AAV9 neutralizing antibody detection method and kit

Abstract

The invention relates to the field of detection of recombinant adeno-associated virus neutralizing antibodies. Specifically, the invention relates to a method for detecting a neutralizing antibody of a recombinant adeno-associated virus AAV9 and a related kit.

Description

Technical Field

[0001] The present invention relates to the field of detection of neutralizing antibodies of recombinant adeno-associated virus, and in particular to a method for detecting neutralizing antibodies of recombinant adeno-associated virus AAV9 and a related kit. Background Art

[0002] Gene therapy has made great progress in recent years, among which gene therapy mediated by adeno-associated virus (AAV) vectors has been widely studied. However, since many adults are exposed to wild-type AAV in childhood and develop humoral and cellular immunity against AAV capsids, they carry anti-AAV neutralizing antibodies in the body that can cross-react with AAV vectors. The presence of these neutralizing antibodies usually blocks gene delivery based on AAV vectors, resulting in limited efficacy of early gene therapy. The body's pre-existing AAV neutralizing antibodies have become one of the key factors affecting the effectiveness of gene therapy.

[0003] In addition, the immunogenicity assessment of AAV vectors is a key content of clinical bioanalysis testing of gene therapy based on AAV vectors. In clinical trials, the titer of AAV neutralizing antibodies in the blood of subjects is the screening criterion for determining whether the subject is included in the group, which is conducive to improving the targetedness and effectiveness of treatment. Preliminary testing of the titer of anti-AAV neutralizing antibodies in subjects can also help improve the clinical efficacy evaluation and market precision development of candidate drugs.

[0004] However, there are many difficulties in the development process of detecting neutralizing antibodies against viral vectors. For example, it is usually necessary to dilute the samples in a gradient to detect the titer in order to determine the test entry threshold, which makes the whole process complicated and time-consuming. In addition, studies have found that serum usually improves the effect of AAV viruses (such as AAV2 / 8 / 9) on cells infected with AAV viruses. Therefore, human serum in the detection system usually interferes with the detection results of neutralizing antibodies. In addition, the sensitivity of existing detection technologies also needs to be improved. Studies have found that for different AAV serotypes, subjects usually have corresponding neutralizing antibodies with different titers and proportions.

[0005] Therefore, there is an urgent need in the art to establish a stable and accurate detection method for detecting neutralizing antibodies of recombinant adeno-associated virus vectors (especially serotype AAV9 vectors) with high sensitivity and good physiological relevance, as well as a kit for detecting neutralizing antibodies of AAV9 vectors.

[0006] The present invention satisfies the above needs to a certain extent. Summary of the invention

[0007] The inventors have developed a highly sensitive detection method for detecting neutralizing antibodies of recombinant adeno-associated virus vector AAV9 through research. The detection method can be used to screen subjects for enrollment in AAV9 vector-mediated gene therapy research and clinical trials; it can also be used to monitor the production, presence and content of anti-AAV9 neutralizing antibodies during and after AAV9 vector-based gene therapy.

[0008] In a first aspect, the present invention provides an optimized method for detecting recombinant adeno-associated virus (AAV) neutralizing antibodies, comprising the following steps:

[0009] 1) Use virus diluent to dilute the AAV virus containing the reporter gene to 2.5×10 7 vg / hole-2×10 8 vg / hole;

[0010] 2) The sample was diluted 2-fold in series starting from 1:20 with the sample diluent to obtain a total of no less than 5 dilutions;

[0011] 3) Take 50 μl of the diluted virus and sample, mix well, and incubate in a 37°C 5% CO2 incubator for 1 hour;

[0012] 4) Dilute the cultured cells to 1×10 5 -3×10 5 cells / mL, and cells were plated into 96-well cell culture plates at 100 μl / well;

[0013] 5) Add the virus and sample mixture from step 3) to the cells from step 4) at 100 μl / well and incubate for 24-72 hours;

[0014] 6) detecting the expression of the reporter gene in the cell culture supernatant after the incubation in step 5);

[0015] 7) Obtain the titer of AAV neutralizing antibodies based on the reporter gene expression obtained in step 6).

[0016] In one embodiment, the reporter gene encodes a protein that provides a colorimetric, fluorescent, luminescent, chemiluminescent or electrochemical signal. In a specific embodiment, the reporter gene is a Gaussia luciferase gene. The luminescence mechanism of Gaussia luciferase is achieved through an oxidation reaction. Under the action of an oxidized substrate, Gaussia luciferase can produce fluorescence.

[0017] In one embodiment, the expression of the Gaussia luciferase gene is detected by the following steps: 20 μl of supernatant from each cell culture well is drawn into a black 96-well flat-bottom plate, 50 μl of substrate reaction solution diluted 1:2000 is added respectively, and the corresponding RLU value is detected on a GLOMAX luminescence detector. In one embodiment, the substrate is native coelenterazine. In a specific embodiment, the substrate is diluted in a PBS buffer containing 0.1%-0.5% EDTA at a ratio of 1:2000 of substrate: substrate diluent, and the final concentration of the substrate is 0.5 μg / ml.

[0018] In one embodiment, in step 7), the transduction rate of serum samples of different dilutions is calculated by using the RLU value obtained in step 6) according to the following formula:

[0019] (RLU value of the sample group - RLU value of the cell control group) / (RLU value of the virus control group - RLU value of the cell control group), and the corresponding titer of the neutralizing antibody was obtained based on the obtained transduction rate and the corresponding serum sample dilution.

[0020] In one embodiment, the transduction rate and the corresponding serum sample dilution are input into GraphPad 8.0 software. The dilution (X) is logarithmically transformed into log10 (X) by the software, and a four-parameter analysis of the logarithmic transformation is performed, the formula is: Log (inhibitor) vs. response-variable slope), the bottom and top limit ranges are set (Bottom = 0, Top = 1), and the IC50 value is calculated according to the fitting formula. The IC50 value is the titer of the AAV9 neutralizing antibody contained in the sample.

[0021] In one embodiment, the AAV virus is an AAV9 virus. In a preferred embodiment, the AAV9 virus is an AAV9 virus comprising a target gene, such as an AAV9-GLUC-2A-EGFP virus.

[0022] In one embodiment, the sample is a blood sample, other body fluids (such as cerebrospinal fluid), etc., such as a serum sample, a plasma sample, such as a human serum sample, or a human plasma sample.

[0023] In one embodiment, the virus diluent is DMEM containing 0.05%-0.1% BSA.

[0024] In one embodiment, the sample diluent is DMEM containing 10% FBS.

[0025] In one embodiment, the cell is selected from Vero, HEK-293, Hela, CHO and BHK-21 cells. In a specific embodiment, the cell is a HEK293 cell. In one embodiment, the HEK293 cell expresses a reporter gene.

[0026] In one embodiment, a sodium butyrate solution is added to the DMEM complete medium used to culture cells to a final concentration of 1 mM.

[0027] In one embodiment, the neutralizing antibody binds to a capsid protein, such as an AAV capsid protein. In a preferred embodiment, the neutralizing antibody binds to an AAV9 capsid protein.

[0028] In one embodiment, the incubation in step 5) is performed in a 37° C. 5% CO 2 incubator for 24-72 hours, preferably 36-72 hours, more preferably 42-72 hours, and more preferably 48-72 hours.

[0029] In one embodiment, the AAV9 virus comprises an AAV9 vector, which comprises one (or more) expression control elements, such as promoter and / or enhancer nucleic acid sequences. In a specific embodiment, the AAV9 vector further comprises one or more flanking sequences, such as one or more AAV inverted terminal repeats (ITRs).

[0030] In a second aspect, the present invention provides a kit for detecting the neutralizing antibodies of the recombinant adeno-associated virus of the first aspect.

[0031] In one embodiment, the kit comprises:

[0032] a. AAV virus, for example, AAV9 virus;

[0033] b. Virus dilution;

[0034] c. Sample diluent.

[0035] In one embodiment, the kit of the present invention further comprises:

[0036] d. AAV vector-sensitive cells, such as BHK-21 cells, HEK-293, Hela, Vero, CHO and / or A549 cells.

[0037] In one embodiment, the AAV vector-sensitive cells are HEK-293 cells.

[0038] In one embodiment, the AAV virus comprises a reporter gene, and in a specific embodiment, the reporter gene is a Gaussia luciferase gene.

[0039] In one embodiment, the virus diluent is DMEM containing 0.05%-0.1% BSA.

[0040] In one embodiment, the sample diluent is DMEM containing 10% FBS.

[0041] In one embodiment, the kit further comprises a culture medium for culturing AAV vector-sensitive cells. In a specific embodiment, the culture medium comprises sodium butyrate at a final concentration of 1 mM.

[0042] Effects of the Invention

[0043] The method for detecting AAV9 virus neutralizing antibodies provided by the present invention has the advantages of high sensitivity, good precision, easy high-throughput, standardized operation and verification. By changing the virus dilution, the stability of the relative luminescence signal value (RLU) is increased, thereby increasing the precision of the detection method.

[0044] The detection methods disclosed in the prior art are prone to inaccuracy when the sample needs to be highly diluted, and have large fluctuations between batches. The present invention effectively improves the R2 of the IC50 fitting curve by optimizing the composition of the sample diluent, and the 95% CI range of IC50 is also significantly narrowed, thereby increasing the sensitivity, detection range and precision of the detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 AAV9-GLUC-2A-EGFP dose-effect curve is shown.

[0046] Figure 2 It was shown that DMEM medium as virus dilution medium affects the binding of AAV9 virus.

[0047] Figure 3 The effect of BSA concentration in virus dilution on fluorescence expression and sample detection results is shown, where RLU 病毒对照 / RLU 细胞对照 It shows the fluorescence expression of virus-infected cells after eliminating experimental operation errors.

[0048] Figure 4 The effect of sample dilution on the test results is shown.

[0049] Figure 5 The effect of sodium butyrate on fluorescence expression and sample IC50 test results are shown.

[0050] Figure 6 The sensitivity of the method developed in this application for detecting AAV9 neutralizing antibodies is shown. DETAILED DESCRIPTION

[0051] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are fully incorporated by reference. In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will be apparent from this specification and the appended claims.

[0052] To interpret this specification, the following definitions will apply. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0053] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0054] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0055] Herein, when the term "comprising" or "including" is used, unless otherwise specified, it also encompasses the case where it consists of the stated elements, integers or steps.

[0056] The terms "recombinant adeno-associated virus vector", "adeno-associated virus vector", "AAV vector" and "rAAV vector" are used interchangeably herein and refer to a highly efficient foreign gene transfer tool transformed from wild-type AAV virus as people understand the life cycle of AAV virus and its related molecular biological mechanisms. The viral vector comprises a vector genome and a protein capsid. The viral vector capsid can be provided by any AAV serotype known in the art, including currently identified human and non-human AAV serotypes and AAV serotypes to be identified (see: Choi VW et al. 2005, Schmidt et al. 2008).

[0057] At present, the construction of recombinant AAV vectors usually uses the target gene to replace the rep gene and cap gene in the wild-type adeno-associated virus genome, and then packages the recombinant genome into infectious viral particles. Recombinant AAV vectors use the homologous or non-homologous recombination mechanism between AAV ITR and the rep gene and cap gene sequences present in trans in auxiliary components or production cells.

[0058] The term "vector genome (vg)" refers to the nucleic acid sequence packaged in the rAAV capsid to form the rAAV vector. The AAV vector genome only contains the ITR sequence of the AAV virus and the expression frame of the exogenous gene to be transported. The Rep and Cap proteins required for AAV virus packaging are provided by other exogenous plasmids or helper viruses, thereby reducing the harm that may be caused by packaging the rep and cap genes into the AAV vector. Furthermore, the AAV virus itself is not pathogenic, which makes the AAV vector one of the most recognized safe viral vectors. Deleting the D sequence and trs sequence in the ITR sequence on one side of the AAV virus can also make the genome carried by the packaged recombinant adeno-associated virus vector self-complementary to form a double strand, significantly improving the in vivo and in vitro transduction efficiency of the AAV vector (Wang Z et al., Gene Ther. 2003; 10(26): 2105-2111; McCarty DM et al., Gene Ther. 2003; 10(26): 2112-2118). The packaged virus is called scAAV (self-complementary AAV), which is a so-called double-stranded AAV virus. It is different from ssAAV (single-stranded AAV), which is a traditional AAV virus, in which both ITRs on both sides are not mutated.

[0059] In the context of antibodies, the term "titer" refers to the concentration of the antibody, also known as the antibody potency.

[0060] In the context of gene therapy, the terms "neutralizing antibodies" to AAV vectors, "neutralizing antibodies" and "anti-AAV vector neutralizing antibodies" are used interchangeably to refer to any antibody or antigen-binding fragment thereof that binds to an AAV vector and interferes with or inhibits the ability or biological activity of the AAV vector, thereby reducing its efficacy.

[0061] In the context of microplate readings and luminescence, the term "RLU" refers to relative light units or relative luminescence signal values. The amount of bioluminescence is measured by a luminometer and the results are given in relative light units.

[0062] The term "subject" refers to an animal, typically a mammal, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domesticated animals (dogs and cats), farm animals (poultry, such as chickens, ducks, horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents, and adult subjects.

[0063] Example

[0064] The experimental methods described in the following examples, unless otherwise specified, are conventional methods in the art using default parameters, steps, etc.; the experimental materials used, unless otherwise specified, are all commercially available products. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art, or the corresponding product instructions, are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0065] The main biological materials and reagents used in the examples of this application are listed as follows:

[0066] The AAV9-GLUC-2A-EGFP viral vector contains a genetic element encoding Gaussia luciferase. AAV9-CMV-GLuc-2A-EGFP is a prepackaged rAAV of serotype 9 (with a capsid from AAV serotype 9 and 2xITRs from AAV serotype 2) that overexpresses Gaussia luciferase and co-expresses EGFP via a 2A linker under the CMV promoter.

[0067] Virus preparation method: The recombinant AAV9 virus vector sample was prepared by the three-plasmid method. Specifically, the AAV9 vector plasmid, the helper plasmid (pHelper) (AAV Helper Free System, Agilent Technologies) and the corresponding Rep and Cap protein expression plasmids (pAAV-RC) were mixed at a molar ratio of 1:1:1, and the suspended HEK293T cells were transfected by the PEI method. After about 48 hours of transfection, the cells and culture supernatant were harvested. Purification was performed according to the AAV vector purification method reported by Wu Xiaobing et al. (Wu, X. et al., A novel method for purification of recombinant adenoassociated virus vectors on a large scale. Chinese Science Bulletin, 2001. 46 (6): p. 485-488) to obtain the recombinant AAV9 virus vector sample AAV9-GLUC-2A-EGFP.

[0068] HEK-293 cells and HEK293T cells were from the ATCC cell bank in the United States.

[0069] DMEM complete medium, from Gibco, catalog number: 11995-040.

[0070] BSA: Bovine serum albumin, from Sigma, catalog number: B2064.

[0071] FBS: Fetal bovine serum, from Hyclone, catalog number: SH30406.05

[0072] Virus dilution: DMEM containing 0.05%-0.1% BSA.

[0073] Sample diluent: DMEM containing 10% FBS.

[0074] The principle of detecting AAV neutralizing antibodies is to incubate the virus and sample, then add them to the target cells, and determine whether the sample contains neutralizing antibodies by detecting whether the virus infects the target cells and expresses the reporter gene. The method of detecting the titer of AAV neutralizing antibodies is usually affected by multiple factors in the test system, thereby affecting the sensitivity of the method, such as the amount of cells added, the amount of virus, the serum in the sample, etc. Since the neutralizing antibodies of different serotypes of AAV vectors have different contents and strengths in the subject, there is an urgent need to optimize and design the detection method of neutralizing antibodies for specific serotypes of AAV vectors in order to obtain a highly sensitive and stable detection method.

[0075] In the early stage of research and development, the inventors found that virus diluents and sample diluents with different compositions would affect the detection results of trace amounts of AAV9 neutralizing antibodies. Therefore, the inventors optimized the above conditions.

[0076] Example 1. Optimization of virus dosage

[0077] With respect to the detection method disclosed in the embodiment, the amount of virus used is selected according to the following principles: (1) the RLU value of the amount of virus added and the RLU value at which 90% of the virus is neutralized are both within the linear range; (2) the amount of virus added is non-toxic to the target cells; (3) the RLU value of the virus control (VC) is at least 5 times the RLU value of the target cell control (background control, CC), that is, VC / CC>5; (4) when the above conditions are met, a lower amount of virus is preferably selected to improve the detection sensitivity.

[0078] First, different amounts of AAV9-GLUC-2A-EGFP virus were added to HEK293 cells, with the virus dosage ranging from 6.25E+06 to 4E+08 vg / well. The RLU value in the supernatant was detected 48 hours after transduction, and a linear regression analysis was performed on the virus dosage and RLU value. The results are shown in Figure 1 The RLU values ​​of the virus within the tested dosage range showed a good linear relationship, with a correlation coefficient R2>0.99, indicating that the method is feasible.

[0079] No changes in cell morphology were observed during the cell culture process, indicating that the AAV9 virus within the scope of this experiment was non-toxic to cells.

[0080] The results showed that when the amount of AAV9-GLUC-2A-EGFP was greater than 2.5E+07 vg / well, VC / CC>5, satisfying the above criterion (3), and when the amount of AAV9-GLUC-2A-EGFP was 2.0E+08 vg / well, VC / CC>10.

[0081] Therefore, in the subsequent neutralizing antibody detection method, the amount of virus used is greater than 2.5E+07 vg / well.

[0082] Example 2: Optimization of virus dilution in the detection method

[0083] Most of the prior art uses DMEM medium (abbreviated as D0) to dilute the AAV9 virus. However, the inventors found that the fitting curve of the sample was poor during the experiment. Figure 2 To this end, it is necessary to adjust the composition of the virus diluent used to dilute the virus in order to improve the sensitivity and stability of the detection method.

[0084] Falese et al. disclosed the use of DMEM containing 1% BSA as a virus diluent (Strategy to detect pre-existing immunity to AAV gene therapy, Gene Therapy (2017) 24, 768-778). The inventors referred to this document and diluted the virus with 1% BSA. The results are shown in Table 1. The IC50 of the two experimental results without adding BSA was quite different, which were 764 / 1150 respectively; while the IC50 of the two experimental results with the addition of BSA was relatively consistent, which were 499 / 471 respectively, indicating that 1% BSA can increase the stability and repeatability of the results.

[0085] Table 1. Effect of 1% BSA in virus dilution on test results

[0086]

[0087]

[0088] However, in subsequent experiments, it was found that after adding 1% BSA, the AAV9 virus transduction rate decreased significantly, reducing the sensitivity of the experiment. Therefore, it is necessary to optimize the concentration of BSA in the virus dilution. The inventors compared the effects of 0.1%, 0.25%, 0.5% and 1% BSA on RLU 病毒对照 / RLU 细胞对照 The results are shown in Figure 3 A, RLU 病毒对照 / RLU 细胞对照When the BSA concentration dropped to 0.1%, the RLU of 1% BSA 病毒对照 / RLU 细胞对照 The fluorescence intensity was increased by two times, thereby improving the sensitivity of the detection. At the same time, the inventors found that the virus dilution containing 0.1% BSA had good repeatability in different experiments, which was consistent with the results in Table 1. Compared with the virus dilution without BSA, it significantly reduced the fluctuation and error between different experimental batches, which was beneficial to improving the stability and sensitivity of the detection method. In addition, the inventors also compared the effect of 0.1% and 1% BSA concentration in the dilution on the transduction rate of serum samples with different dilutions. The results are shown in Table 1. Figure 3 B, The transduction rates were basically the same under the two conditions, indicating that both 0.1% and 1% BSA had good stability.

[0089] In summary, the virus dilution prepared by adding 0.05%-0.1% BSA to DMEM improves the sensitivity and stability of the detection method of AAV neutralizing antibodies.

[0090] Example 3: Optimizing the sample dilution in the detection method

[0091] Most of the existing technologies use DMEM basal medium (abbreviated as D0) to dilute serum samples. When we use this method to detect serum samples with high dilution, we find that the transduction rate of the sample increases sharply at a certain dilution ( Figure 4 A), the R2 value of the fitting curve is very low, and the 95% CI range of IC50 is very wide (Table 2). The inventors then changed the sample diluent to DMEM containing 10% FBS (abbreviated as D10) to dilute the sample. It was found that for high titer samples (which need to be highly diluted during detection), after highly diluting them with the sample diluent added with FBS, the transduction rate showed a good gradient change. The results are shown in Figure 4 B, the R2 values ​​of all samples were above 0.9, and the 95% CI range of IC50 was also significantly narrowed (Table 2).

[0092] The above results indicate that diluting blood samples with DMEM containing 10% FBS can more accurately detect AAV9 neutralizing antibodies in blood samples with high dilutions (dilution range 1:20-1:163840), thereby improving the detection range of the detection method.

[0093] Table 2. Effect of sample diluent on test results

[0094]

[0095] Example 4: Effect of Sodium Butyrate on RLU Value and IC50

[0096] Sodium butyrate can promote the expression level of various viruses in cells. In order to increase the expression and sensitivity, we systematically tested the effect of adding sodium butyrate during culture to make the final concentration of sodium butyrate 1mM on the expression level of fluorescence (Gluc). The cells tested were HEK293 cells. The inoculation density of HEK293 cells was 2E+04 cells / well, and the dosage of AAV9-GLUC-2A-EGFP virus was doubled from 2.56E+10vg / well to 1E+8vg / well. The RLU value in the supernatant was detected 48h after transduction. The results are shown in Figure 5 A. Compared with the culture wells without sodium butyrate, the RLU values ​​in the culture supernatant of all virus amounts increased by more than 50% after the addition of sodium butyrate. The virus amount and the RLU signal value were linearly fitted, and the two reaction curves were basically parallel, indicating that the addition of sodium butyrate can increase the expression level of the gene and increase the reaction sensitivity, but does not change the dose-effect relationship.

[0097] Example 5. Optimized method for detecting trace amounts of AAV9 neutralizing antibodies

[0098] Therefore, the present invention has developed an optimized method for detecting AAV9 neutralizing antibodies, which specifically comprises the following steps:

[0099] 1) Use virus diluent (DMEM medium containing 0.05%-0.1% BSA) to dilute AAV9-GLUC-2A-EGFP virus to 2.5×10 7 -2×10 8 vg / hole.

[0100] 2) The sample is diluted in a two-fold series starting from 1:20 using a sample diluent (DMEM culture medium containing 10% FBS) to obtain a total of 6 dilutions. The sample can be a plasma sample, a serum sample, or other body fluids (such as cerebrospinal fluid).

[0101] 3) Take 50 μl of the diluted virus and sample, mix well, and place in a 37°C 5% CO2 incubator for 1 hour.

[0102] 4) Dilute the cultured HEK293 cells to 2×10 5 To the HEK293-C340 single cell suspension with adjusted concentration, add sodium butyrate storage solution (final sodium butyrate concentration 1 mM), and plate the cells into a 96-well cell culture plate at 100 μl / well.

[0103] 5) Add the virus and sample mixture after incubation to each cell well at 100 μl / well and mix well. Place in a 37°C 5% CO2 incubator for 48 hours. This experiment includes the following groups: blank HEK293 cell control group, virus control group (including virus + HEK293 cells), sample group (including sample + virus + HEK293 cells).

[0104] 6) Take out the 96-well plate, pipette 20 μl of supernatant from each well into a black 96-well flat-bottom plate, add 50 μl of substrate reaction solution diluted 1:2000 (the substrate natural enteroluminal was purchased from Nanolight, item number 303-1, and PBS buffer containing 0.2% EDTA was used as the substrate diluent, and the substrate was diluted at a ratio of 1:2000 for substrate: substrate diluent), and detect the corresponding RLU value on the GLOMAX luminescence detector.

[0105] 7) Use the RLU values ​​obtained from the test to calculate the transduction rate of serum samples of different dilutions according to the following formula:

[0106] (RLU value of the sample group - RLU value of the cell control group) / (RLU value of the virus control group - RLU value of the cell control group)

[0107] 8) The transduction rate and the corresponding serum sample dilution were input into GraphPad 8.0 software. The dilution (X) was logarithmically transformed into log10 (X) using the software, and a four-parameter analysis of the logarithmic transformation was performed using the formula: Log (inhibitor) vs. response-variable slope), the bottom and top limits were set (Bottom = 0, Top = 1), and the IC50 value was calculated according to the fitting formula. The IC50 value is the titer of the AAV9 neutralizing antibody contained in the sample.

[0108] Example 6: Determination of the sensitivity of the AAV9 neutralizing antibody detection method

[0109] Using the method disclosed in Example 5, the amount of virus per well was adjusted to 5×10 7 The results for the sample containing neutralizing antibodies at a concentration of 1.56 ng / mL (pure antibody preparation diluted with AAV9 antibody negative serum) were shown in Figure 2. Figure 6 , IC50=87>20, and R2=0.98>0.8, so the sensitivity of this experimental method is 1.56ng / mL. Compared with the existing technology, the sensitivity has been greatly improved.

[0110] Example 7. Precision of the AAV9 Neutralizing Antibody Detection Method

[0111] In order to test the stability and repeatability of the detection method developed in this application, the inventors used the method disclosed in Example 5 to repeatedly test the AAV9 mouse neutralizing antibodies prepared by themselves. Two experimenters conducted a total of 6 tests at different time points. The results are shown in Table 3. The R2 of the fitting curves of the 6 tests was greater than 0.9, the mean IC50 was 6.98 ng / mL, and the CV was 11%, indicating that the test results had good consistency and repeatability. The present invention greatly improves the precision of AAV9 neutralizing antibody detection.

[0112] Table 3. Precision evaluation of AAV9 neutralizing antibodies

[0113]

Claims

1. A method for detecting neutralizing antibodies of a recombinant adeno-associated virus (AAV), comprising: 1) Use virus diluent to dilute the AAV virus containing the reporter gene to 2.5×10 7 vg / hole-2×10 8 vg / hole; 2) The sample was diluted 2-fold in series starting from 1:20 with the sample diluent to obtain a total of no less than 5 dilutions; 3) Take 50 μl of the diluted virus and sample, mix well, and incubate in a 37°C 5% CO2 incubator for 1 hour; 4) Dilute the cultured cells to 1×10 5 -3×10 5 cells / mL, and cells were plated into 96-well cell culture plates at 100 μl / well; 5) Add the virus and sample mixture from step 3) to the cells from step 4) at 100 μl / well and incubate for 24-72 hours; 6) detecting the expression of the reporter gene in the cell culture supernatant after the incubation in step 5); 7) Obtain the titer of AAV neutralizing antibodies based on the reporter gene expression obtained in step 6).

2. The method according to claim 1, wherein the AAV virus is an AAV9 virus, preferably, the AAV9 virus is an AAV9 virus comprising a target gene, and more preferably, the AAV9 virus is an AAV9-GLUC-2A-EGFP virus.

3. The method according to claim 1 or 2, wherein the reporter gene encodes a protein that provides a colorimetric, fluorescent, luminescent, chemiluminescent or electrochemical signal, preferably, the reporter gene is a Gaussia luciferase gene.

4. The method according to claim 3, wherein the expression level of the Gaussia luciferase gene is determined by detecting the RLU value of the Gaussia luciferase gene.

5. The method according to any one of claims 1 to 4, wherein The transduction rate of serum samples with different dilutions was calculated using the RLU value according to the following formula: (RLU value of the sample group - RLU value of the cell control group) / (RLU value of the virus control group - RLU value of the cell control group), And the corresponding titer of neutralizing antibodies is obtained according to the obtained transduction rate and the corresponding serum sample dilution; Optionally, the obtained transduction rate and the corresponding serum sample dilution are calculated by software to obtain the titer of AAV neutralizing antibodies.

6. The method of any one of claims 1-5, wherein the sample is a blood sample or other body fluid (such as cerebrospinal fluid), such as a serum sample, a plasma sample, such as a human serum sample, or a human plasma sample.

7. The method of any one of claims 1 to 6, wherein the virus diluent is DMEM complete medium containing 0.05%-0.1% BSA.

8. The method according to any one of claims 1 to 7, wherein the sample diluent is DMEM complete medium containing 10% FBS.

9. The method of any one of claims 1-8, wherein the cell is selected from Vero, HEK-293, Hela, CHO and BHK-21 cells, preferably, the cell is a HEK293 cell, more preferably, the HEK293 cell expresses a reporter gene.

10. The method according to any one of claims 1 to 9, wherein the medium used to culture the cells is a DMEM complete medium.

11. The method according to any one of claims 1 to 10, wherein a sodium butyrate solution having a final concentration of 1 mM is added to the DMEM complete medium used for culturing cells.

12. The method of any one of claims 1-11, wherein the neutralizing antibody binds to a capsid protein, such as an AAV capsid protein, preferably, the neutralizing antibody binds to an AAV9 capsid protein.

13. The method of any one of claims 1-12, wherein the incubation in step 5) is performed in a 37°C 5% CO2 incubator for 24-72 hours, preferably 36-72 hours, more preferably 42-72 hours, and more preferably 48-72 hours.

14. A kit for detecting the neutralizing antibody of the recombinant adeno-associated virus according to any one of claims 1 to 13, the kit comprising: a. AAV virus, for example, AAV9 virus; b. Virus dilution; c. Sample diluent.

15. The kit of claim 14, further comprising: d. AAV vector-sensitive cells, such as BHK-21 cells, HEK-293, Hela, Vero, CHO and / or A549 cells.

16. The kit of claim 14 or 15, wherein the AAV virus comprises a reporter gene, preferably, the reporter gene is a Gaussia luciferase gene.

17. The kit according to any one of claims 14 to 16, wherein the virus diluent is DMEM containing 0.05%-0.1% BSA; and the sample diluent is DMEM containing 10% FBS.

18. The kit according to any one of claims 14 to 17, wherein the kit further comprises a culture medium for culturing AAV vector-sensitive cells, preferably, the culture medium comprises sodium butyrate at a final concentration of 1 mM, preferably, the culture medium is a DMEM complete medium comprising 1 mM sodium butyrate.

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