Drug-resistant antibody detection kit of gene therapy drug based on AAV9 carrier and detection method and application of drug-resistant antibody detection kit

Abstract

The invention discloses an anti-drug antibody detection kit of a gene therapy drug based on an AAV9 carrier and a detection method and application of the anti-drug antibody detection kit, the kit comprises a capture plate and a second antibody solution, and the capture plate is prepared by coating an AAV9 antigen and sealing with a sealing solution; and the second antibody solution is a solution of an HRP-labeled IgG antibody for resisting the species of the sample to be detected. By determining a screening critical value, a confirmation critical value and a titer critical value, whether a to-be-detected sample contains the anti-AAV9 antibody or not and the antibody titer are determined according to the detection absorbance or inhibition ratio of the to-be-detected sample. The kit and the detection method have the characteristics of short time consumption, wide sample species applicability, high sensitivity, high result judgment accuracy and high automation level, so that the kit and the detection method are more suitable for large-scale, high-throughput and standardized serum detection, and can be widely applied to detection of anti-AAV9 antibodies in serum samples of various species.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological analysis and detection, and in particular relates to an anti-drug antibody detection kit for gene therapy drugs based on an AAV9 vector, and a detection method and application thereof. Background Art

[0002] Adeno-associated virus (AAV) is a member of the Parvoviridae family. It is a small, non-enveloped, icosahedral virus. The diameter of the virus particle is between 20 and 26 nm, and it contains a linear single-stranded DNA genome of about 4.70 kb. AAV can be used as a vector to transduce cells for long-term transgene expression. To date, about 12 natural serotypes and more than 100 AAV variants have been isolated and studied as gene delivery vectors.

[0003] Adeno-associated virus 9 (AAV9) is isolated from non-human primates and has multiple advantages, such as weak immunogenicity, high safety, stable expression, wide range of infected cell types, and no integration with the target cell genome. AAV9 can mediate long-term and effective expression of exogenous genes, and has a high transduction efficiency in tissues and organs such as myocardium, skeletal muscle, liver and pancreas. It has now become one of the main vectors in gene therapy research. Studies have shown that AAV9 also has the ability to cross the blood-brain barrier and efficiently transduce neural cells, opening up a new path for gene therapy of the nervous system.

[0004] Anti-drug antibodies (ADA) refer to the production of antibodies specific to drugs during treatment, which are caused by the body's immune response to drugs due to drug stimulation of the body's immune system. These antibodies may affect the pharmacokinetic and pharmacodynamic properties of drugs, reduce drug efficacy, and even cause adverse reactions.

[0005] Anti-drug antibody analysis is carried out throughout the non-clinical and clinical stages. In the immunogenicity evaluation test and toxicokinetic test in the non-clinical safety evaluation research test, animals must be screened for anti-drug antibodies before enrolling; the anti-drug antibody level must be tested during the test. In the pharmacokinetic test in the non-clinical stage, animals must be screened for anti-drug antibodies before enrolling. In the pharmacokinetic test in the clinical stage, subjects must be screened for anti-drug antibodies before enrolling; the anti-drug antibody level must be continuously tested during the test.

[0006] The anti-drug antibody detection method for AAV9 vector-based gene therapy drugs can be used to evaluate whether the sample contains anti-AAV9 antibodies (distinguishing between negative and positive samples) and its anti-AAV9 antibody titer level, providing a simple and rapid detection tool for the evaluation of anti-drug antibodies for AAV9 vector-based gene therapy drugs. Summary of the invention

[0007] The purpose of the present invention is to provide an anti-drug antibody detection kit for gene therapy drugs based on AAV9 vectors, and its detection method and application. The method of the present invention has the characteristics of short time consumption, wide applicability of sample species, high sensitivity, high accuracy of result judgment, and high level of automation. Therefore, it is more suitable for large-scale, high-throughput, standardized serum detection, and can be widely used in the detection of anti-AAV9 antibodies in serum samples of various species.

[0008] In order to achieve the above purpose, this application adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a kit for detecting anti-drug antibodies of gene therapy drugs based on AAV9 vectors, the kit comprising a capture plate and a secondary antibody solution,

[0010] The capture plate is prepared by coating with AAV9 antigen and blocking with blocking solution;

[0011] The secondary antibody solution is a solution of HRP-labeled anti-IgG antibody of the sample species to be tested.

[0012] In the above technical solution, the method for preparing the capture plate comprises the following steps:

[0013] S11, AAV9 was diluted to a concentration of 0.5×10 10 ~2×10 10 vg / mL solution was evenly added to the ELISA plate, sealed with sealing film and incubated at 2-8°C for 16-20 hours;

[0014] S12, discarding the coating solution in the ELISA plate and washing the plate twice;

[0015] S13, add 1# blocking solution and block at 25-37°C for 1-4 hours;

[0016] S14, discarding the 1# blocking solution in the ELISA plate, and placing the ELISA plate at 25-37° C. to dry for 1-4 hours;

[0017] S15, after drying is completed, the coated ELISA plate is vacuum packed to obtain the capture plate.

[0018] In the above technical solution, the preparation method of the secondary antibody solution is:

[0019] The corresponding HRP-labeled IgG antibody of the sample species to be tested is diluted to a working solution of 10-100 ng / mL using a sample diluent to obtain the secondary antibody solution.

[0020] In the above technical scheme, the HRP-labeled IgG antibody of the sample species to be tested is one of HRP-labeled goat anti-rat IgG antibody, HRP-labeled rabbit anti-rat IgG antibody, HRP-labeled goat anti-mouse IgG antibody, HRP-labeled rabbit anti-mouse IgG antibody, HRP-labeled goat anti-monkey IgG antibody, HRP-labeled rabbit anti-monkey IgG antibody, HRP-labeled donkey anti-monkey IgG antibody, HRP-labeled goat anti-human IgG antibody, and HRP-labeled donkey anti-human IgG antibody.

[0021] In the above technical solution, the kit also includes 1× washing solution, sample diluent, negative mixed serum solution, TMB substrate color developing solution and stop solution.

[0022] In the above technical solution, the sample diluent is prepared by accurately weighing 2.27 g Na 2 HPO 4 , 0.54gKH 2 PO 4 , 16.01g NaCl, 0.40g KCl, 40.00g sucrose, and 20.00g BSA are placed in a preparation container, and appropriate amount of ultrapure water is added, and stirred to dissolve. After dissolution, 1.00mL Tween-20 and 1.00mL ProClin 300 are added, and stirred to mix. The volume is made up to 2.00L with ultrapure water, and filtered through a filter with a membrane pore size of 0.22μm.

[0023] In the above technical solution, the preparation method of the negative mixed serum solution is: dilute the negative mixed serum 10 to 50 times with the sample diluent, and filter it with a filter with a membrane pore size of 0.22 μm;

[0024] The negative mixed serum includes but is not limited to one of the negative mixed serums of rat, mouse, rabbit, monkey, dog, human, etc.

[0025] The preparation method of the 1× washing solution is as follows: accurately weigh 5.68 g Na 2 HPO 4 , 1.36g KH 2 PO 4, 40.03g NaCl, 1.01g KCl to a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 2.5mL Tween-20, 2.5mL ProClin 300 after dissolution, stir to mix, make up to 5.00L with ultrapure water, and filter through a filter with a membrane pore size of 0.22μm;

[0026] The preparation method of the stop solution is: accurately measure 270 mL of ultrapure water into a preparation container, add 30 mL of 10 mol / L hydrochloric acid solution to prepare a working solution with a concentration of 1 mol / L, and filter it using a filter with a membrane pore size of 0.22 μm.

[0027] In the above technical solution, the coating solution is prepared by accurately weighing 1.14 g Na 2 HPO 4 , 0.27gKH 2 PO 4 , 8.01g NaCl, 0.20g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, dilute to 1.00L, and filter through a filter with a membrane pore size of 0.22μm;

[0028] The preparation method of the 1# blocking solution is as follows: accurately weigh 475 g of the coating solution into a preparation container, add 25.00 g of skimmed milk powder, and prepare a working solution with a concentration of 5%.

[0029] In a second aspect, the present invention provides a method for detecting anti-drug antibodies of gene therapy drugs based on AAV9 vectors, wherein the detection method is performed using the above-mentioned kit and comprises the following steps:

[0030] S21, restore all reagents to room temperature;

[0031] S22, returning all samples to room temperature;

[0032] S23, experimental steps:

[0033] Add the samples to be tested diluted with the negative mixed serum solution to the corresponding wells of the kit, seal the plate with a sealing film, and incubate at 37° C. for 20-90 minutes;

[0034] Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper;

[0035] Add 100 μL of secondary antibody solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes;

[0036] Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper;

[0037] Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 5-25 minutes;

[0038] Add 100 μL of stop solution to each well to terminate the reaction;

[0039] Complete OD in 15 minutes 450nm reading;

[0040] S24, absorbance calculation:

[0041] Calculate the average absorbance or inhibition rate of duplicate wells of samples;

[0042] S25, sample test result determination:

[0043] Screening test result determination: Samples with test results greater than or equal to the screening critical value are considered positive samples, and samples with test results less than the screening critical value are considered negative samples;

[0044] Determination of confirmation test results: Sample test results greater than or equal to the confirmation critical value are considered true positive samples, and sample test results less than the confirmation critical value are considered false positive samples;

[0045] S26, sample anti-drug antibody titer detection:

[0046] The negative mixed serum solution was used to perform a 2-fold gradient dilution of the true positive samples determined in the confirmatory test, and the anti-drug antibody titer of the sample to be tested was calculated based on the test results of the diluted samples.

[0047] In a third aspect, the present invention provides the use of the above-mentioned kit in the detection of anti-AAV9 antibodies in serum samples of rats, mice, rabbits, monkeys, dogs and humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The working principle diagram of the kit and the detection method of the present invention;

[0049] Figure 2 The results of the coating concentration and blocking solution optimization experiment in Example 1;

[0050] Figure 3 The results of the secondary antibody concentration optimization experiment in Example 1 are shown. DETAILED DESCRIPTION

[0051] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0052] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0053] Unless otherwise specified, the reagents used in the following examples can be obtained from commercial sources.

[0054] The solutions in the following examples were prepared as follows:

[0055] Coating solution preparation: Accurately weigh 1.14g Na 2 HPO 4 , 0.27g KH 2 PO 4 , 8.01g NaCl, 0.20g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, dilute to 1.00L, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stored stably at 2-8℃ for 1 year.

[0056] Preparation of 1# blocking solution: Accurately weigh 475g of coating solution into the preparation container, add 25.00g skim milk powder (Sangon Biotechnology (Shanghai) Co., Ltd., A600669) to prepare a working solution with a concentration of 5%. The solution can be stored stably at 2-8°C for 2 months.

[0057] Preparation of 2# blocking solution: weigh 1.14g Na 2 HPO 4 , 0.27g KH 2 PO 4 , 8.01g NaCl, 0.20g KCl, 10.00g BSA into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20, 0.50mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stably stored at 2-8℃ for 1 year.

[0058] Sample diluent preparation: weigh 1.14g Na 2 HPO 4 , 0.27g KH 2 PO 4, 8.01g NaCl, 0.20g KCl, 20.00g sucrose, 10.00g BSA into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20, 0.50mL ProClin 300 after dissolution, stir to mix, dilute to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stably stored at 2-8℃ for 2 years.

[0059] Preparation of washing solution: Accurately weigh 5.68g Na 2 HPO 4 , 1.36g KH 2 PO 4 , 40.03g NaCl, 1.01g KCl to the preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 2.50mL Tween-20, 2.50mL ProClin 300 after dissolution, stir to mix, make up to 5.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stored stably at room temperature for 2 months.

[0060] Preparation of secondary antibody solution: Accurately measure 300mL of sample diluent into the preparation container, add a certain volume of HRP-labeled anti-test sample species IgG antibody, and prepare a working solution with a concentration of 100ng / mL. The solution can be stored stably at 2-8℃ for 1 year.

[0061] Preparation of stop solution: Accurately measure 270 mL of ultrapure water into the preparation container, add 30 mL of 10 mol / L hydrochloric acid solution (Sinopharm Chemical Reagent Co., Ltd., 53100258), prepare a working solution with a concentration of 1 mol / L, and filter it with a filter with a membrane pore size of 0.22 μm. The solution can be stored stably at 2-8°C for 3 years.

[0062] AAV9: AAV2 / 9-pAAV-CMV-EGFP-P2A-3xFLAG-WPRE, Heyuan Biotechnology (Shanghai) Co., Ltd., catalog number AOV024.

[0063] Goat anti-rabbit IgG antibody: Peroxidase AffiniPure GoatAnti-Rat IgG(H+L)(min X Hu,Bov,Hrs SrProt), Jackson ImmunoResearch Inc., catalog number 112-035-062.

[0064] TMB substrate colorimetric solution: TMB Super Sensitive One Component HRP MicrowellSubstrate, Surmodics, Inc., Cat. No. TMBS-1000-01.

[0065] The preparation method of the capture plate in the following examples is:

[0066] S1, coating

[0067] AAV9 was diluted to a concentration of 2×10 10 vg / mL solution was evenly added to the ELISA plate at 100 μL / well, sealed with sealing film and incubated at 8°C for 20 hours;

[0068] S2, Washing plate

[0069] Discard the coating solution in the ELISA plate and wash the plate twice (260 μL / well of washing solution, 60 seconds for each wash);

[0070] S3, closed

[0071] Add blocking solution evenly at 200 μL / well and block at 37°C for 4 hours;

[0072] S4. Drying

[0073] Discard the blocking solution in the ELISA plate, pat dry on absorbent paper, and place the ELISA plate at 37°C to dry for 8 hours;

[0074] S5. Packaging

[0075] After drying, the coated ELISA plate is vacuum packed in an aluminum foil bag (a desiccant needs to be placed in the aluminum foil bag) to form a capture plate.

[0076] Example 1 Indirect ELISA detection method establishment

[0077] 1. Operation steps of indirect ELISA detection method

[0078] 1) Sample addition: Add 100 μL of the sample to be tested to the corresponding wells (the minimum dilution factor is not less than 10 times), seal the plate with a sealing film, and incubate at 37°C for 60 minutes. Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate, and wash 4 times (pat dry on absorbent paper each time).

[0079] 3) Add secondary antibody: Add 100 μL of secondary antibody of appropriate concentration to each well (the secondary antibody should be HRP-labeled antibody against IgG of the corresponding species according to the species of the sample to be tested), seal the plate with a new sealing film, and incubate at 37°C for 30 minutes. Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate, and wash 4 times (pat dry on absorbent paper each time).

[0080] 4) Color development: Add 100 μL TMB substrate color development solution to each well, protect from light, and incubate at room temperature for 15 minutes.

[0081] 5) Termination of reaction: Add 100 μL of stop solution to each well to terminate the reaction.

[0082] 6) Reading: Complete OD within 15 minutes 450nm reading.

[0083] 2. Optimization of various conditions during the establishment of the indirect ELISA detection method

[0084] 1) Optimization of coating concentration and blocking solution

[0085] AAV9 (AAV2 / 9-pAAV-CMV-EGFP-P2A-3xFLAG-WPRE, Heyuan Biotechnology (Shanghai) Co., Ltd., Catalog No. AOV024) was prepared with coating solution to a concentration of 6.64×10 10 vg / mL and 1.66×10 10 vg / mL solution, and blocked with 1# blocking solution and 2# blocking solution respectively to prepare capture plates. During the experiment, the concentration of secondary antibody was 50ng / mL.

[0086] The results are as follows Figure 2 As shown in the figure, the combination of low coating concentration and 1# blocking solution is beneficial to reduce the background of the detection method. 10 vg / mL coating concentration and 1# blocking solution were used for subsequent optimization experiments.

[0087] 2) Optimization of secondary antibody concentration

[0088] AAV9 (AAV2 / 9-pAAV-CMV-EGFP-P2A-3xFLAG-WPRE, Heyuan Biotechnology (Shanghai) Co., Ltd., Catalog No. AOV024) was prepared with coating solution to a concentration of 1.66×10 10 vg / mL solution was blocked with 1# blocking solution to prepare a capture plate. During the experiment, the secondary antibody concentration was set to 50ng / mL, 25ng / mL and 10ng / mL to study the effect of different secondary antibody concentrations on the reaction results.

[0089] The results are as follows Figure 3 As shown in the figure, as the concentration of the secondary antibody decreases, the reaction background gradually decreases. Considering the sensitivity of the detection method and the detection rate of positive samples, it was determined that a 25 ng / mL secondary antibody solution was used for the development of the detection method.

[0090] Example 2 Screening critical value & titer critical value determination

[0091] AAV9 (AAV2 / 9-pAAV-CMV-EGFP-P2A-3xFLAG-WPRE, Heyuan Biotechnology (Shanghai) Co., Ltd., Catalog No. AOV024) was prepared with coating solution to a concentration of 1.66×10 10 vg / mL solution was blocked with 1# blocking solution to prepare a capture plate. 16 negative serum samples were tested at different times, the sample dilution multiple was 25 times, and the secondary antibody concentration was 25ng / mL. A total of 4 analysis batches were tested, and the screening critical value and titer critical value of the detection method were determined through 64 sets of valid data.

[0092] The test results are shown in Table 1. The screening critical value is 0.1317 (OD 450nm ), the critical titer value was 0.1566 (OD 450nm ).

[0093] Table 1 Screening critical value & titer critical value determination experiment

[0094]

[0095]

[0096] Remark:

[0097] Ratio = absorbance of a single sample / mean absorbance of negative serum samples;

[0098] Screening critical value factor (SCPF) = Mean Ratio + 1.645 × SD;

[0099] Titer critical value factor (TCPF) = Mean Ratio + 3.09 × SD;

[0100] Screening critical value (SCP) = Mean NC × SCPF;

[0101] Titer critical value (TCP) = Mean NC × TCPF.

[0102] Example 3 Confirmation of critical value determination

[0103] AAV9 (AAV2 / 9-pAAV-CMV-EGFP-P2A-3xFLAG-WPRE, Heyuan Biotechnology (Shanghai) Co., Ltd., Catalog No. AOV024) was prepared with coating solution to a concentration of 1.66×10 10 vg / mL solution, blocked with 1# blocking solution, and prepared into capture plates. The 16 negative serum samples were divided into two groups, one without drug addition (no AAV9), and one with drug addition (AAV9 was added so that the final concentration of AAV9 in the sample was 1.66×1010 vg / mL), both groups of samples were diluted 25 times, and the secondary antibody concentration was 25ng / mL. One analytical batch was tested, and the confirmation critical value was determined by the inhibition rate of the drug-added group to the non-drug-added group.

[0104] The test results are shown in Table 2, and the critical value for confirmation is 6.768%.

[0105] Table 2 Confirmation critical value determination experiment

[0106]

[0107] Remark:

[0108] Inhibition = (test results of drug-added samples - test results of non-drug-added samples) / test results of non-drug-added samples × 100%;

[0109] Confirmation critical value = Mean Inhibition + 2.335 × SD.

[0110] Example 4 Application of the Anti-drug Antibody Detection Method for Gene Therapy Drugs Based on AAV9 Vectors

[0111] In order to clearly illustrate the specific application of the detection method of the present invention, Example 4 takes the detection method for anti-drug antibodies in SD rat serum samples as an example.

[0112] 1. Animal Experimental Protocol

[0113] AAV2 / 9-pAAV-CMV-EGFP-P2A-3xFLAG-WPRE (Heyuan Biotechnology (Shanghai) Co., Ltd., Catalog No. AOV024) was used as the vector of gene therapy drugs, and SPF-grade SD rats were selected as experimental animals. The animal experiment scheme is detailed in Table 3.

[0114] Table 3 Animal experiment plan

[0115]

[0116]

[0117] 2. Screening test results

[0118] The detection method described in Example 2 was used to detect serum samples of each animal at each time point in the low-dose group (Rat 1, Rat 2, Rat 3, Rat 4) and the high-dose group (Rat 5, Rat 6, Rat 7, Rat 8).

[0119] The test results are shown in Table 4. On the 4th day after administration, anti-AAV9 antibodies were detected in both the low-dose group and the high-dose group through the screening test (the test results were greater than the screening critical value).

[0120] Table 4 Screening test results

[0121]

[0122] Note: The screening threshold is 0.1317 (OD 450nm ).

[0123] 3. Confirmation of test results

[0124] The detection method described in Example 3 was used to detect the samples with positive screening test results in Example 4.

[0125] The test results are shown in Table 5. Through the confirmation test, it is possible to further confirm which positive samples obtained from the screening test are true positive samples (the test results are greater than or equal to the confirmation critical value) and which are false positive samples (the test results are less than the confirmation critical value).

[0126] Table 5 Confirmation test results

[0127]

[0128]

[0129] Note: The confirmation critical value is 6.768%.

[0130] 4. Titer test results

[0131] The samples with positive test results in Example 4 were tested respectively using the detection method described in Example 2.

[0132] The test results are shown in Table 6. Through the titer test, the titer value of the true positive sample of the confirmation test can be further obtained. No anti-drug antibodies were detected in the low-dose group 14 days after administration, while anti-drug antibodies were still detected in some animals in the high-dose group on the 14th day after administration with a high antibody titer.

[0133] Table 6 Titer test results

[0134]

[0135] Note: The critical titer value is 0.1566 (OD 450nm).

[0136] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A kit for detecting anti-drug antibodies of gene therapy drugs based on AAV9 vectors, the kit comprising a capture plate and a secondary antibody solution, characterized in that: The capture plate is prepared by coating with AAV9 antigen and blocking with blocking solution; The secondary antibody solution is a solution of HRP-labeled anti-IgG antibody of the sample species to be tested.

2. The kit according to claim 1, characterized in that: The method for preparing the capture plate comprises the following steps: S11, AAV9 was diluted to a concentration of 0.5×10 10 ~2×10 10 vg / mL solution was evenly added to the ELISA plate, sealed with sealing film and incubated at 2-8°C for 16-20 hours; S12, discarding the coating solution in the ELISA plate and washing the plate twice; S13, add 1# blocking solution and block at 25-37°C for 1-4 hours; S14, discarding the 1# blocking solution in the ELISA plate, and placing the ELISA plate at 25-37° C. to dry for 1-4 hours; S15, after drying is completed, the coated ELISA plate is vacuum packed to obtain the capture plate.

3. The kit according to claim 1, characterized in that: The preparation method of the secondary antibody solution is: The corresponding HRP-labeled IgG antibody of the sample species to be tested is diluted to a working solution of 10-100 ng / mL using a sample diluent to obtain the secondary antibody solution.

4. The kit according to claim 1 or 3, characterized in that: The HRP-labeled IgG antibody of the sample species to be tested is one of HRP-labeled goat anti-rat IgG antibody, HRP-labeled rabbit anti-rat IgG antibody, HRP-labeled goat anti-mouse IgG antibody, HRP-labeled rabbit anti-mouse IgG antibody, HRP-labeled goat anti-monkey IgG antibody, HRP-labeled rabbit anti-monkey IgG antibody, HRP-labeled donkey anti-monkey IgG antibody, HRP-labeled goat anti-human IgG antibody, and HRP-labeled donkey anti-human IgG antibody.

5. The kit according to claim 1, characterized in that: The kit also includes 1× washing solution, sample diluent, negative mixed serum solution, TMB substrate color developing solution and stop solution.

6. The kit according to claim 3 or 5, characterized in that: The preparation method of the sample diluent is as follows: accurately weigh 2.27g Na2HPO4, 0.54g KH2PO4, 16.01g NaCl, 0.40g KCl, 40.00g sucrose, and 20.00g BSA into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, add 1.00mL Tween-20 and 1.00mL ProClin 300 after dissolution, stir to mix, make up to 2.00L with ultrapure water, and filter using a filter with a membrane pore size of 0.22μm.

7. The kit according to claim 5, characterized in that: The negative mixed serum solution is prepared by diluting the negative mixed serum by 10 to 50 times using the sample diluent, and filtering the solution using a filter with a membrane pore size of 0.22 μm. The preparation method of the 1× washing solution is as follows: accurately weigh 5.68g Na2HPO4, 1.36g KH2PO4, 40.03g NaCl, and 1.01g KCl into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, add 2.5mL Tween-20 and 2.5mL ProClin300 after dissolution, stir to mix, make up to 5.00L with ultrapure water, and filter through a filter with a membrane pore size of 0.22μm to obtain the solution; The preparation method of the stop solution is: accurately measure 270 mL of ultrapure water into a preparation container, add 30 mL of 10 mol / L hydrochloric acid solution to prepare a working solution with a concentration of 1 mol / L, and filter it using a filter with a membrane pore size of 0.22 μm.

8. The kit according to claim 2, characterized in that: The coating solution is prepared by accurately weighing 1.14 g Na2HPO4, 0.27 g KH2PO4, 8.01 g NaCl, and 0.20 g KCl into a preparation container, adding an appropriate amount of ultrapure water, stirring to dissolve, and adjusting the volume to 1.00 L. The solution is filtered through a filter with a membrane pore size of 0.22 μm. The preparation method of the 1# blocking solution is as follows: accurately weigh 475 g of the coating solution into a preparation container, add 25.00 g of skimmed milk powder, and prepare a working solution with a concentration of 5%.

9. A method for detecting anti-drug antibodies of gene therapy drugs based on AAV9 vectors, characterized in that: The detection method is carried out using the kit according to any one of claims 1 to 8, comprising the following steps: S21, restore all reagents to room temperature; S22, returning all samples to room temperature; S23, experimental steps: Add the samples to be tested diluted with the negative mixed serum solution to the corresponding wells of the kit, seal the plate with a sealing film, and incubate at 37° C. for 20-90 minutes; Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper; Add 100 μL of secondary antibody solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes; Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper; Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 5-25 minutes; Add 100 μL of stop solution to each well to terminate the reaction; Complete OD in 15 minutes 450nm reading; S24, absorbance calculation: Calculate the average absorbance or inhibition rate of duplicate wells of samples; S25, sample test result determination: Screening test result determination: Samples with test results greater than or equal to the screening critical value are considered positive samples, and samples with test results less than the screening critical value are considered negative samples; Determination of confirmation test results: Sample test results greater than or equal to the confirmation critical value are considered true positive samples, and sample test results less than the confirmation critical value are considered false positive samples; S26, sample anti-drug antibody titer detection: The negative mixed serum solution was used to perform a 2-fold gradient dilution of the true positive samples determined in the confirmatory test, and the anti-drug antibody titer of the sample to be tested was calculated based on the test results of the diluted samples.

10. Use of the kit according to any one of claims 1 to 8 in detecting anti-AAV9 antibodies in serum samples of rats, mice, rabbits, monkeys, dogs and humans.

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