTTLTVSA.

[0082] CDR annotation:

[0083] The amino acid sequences of the complementarity determining region CDR-H1 of the heavy chain variable region of the monoclonal antibody 1E12 are shown in SEQ ID NO. 1: GYWIN;

[0084] The amino acid sequences of the complementarity determining region CDR-H2 of the heavy chain variable region of the monoclonal antibody 1E12 are shown in SEQ ID NO. 2: NIYPADSDSFGDCLFKD;

[0085] The amino acid sequence of the complementarity determining region CDR-H3 of the heavy chain variable region of the monoclonal antibody 1E12 is shown in SEQ ID NO. 3: SRRELDY.

[0086] Monoclonal antibody 1E12 monoclonal antibody light chain:

[0087] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 1E12 is as SEQ ID NO.10 shows: AACATCGTGATGACCCAGAGCCCCGCCCTGATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGCGCCCAGCAGCTGAGCGTGTACATGTACTGGTACCAGCAGAAGCCCAGGAGCAGCCCCAAGCCCTGGATCTTCAGGAGCACCAACCAGAAGGA GGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCTGGTACCCCCGCTTCAGGAGCCCCATGCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0088] The amino acid sequence of the light chain variable region of monoclonal antibody 1E12 is shown in SEQ ID NO.8:

[0089] NIVMTQSPALMSASPGEKVTMTCSAQQLSVYMYWYQQKPRSSPKPWIFRSTNQKEGVPARFSGSGSGTSYSLTISSMEAEDAATYYCWYPPAFRSPMLTFGAGTKLEIKRTV.

[0090] CDR annotation:

[0091] The amino acid sequences of the complementarity determining region CDR-L1 of the light chain variable region of monoclonal antibody 1E12 are shown in SEQ ID NO. 4: SAQQLSVYMY;

[0092] The amino acid sequences of the complementarity determining region CDR-L2 of the light chain variable region of monoclonal antibody 1E12 are shown in SEQ ID NO. 5: RSTNQKE;

[0093] The amino acid sequence of the complementarity determining region CDR-L3 of the light chain variable region of monoclonal antibody 1E12 is shown in SEQ ID NO. 6: WYPPAFRSPMLT.

[0094] So far, the various embodiments of the present application have been described in detail. To avoid obscuring the concept of the present application, some details well known in the art have not been described. Based on the above description, those skilled in the art can fully understand how to implement the technical solutions disclosed herein.

[0095] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art will understand that the above examples are for illustration only and are not intended to limit the scope of the present application. Those skilled in the art will understand that the above embodiments may be modified or some technical features may be replaced with equivalents without departing from the scope and spirit of the present application.

Claims

1. A monoclonal antibody 1E12 for specific detection of AAV6, characterized in that: The heavy chain variable region of the monoclonal antibody 1E12 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively; The light chain variable region of the monoclonal antibody 1E12 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 4 to SEQ ID NO. 6, respectively.

2. The monoclonal antibody 1E12 for specific detection of AAV6 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E12 is shown in SEQ ID NO.

7.

3. The monoclonal antibody 1E12 for specific detection of AAV6 according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the monoclonal antibody 1E12 is shown in SEQ ID NO.

8.

4. The monoclonal antibody 1E12 for specific detection of AAV6 according to claim 3, characterized in that The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1E12 is shown in SEQ ID NO.

9.

5. The monoclonal antibody 1E12 for specific detection of AAV6 according to claim 4, characterized in that The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1E12 is shown in SEQ ID NO.

10.

6. Use of the monoclonal antibody 1E12 according to claim 1 in preparing a tool for specifically detecting AAV6.

7. The use according to claim 6, characterized in that The tools include reagents, test kits, test strips and antibody chips.

8. The use according to claim 7, characterized in that The kit includes a double antibody sandwich ELISA kit.

9. The use according to claim 8, characterized in that The kit comprises a double antibody sandwich ELISA kit of a biotin-avidin amplification system.

10. The use according to claim 9, characterized in that The kit is coated and labeled using the monoclonal antibody 1E12.

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