AAV neutralizing antibody detection method

Abstract

The invention discloses a method for detecting an AAV neutralizing antibody. The method comprises the following steps: preparing a recombinant AAV vector AAV-Gluc carrying a Gaussian luciferase reporter gene; the method comprises the following steps: diluting AAV-Gluc according to a certain dilution step to prepare an AAV-Gluc working solution; diluting the sample to be detected; carrying out cell planking, culturing the target cells to form a uniform cell suspension, and adding the cell suspension into a cell culture plate; mixing the sample diluent with the AAV-Gluc diluent, and carrying out incubation; adding the incubated sample mixed solution into the cell culture plate, and carrying out virus infection and culture; obtaining a cultured cell supernatant, adding a detection solution, and detecting signal intensity; and analyzing and sorting the data to obtain the neutralization titer. The secreting type Gaussian luciferase reporter vector is used, target protein is secreted outside cells after being expressed, and detection is facilitated; the infected supernatant is directly sucked for detection, and the operation is convenient and fast; cell lysis and extraction are not needed, protein loss is reduced, and detection accuracy and precision are improved.

Description

Technical Field

[0001] The present invention relates to the field of clinical testing technology, and in particular to a method for detecting AAV neutralizing antibodies, which is applied to the determination of AAV neutralizing antibody levels. Background Art

[0002] Recombinant adeno-associated virus (rAAV) has been developed as a successful vector for both basic research and human gene therapy. However, neutralizing antibodies (NAbs) against the AAV capsid can eliminate AAV infectivity in target cells, thereby reducing transduction efficacy. The absence of AAV NAbs has become a prerequisite for patient enrollment in gene therapy trials. Despite this, accurately assessing AAV NAbs remains a challenging task. Existing methods for detecting AAV neutralizing antibodies include enzyme-linked immunosorbent assays (ELISAs) using binding antibodies and assays based on cell-infected reporter vectors.

[0003] ELISA-based capture assays are designed to measure the amount of immunoglobulin G (IgG) bound to AAV capsids. They detect total antibodies, meaning all antibodies capable of binding to AAV. Total antibodies are divided into neutralizing antibodies (those with the ability to neutralize AAV) and other antibodies that do not. Therefore, antibody-based assays have limitations and cannot truly distinguish between target neutralizing antibodies.

[0004] Existing AAV neutralizing antibody detection methods based on cell infection, or using green fluorescent protein GFP reporter gene, using fluorescence detection equipment such as fluorescence microscopes, cannot accurately quantify. Or based on the firefly luciferase FireflyLuciferase reporter gene. Because firefly luciferase is expressed intracellularly, cells need to be lysed and luciferase extracted during detection. The detection steps are cumbersome, the lysis and extraction efficiency is uncontrollable, and the lysis solution is inconvenient to use. After the extraction step, the accuracy and precision of protein detection are greatly affected.

[0005] U.S. Patent No. US20180356394A1 discloses a method for detecting AAV neutralizing antibodies, comprising: (a) incubating an AAV vector with a dilution of a serum sample to form a mixture; (b) exposing AAV-permissive cells to the mixture of step (a); (c) measuring the number or quantity of transduced AAV-permissive cells; and (d) determining the AAV neutralizing antibody titer in the serum sample based on the determination in (c). However, the AAV vector in this patent is tagged with a GFP signal, and the method is based on a GFP reporter gene, with GFP expression signals detected by flow cytometry. The use of GFP as a reporter requires expensive flow cytometers, making the method less versatile. Furthermore, using flow cytometry to detect GFP signals as a quantitative method has several drawbacks: the detection process is cumbersome, the assay targets the cells themselves, and the sample cannot be frozen to temporarily pause the assay. It requires prompt operation and high technical proficiency. Furthermore, the method suffers from poor reproducibility and precision, and limited sensitivity. Summary of the Invention

[0006] In view of the above-mentioned defects in the prior art, the purpose of the present invention is to provide a method for detecting AAV neutralizing antibodies, which uses a cell infection-based method to detect AAV neutralizing antibodies, specifically targeting a target antibody group with neutralizing effect, and can avoid the limitation of the ELISA-based method for total antibodies.

[0007] The present invention provides a method for detecting AAV neutralizing antibodies, comprising the following steps: Preparation of recombinant AAV vector AAV-Gluc carrying Gaussia luciferase reporter gene; AAV-Gluc is diluted according to a certain dilution step to prepare an AAV-Gluc working solution, wherein the titer of the AAV-Gluc working solution meets the requirements of an appropriate multiplicity of infection (MOI) during infection; diluting the sample to be tested to obtain a sample dilution solution; Cell plating: culturing target cells to form a uniform cell suspension, and adding the cell suspension to the cell culture plate; The sample dilution was mixed with the AAV-Gluc dilution and incubated; adding the incubated sample mixture to the cell culture plate for virus infection and culturing; Obtain the cultured cell supernatant, add the detection solution, and detect the signal intensity; Analyze and organize the data to obtain the neutralization titer.

[0008] Furthermore, the cell plating method of the present invention comprises the following steps: Cultivate host cells to a cell density of 90% to 100%; Discard the original culture medium, add 37℃ preheated PBS to wash once, and discard the PBS; Add 0.25% Trypsin-EDTA preheated at 37°C, wait until 90% of the cells have detached, and then add fresh complete medium preheated at 37°C to terminate the digestion; Transfer to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in fresh complete medium preheated at 37°C. Take the cell suspension to count the cells and adjust the cell density to a uniform standard; The cell suspension was added to the cell culture plate for plating, and the plated cells were placed in a 37°C, 8% CO2 incubator for 24 h.

[0009] Furthermore, the steps of the present invention dilute AAV-Gluc according to a certain dilution step to prepare AAV-Gluc working solution, including: mixing AAV-Gluc and wtAdV dilution solution in proportion, and diluting them multiple times according to the same gradient.

[0010] Furthermore, the dilution ratio of the present invention is 1:9; multiple dilutions are performed according to the same gradient, as follows: For the first dilution, 1 part AAV-Gluc was mixed with 9 parts wtAdV dilution to obtain AAV-Gluc dilution No. 1; For the second dilution, one part of the AAV-Gluc dilution No. 1 was mixed with nine parts of the wtAdV dilution to obtain the AAV-Gluc dilution No. 2. For the third dilution, one part of the second AAV-Gluc dilution was mixed with nine parts of the wtAdV dilution to obtain the third AAV-Gluc dilution. And so on.

[0011] Furthermore, the present invention also provides a positive antibody control group, comprising the steps of: The positive antibody is diluted according to multiple identical gradients to obtain multiple groups of positive antibody dilution solutions with different concentrations; The positive antibody dilution was mixed with the AAV-Gluc dilution and incubated; adding the incubated positive antibody mixture to the cell culture plate for virus infection and culture; Obtain the cell supernatant after culture, add the detection solution, and detect the signal intensity.

[0012] Furthermore, the present invention also provides a 100% AAV-Gluc diluent control group, in which equal amounts of FBS are mixed with the AAV-Gluc diluent.

[0013] Furthermore, the present invention also provides a 0% AAV-Gluc dilution control group, in which equal amounts of FBS and DMEM are mixed.

[0014] Furthermore, the preparation method of the detection solution of the present invention is as follows: Remove the coelenterazine solution from the -20°C freezer and place it on ice to preheat and dissolve. Once dissolved, transfer it all to the assay buffer. Perform the above steps on ice. Aliquot the amount used for each test and freeze it at -80°C. Thaw it on ice before each use.

[0015] Furthermore, the steps of the present invention for analyzing and collating data to obtain neutralization titers include: , Luciferase inhibition rate = 100% - luciferase expression rate Result judgment standard: The neutralization titer of the standard assay sample is the first dilution that reaches 50% luciferase inhibition.

[0016] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: 1. Use a secretory Gaussia luciferase reporter vector. Gaussia luciferase is a secreted protein. After the target protein is expressed, it is secreted outside the cell, which is conducive to detection. Only the cell supernatant needs to be directly aspirated for detection, which is convenient and fast. Using Gaussia luciferase as a reporter gene makes the detection operation convenient, simple, and fast, greatly improving the detection stability and precision. At the same time, when used with a luciferase detection kit, only a microplate reader is needed to detect the luminescence signal, eliminating the need for a flow cytometer with high preparation costs and cumbersome operation, greatly reducing the detection cost. 2. The chemiluminescence signal has high sensitivity, which can reach multiple orders of magnitude and has a high dynamic range of detection; 3. No need for cell lysis and extraction, reducing protein loss and improving detection accuracy and precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the positive control standard curve of the present invention. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below with reference to the examples. The following examples are intended to explain the present invention but the present invention is not limited to the following examples.

[0019] This embodiment discloses a method for detecting AAV neutralizing antibodies, comprising the following steps: S1. Preparation of recombinant AAV vector AAV-Gluc carrying Gaussia luciferase reporter gene; S2, cell plating, culturing the target cells to form a uniform cell suspension, and adding the cell suspension to the cell culture plate; S3. Diluting AAV-Gluc according to a certain dilution step to prepare an AAV-Gluc working solution, wherein the titer of the AAV-Gluc working solution meets the requirements of an appropriate multiplicity of infection (MOI) during infection; S4, diluting the positive antibody according to multiple identical gradients to obtain multiple groups of positive antibody dilutions with different concentrations; S5, diluting the sample to be tested to obtain a sample dilution solution; S6. Mix the sample diluent and the positive antibody diluent with the AAV-Gluc diluent, respectively, and set up a 100% AAV-Gluc diluent control group and a 0% AAV-Gluc diluent control group for incubation; S7, adding all the diluted solutions after incubation to the cell culture plate for virus infection and culture; S8. Obtain the cultured cell supernatant, add the detection solution, and detect the signal intensity; S9. Analyze and organize the data to obtain the neutralization titer.

[0020] In step S1, the traditional three-plasmid transfection method can be used to package and prepare an AAV vector (AAV-Gluc) carrying the Gaussia luciferase reporter gene.

[0021] Before the test, the solution is prepared. The solution in this example includes complete culture medium and assay solution.

[0022] Preparation of complete culture medium: Take 50 ml of FBS (fetal bovine serum), add 500 ml of DMEM (basal culture medium), add 5.5 ml of 100× Pen-strep (penicillin-streptomycin solution), and mix well.

[0023] FBS can provide key nutrients such as proteins, hormones, lipids, etc. required for cell growth; DMEM contains basic metabolic substances such as glucose, amino acids, and vitamins; Pen-strep inhibits bacterial contamination and ensures a sterile environment for cell culture.

[0024] Complete culture medium provides optimal growth conditions for target cells, ensuring normal cell viability and function during infection experiments. The synergistic effect of FBS and antibiotics provides nutrients to cells and prevents contamination, which is essential for successful experiments.

[0025] To prepare the assay solution, remove the coelenterazine solution from a -20°C freezer and preheat on ice to dissolve. Once dissolved, transfer the solution to the assay buffer. Perform the entire procedure on ice. Aliquot the amount needed for each assay and freeze at -80°C. Thaw on ice before each use.

[0026] Coelenterazine is a luminescent substrate extracted from marine organisms. Its oxidation reaction, catalyzed by Gaussia luciferase (Glu), produces blue light. Coelenterazine exhibits strong photostability and low background, making it a popular choice for in vitro chemiluminescence assays.

[0027] Coelenterazine is a substrate for Gaussia luciferase (Gluc) and must be dissolved before participating in the chemiluminescent reaction. Operating on ice prevents its degradation or premature reaction.

[0028] Assay buffer can provide a stable pH and ionic environment to ensure the uniformity of the Gluc catalytic reaction.

[0029] Aliquoting and freezing can avoid substrate inactivation caused by repeated freezing and thawing, ensuring the sensitivity and repeatability of each test.

[0030] The Assay Solution ensures high sensitivity and reproducibility of chemiluminescent signals through precise substrate dissolution and buffer system design.

[0031] The preparation of complete culture medium and Assay Solution both revolve around "control variables". The former optimizes the cell environment, and the latter optimizes the detection environment, jointly improving the accuracy of neutralizing antibody detection.

[0032] Step S2: Cell plating: culturing the target cells to form a uniform cell suspension, and adding the cell suspension to a cell culture plate.

[0033] Specifically, the target cells in this embodiment are HeLaRC32 cells. Cell plating includes the following steps: S21. Observe the HeLaRC32 cells cultured in T75 flasks. The cell density is between 90% and 100%.

[0034] It can ensure that the cells are in the late logarithmic growth phase and in good condition, avoiding the impact of overcrowding or sparseness on subsequent experiments. At the same time, choosing the appropriate cell state can improve cell survival rate and uniformity after plating.

[0035] S22. Discard the original culture medium, add 5 ml of PBS preheated at 37°C to wash once, and discard the PBS.

[0036] PBS (Phosphate-Buffered Saline): Phosphate buffered saline, used to maintain cell osmotic pressure and remove residual culture medium.

[0037] This method removes metabolic waste and components that may inhibit digestion (such as serum) from old culture medium, preventing serum from interfering with subsequent trypsin digestion while keeping cells moist and preventing desiccation damage.

[0038] S23. Add 3 ml of 0.25% Trypsin-EDTA preheated at 37°C. After 90% of the cells have detached, add 7 ml of fresh complete medium preheated at 37°C to terminate the digestion.

[0039] Trypsin-EDTA: A mixture of trypsin and EDTA (ethylenediaminetetraacetic acid) used to break down intercellular adhesion proteins. EDTA: A chelating agent that enhances trypsin activity and prevents cell aggregation.

[0040] Enzymatically dissociate cells from the culture flask surface to form a single-cell suspension. Gentle digestion avoids mechanical damage and ensures cell viability; EDTA can help improve digestion efficiency.

[0041] S24. Transfer the cells to a 50 ml centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant and add 5.5 ml of fresh complete medium preheated at 37°C to resuspend the cells.

[0042] Removes trypsin and residual culture medium, preventing them from interfering with subsequent experiments. Concentrates cells and removes harmful components in preparation for cell counting and plating.

[0043] S25. Take 500 μl of cell suspension for cell counting and adjust the cell density to a uniform standard of 4E+5 cells / ml.

[0044] Standardize the cell seeding volume to ensure the same number of cells per well and comparable experimental results. Avoid experimental errors caused by uneven cell density, such as excessive cell death in some wells or insufficient cell population in others to form a monolayer.

[0045] S26. Add the cell suspension to the cell culture plate and spread it in the wells three times using a dispenser at a rate of 100 μl / well. Place the plated cells in a 37°C, 8% CO2 incubator for 24 hours, and record the placement time and cell generation number on the plate.

[0046] Allow cells to attach and resume growth in preparation for infection experiments. A uniformly distributed cell monolayer is crucial for viral infection experiments, directly impacting infection efficiency and data consistency.

[0047] Cell plating is the cornerstone of experimental standardization and result reliability. By precisely controlling cell status, digestion conditions, and culture environment, highly consistent starting conditions are provided for subsequent AAV infection experiments, ensuring accurate and reproducible neutralizing antibody detection.

[0048] Step S3: dilute AAV-Gluc according to a certain dilution step to prepare an AAV-Gluc working solution, wherein the titer of the AAV-Gluc working solution meets the requirements of an appropriate multiplicity of infection (MOI) during infection.

[0049] Specifically, according to the titer of the AAV-Gluc stock solution, the AAV-Gluc stock solution is diluted with a wtAdV diluent of appropriate concentration to obtain an AAV-Gluc working solution of appropriate titer.

[0050] First, prepare wtAdV as a diluent: dilute the wAdV stock solution with DMEM medium to an appropriate titer range, such as 1-5E+9 vp / mL, to obtain a diluent carrying wtAdV (Component B), and use Component B to dilute the AAV vector.

[0051] The multiplicity of infection (MOI) of wtAdV can be controlled within an appropriate range, such as MOI = 2-10, to ensure that most cells are infected with the helper virus without excessively interfering with the experimental results.

[0052] wtAdV, a wild-type adenovirus, acts as a "helper virus," providing the coreceptors necessary for viral entry and enhancing AAV infection efficiency. Adding a helper adenovirus poses no additional operational risks in a biosafety laboratory setting, yet significantly improves AAV transduction efficiency, enabling AAV reporter vectors with low infection efficiency to successfully transduce, thus preventing the appearance of falsely elevated neutralizing antibody titers.

[0053] The preparation of AAV-Gluc dilution is shown in Table 1 below.

[0054] Table 1 AAV-Gluc dilution series AAV-Gluc dilution series Component A Component B AAV-Gluc - dilution 1 10 μl AAV-Gluc stock solution (e.g. titer 3.88E+12 vg / ml) 90 μl wtAdV dilution AAV-Gluc - dilution 2 50 μl AAV-Glu-dilution 1 450 μl wtAdV dilution AAV-Gluc-working solution 200 μl AAV-Gluc-dilution 2 1800 μl wtAdV dilution Step S4: diluting the positive antibody according to multiple identical gradients to obtain multiple groups of positive antibody dilutions with different concentrations.

[0055] Specifically, the present invention also provides a positive antibody control group, in which a positive antibody with known neutralizing activity (such as an anti-AAV monoclonal antibody) is continuously diluted from 1:1 to 1:5 in 8 gradients (1:1→1:78125), and the dilution solution is PBS (phosphate buffered saline).

[0056] See also Figure 1, generating a positive control standard curve to verify experimental accuracy. The positive sample is used to mix with the reporter vector (AAV-Gluc) working solution. The antibodies in the positive sample neutralize the reporter vector. The neutralized vector will be unable to express the reporter signal during subsequent infection and culture, thus becoming "inhibited." The degree of reporter vector inhibition is expressed as a percentage and depends on the antibody concentration in the positive sample. When the antibody concentration in the positive sample is high, the inhibition rate will reach 100%. At very low concentrations, the inhibition rate will be very low, even close to 0%.

[0057] The assay system was calibrated with a known titer of positive antibody to ensure comparable results. A standard curve was established by serial dilution of the positive antibody to ensure the linear response of the assay system.

[0058] If the sample to be tested contains antibodies, a similar sample curve will be generated, which can be compared with Figure 1 If the sample does not contain antibodies, no similar curve will be formed. For samples without antibodies, the inhibition rate will be a horizontal line that is always 0%.

[0059] The preparation of positive antibody dilution is shown in Table 2 below.

[0060] Table 2 Serial dilution of positive antibody

[0061] Step S5: dilute the sample to be tested to obtain a sample dilution solution.

[0062] Specifically, the sample to be tested was diluted in multiple identical gradients to obtain multiple sets of sample dilutions of different concentrations. The serum sample to be tested was diluted in 8 gradients starting from 1:1 and continuously diluted in 1:5 ratio (1:1→1:78125) using PBS as the dilution solution.

[0063] The core purpose is to screen samples for the presence of neutralizing antibodies and quantify their neutralizing potency. Neutralizing antibodies block viral binding to cell receptors, reducing the efficiency of subsequent infection. Using multiple gradients of dilution can accommodate differences in neutralizing capacity among different samples, expanding the scope of testing.

[0064] The preparation of sample dilutions is shown in Table 3 below.

[0065] Table 3 Sample dilution gradient

[0066] Step S6: The sample diluent and the positive antibody diluent are mixed with the AAV-Gluc diluent, respectively, and a 100% AAV-Gluc diluent control group and a 0% AAV-Gluc diluent control group are set up for incubation.

[0067] Specifically, the test sample, positive antibody, and AAV-Gluc diluent were mixed in a 1:1 ratio, as shown in Table 4. After mixing, the mixture was placed in a 37°C, 8% CO2 incubator and incubated for 1 hour. This simulates the in vivo environment and allows the neutralizing antibodies to fully bind to the AAV capsid, forming an antibody-virus complex.

[0068] Table 4 Mixing methods of samples of different categories category Preparation method Samples to be tested 100 μl sample + 100 μl AAV-Gluc-2000 Positive antibodies 100 μl positive control + 100 μl AAV-Gluc-2000 100% control 100μl FBS+100μl AAV-Gluc-2000 0% control 100 μl FBS + 100 μl DMEM Step S7: adding all the diluted solutions after incubation to the cell culture plate for virus infection and culture.

[0069] Specifically, the incubated mixture (50 μl / well) was added to a 96-well plate seeded with HeLaRC32 cells and cultured in a 37°C, 8% CO2 incubator for 24 hours.

[0070] Used to observe the inhibitory effect of neutralizing antibodies on AAV-infected cells, and indirectly reflect the activity of neutralizing antibodies through the degree of cell infection.

[0071] The addition positions of each dilution are shown in Table 5 below.

[0072] Table 5 Layout of each dilution in the well plate

[0073] Due to the limited number of wells, the dilution gradient of the positive control depends on the original concentration. The first dilution point only needs to meet the inhibition rate of 100%. Generally, it is not necessary to start from 1:1. This example starts directly from a dilution of 1:25.

[0074] Cell culture was repeated in multiple groups to make the data more accurate and reduce systematic errors.

[0075] Step S8: Obtain the cultured cell supernatant, add the detection solution, and detect the signal intensity.

[0076] Specifically, remove the Assay Solution from -80°C and thaw in an ice bath. Use a pure white ELISA plate, add 50 μl of the assay solution and 25 μl of the cell supernatant to each well, and use the same spotting layout as in Table 5.

[0077] The bottom of the white ELISA plate is white, which can enhance the sensitivity of chemiluminescent signal detection.

[0078] Repeated wells can reduce experimental errors and ensure data reliability.

[0079] The effectiveness of the reagent can be verified through the positive control; the negative control (0% control) is a control without AAV-Gluc working solution, which is used to eliminate background interference; the 100% control is a control without positive antibody, which is equivalent to 100% AAV expression, and is later used to obtain 100% signal to calculate the inhibition rate.

[0080] Then, the ELISA plate was immediately placed into a multifunctional ELISA reader, the chemiluminescence detection mode was set, and the luminescence intensity of each well was recorded.

[0081] Chemiluminescence: The light signal produced by the Gluc-catalyzed oxidation reaction of Coelenterazine, the intensity of which is positively correlated with the Gluc expression level.

[0082] Gluc secretion can indirectly reflect AAV infection efficiency. The data can be quantified, and the luminescence signal is negatively correlated with the neutralizing antibody titer (the more neutralizing antibodies, the weaker the signal).

[0083] The present invention detects secreted proteins in the supernatant directly without lysing the cells, thus avoiding protein loss caused by the lysis step in traditional methods.

[0084] In addition, chemiluminescence detection has high sensitivity and is suitable for low-concentration sample analysis.

[0085] Step S9: Analyze and organize the data to obtain the neutralization titer.

[0086] Specifically, when the positive antibody titer meets expectations during the experiment, and the 0% control fluorescence intensity is close to the background reading, the experimental data is accurate and the results can be calculated. The luminescent signal is converted into the neutralizing antibody titer to facilitate the interpretation of the results.

[0087] , Wherein, Sample Reading: the signal of the sample to be tested; 100% Control Reading: the positive control without added neutralizing antibody (maximum signal); Blank Reading: the background signal containing only culture medium, i.e., the negative control (0% control).

[0088] Luciferase inhibition rate = 100% - luciferase expression rate Luciferase inhibition rate is an indicator of the ability of neutralizing antibodies to block AAV infection.

[0089] Result determination criteria: The neutralization titer of the standard assay sample is the first dilution at which luciferase inhibition reaches 50%. For example, if 50% luciferase inhibition is observed at a 1:25 dilution, the neutralization titer of the sample is reported as 1:25, meaning that after the sample is diluted 25 times, the neutralizing antibodies in the sample can inhibit 50% of the virus in the assay system.

[0090] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for detecting AAV neutralizing antibodies, characterized in that: The following steps are involved: Preparation of recombinant AAV vector AAV-Gluc carrying Gaussia luciferase reporter gene; AAV-Gluc is diluted according to a certain dilution step to prepare an AAV-Gluc working solution, wherein the titer of the AAV-Gluc working solution meets the requirements of an appropriate multiplicity of infection (MOI) during infection; diluting the sample to be tested to obtain a sample dilution solution; Cell plating: culturing target cells to form a uniform cell suspension, and adding the cell suspension to the cell culture plate; The sample dilution was mixed with the AAV-Gluc dilution and incubated; adding the incubated sample mixture to the cell culture plate for virus infection and culturing; Obtain the cultured cell supernatant, add the detection solution, and detect the signal intensity; Analyze and organize the data to obtain the neutralization titer.

2. The AAV neutralizing antibody detection method according to claim 1, characterized in that: The cell plating comprises the following steps: Cultivate host cells to a cell density of 90% to 100%; Discard the original culture medium, add 37℃ preheated PBS to wash once, and discard the PBS; Add 0.25% Trypsin-EDTA preheated at 37°C, wait until 90% of the cells have detached, and then add fresh complete medium preheated at 37°C to terminate the digestion; Transfer to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in fresh complete medium preheated at 37°C. Take the cell suspension to count the cells and adjust the cell density to a uniform standard; The cell suspension was added to the cell culture plate for plating, and the plated cells were placed in a 37°C, 8% CO2 incubator for 24 h.

3. The AAV neutralizing antibody detection method according to claim 1 or 2, characterized in that: The step dilutes AAV-Gluc according to a certain dilution step to prepare an AAV-Gluc working solution, including: mixing AAV-Gluc and wtAdV diluent in proportion, and diluting them multiple times according to the same gradient.

4. The AAV neutralizing antibody detection method according to claim 3, characterized in that: The dilution ratio is 1:9; dilute multiple times in the same gradient as follows: For the first dilution, 1 part AAV-Gluc was mixed with 9 parts wtAdV dilution to obtain AAV-Gluc dilution No. 1; For the second dilution, one part of the AAV-Gluc dilution No. 1 was mixed with nine parts of the wtAdV dilution to obtain the AAV-Gluc dilution No.

2. For the third dilution, one part of the second AAV-Gluc dilution was mixed with nine parts of the wtAdV dilution to obtain the third AAV-Gluc dilution. And so on.

5. The AAV neutralizing antibody detection method according to claim 1, characterized in that: A positive antibody control group is also provided, comprising the steps of: The positive antibody is diluted according to multiple identical gradients to obtain multiple groups of positive antibody dilution solutions with different concentrations; The positive antibody dilution was mixed with the AAV-Gluc dilution and incubated; adding the incubated positive antibody mixture to the cell culture plate for virus infection and culture; Obtain the cell supernatant after culture, add the detection solution, and detect the signal intensity.

6. The AAV neutralizing antibody detection method according to claim 5, characterized in that: A 100% AAV-Gluc diluent control group was also set up, and an equal amount of FBS was mixed with the AAV-Gluc diluent.

7. The AAV neutralizing antibody detection method according to claim 5, characterized in that: A 0% AAV-Gluc dilution control group was also set up, and an equal amount of FBS was mixed with DMEM.

8. The AAV neutralizing antibody detection method according to claim 1, characterized in that: The preparation method of the detection solution is as follows: Remove the coelenterazine solution from the -20°C freezer and place it on ice to preheat and dissolve. Once dissolved, transfer it all to the assay buffer. Perform the above steps on ice. Aliquot the amount used for each test and freeze it at -80°C. Thaw it on ice before each use.

9. The AAV neutralizing antibody detection method according to claim 1, characterized in that: The steps of analyzing and collating data to obtain neutralization titers include: ， Luciferase inhibition rate = 100% - luciferase expression rate; Result judgment standard: The neutralization titer of the standard assay sample is the first dilution that reaches 50% luciferase inhibition.

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