Universal anti-AAV9 antibody ELISA kit and anti-AAV9 antibody detection method thereof

Abstract

The invention relates to a universal type anti-AAV9 antibody ELISA kit and an anti-AAV9 antibody detection method, AAV9 in the kit is pre-coated on a high-affinity ELISA plate, a quality control product and a sample to be detected are added into the holes of the ELISA plate, and the anti-AAV9 antibody in the sample is combined with the AAV9 coated on the ELISA plate through incubation. According to the detection method disclosed by the invention, the sample is judged to be negative / positive according to the U value relative unit (Units, U) (U = sample OD 450nm / CC OD 450nm), so that the effect of qualitatively detecting whether the serum contains the anti-AAV9 antibody in vitro is achieved. The kit disclosed by the invention has good specificity, durability, precision, negative / positive control coincidence rate and stability, and also has the advantages of simplicity and convenience in operation, short detection period and the like.

Description

Technical Field

[0001] The present invention relates to virus antibody detection in the field of biological analysis and detection, and in particular to a universal anti-AAV9 antibody ELISA kit and a detection method of the anti-AAV9 antibody thereof. Background Art

[0002] Adeno-associated virus (AAV) is a member of the Parvoviridae family, a small, non-enveloped virus with an icosahedral structure. The diameter of the virus particle is between 20 and 26 nm, and contains a linear single-stranded DNA genome of about 4.70 kb.

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

[0007] The purpose of the present invention is to provide a universal anti-AAV9 antibody ELISA kit, which has good specificity, durability, precision, negative / positive control compliance rate and stability by detecting serum samples in vitro, and has the advantages of simple operation and short detection cycle.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] A universal anti-AAV9 antibody ELISA kit, the universal anti-AAV9 antibody ELISA kit comprises a capture plate, a positive quality control, a critical value quality control and a negative quality control, the capture plate is prepared by coating AAV9 and blocking with a blocking solution, and the preparation method of the capture plate is:

[0010] AAV9 was diluted to a concentration of 2×10 using 50 mM CBS solution with a pH of 9.6±0.2 as the coating solution. 10 vg / mL, add to the ELISA plate, seal with sealing film and incubate at 2-8℃ for 16-20 hours;

[0011] Discard the coating solution in the ELISA plate and wash the plate;

[0012] Add a blocking solution containing 5% to 6% by weight of skimmed milk powder, and block at 25 to 37° C. for 1 to 4 hours;

[0013] Discard the blocking solution in the ELISA plate and place the plate at 25-37°C to dry for 1-4 hours;

[0014] After drying, the coated ELISA plate is vacuum packed to become the capture plate of the test kit.

[0015] Preferably, the coating solution is prepared by: taking 1.59g Na2CO3 and 2.94g NaHCO3 into a preparation container, adding an appropriate amount of ultrapure water, stirring to dissolve, making the volume to 1.00L with ultrapure water, and filtering with a filter with a membrane pore size of 0.22μm; the blocking solution is prepared by: taking 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, and 0.20g KCl into a preparation container, adding an appropriate amount of ultrapure water, stirring to dissolve, making the volume to 1.00L, and filtering with a filter with a membrane pore size of 0.22μm to obtain a mixed solution; taking 475mL of the mixed solution into a preparation container, adding 25.00g of skimmed milk powder to obtain a blocking solution.

[0016] Preferably, the universal anti-AAV9 antibody ELISA kit also includes 10× washing solution, sample diluent, detection antigen solution, HRP labeling solution, TMB substrate color development solution and stop solution.

[0017] Preferably, the negative quality control is a dilution of cynomolgus monkey AAV9 NAb negative mixed serum, beagle dog AAV9 NAb negative mixed serum, SD rat AAV9 NAb negative mixed serum, BALB / c mouse AAV9 NAb negative mixed serum, and hamster AAV9 NAb negative mixed serum;

[0018] The critical value quality control is 20ng / mL AAV9 antibody prepared using negative quality control;

[0019] The positive quality control is 500 ng / mL AAV9 antibody prepared using the negative quality control.

[0020] Preferably, the components of the universal anti-AAV9 antibody ELISA kit are: a capture plate coated with AAV9 antigen, a negative quality control containing 1 / 25 negative mixed serum, a critical value quality control containing 20 ng / mAAV9 antibody, a positive quality control containing 500 ng / mAAV9 antibody, 10× washing solution, sample diluent, detection antigen solution, HRP labeling solution, TMB substrate color development solution and stop solution.

[0021] Preferably, the reagents in the kit are configured as follows:

[0022] Preparation of 10× washing solution: weigh 11.36g Na2HPO4, 2.72g KH2PO4, 80.06g NaCl, 2.01g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 5.00mL Tween-20 and 5.00mL ProClin300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter through a filter with a membrane pore size of 0.22μm;

[0023] Preparation of sample diluent: weigh 2.27g Na2HPO4, 0.54g KH2PO4, 16.01g NaCl, 0.40g KCl, 40.00g sucrose, 20.00g BSA into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 1.00mL Tween-20, 1.00mL ProClin 300 after dissolution, stir to mix, make up to 2.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm;

[0024] Preparation of detection antigen solution: Measure 300 mL of sample diluent into the preparation container, add 30 μL of biotin-labeled AAV9, and prepare a working solution with a concentration of 1.2 μg / mL;

[0025] Preparation of HRP labeled solution: Measure 300 mL of sample dilution solution into the preparation container, add 30 μL of HRP labeled streptavidin to prepare a working solution with a concentration of 100 ng / mL;

[0026] Preparation of stop solution: 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] Preferably, the preparation method of the biotin-labeled AAV9 is as follows: Biotin-LC-LC-NHS can react with primary amino groups (NH2-) to form a stable and irreversible amide bond, thereby achieving the effect of labeling AAV9 containing primary amino groups (NH2-), according to 1×10 13 vg AAV9 was added with 6 μL of 10 mM Biotin-LC-LC-NHS test solution, labeled in an ultrafiltration tube, free biotin was removed, the concentration of biotin labeled protein was determined, and an equal volume of label protection solution was added to obtain the product.

[0028] A method for detecting anti-AAV9 antibodies of a universal anti-AAV9 antibody ELISA kit, the method comprising the following steps:

[0029] S1 Preparation of experimental reagents

[0030] Return all reagents to room temperature before testing;

[0031] If the concentrated reagent crystallizes, it needs to be warmed at 37°C until all the crystals are dissolved;

[0032] Dilute the 10× wash solution to 1× wash solution with water, gently mix the 1× wash solution and set aside;

[0033] S2 Experimental Sample Preparation

[0034] Before testing, all samples were returned to room temperature and the serum samples to be tested were diluted 1:25;

[0035] S3 Experimental Procedure

[0036] Add 100 μL of quality control (positive quality control, critical value quality control, negative quality control) and sample to the corresponding wells; seal the plate with a sealing film and incubate at 37°C for 1 hour;

[0037] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time);

[0038] Add 100 μL of the detection antigen solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 1 hour;

[0039] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time);

[0040] Add 100 μL of HRP labeling solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes;

[0041] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time);

[0042] Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 15 min;

[0043] Add 100 μL of stop solution to each well to terminate the reaction;

[0044] Complete OD in 15 minutes 450nm reading;

[0045] S4 absorbance calculation

[0046] Calculate the average absorbance of the quality control and sample duplicate wells;

[0047] Calculate the absorbance ratio of each sample to the critical value quality control (Cut-off Control, CC);

[0048] S5 Result Determination

[0049] Experimental validity: positive control absorbance (OD 450nm ):>1.000,Cut-offControl absorbance (MeanOD 450nm ): 0.150~0.500, Negative Control absorbance (Mean OD 450nm ):<0.150;

[0050] Sample result determination: Sample results are determined in relative units (Units, U), sample OD 450nm / CC OD 450nm =U;

[0051] When the sample test result is >1.1U, it is positive and anti-AAV9 antibodies are detected in the sample;

[0052] When the sample test result is between 0.9U and 1.1U, it is a suspicious result and needs to be retested;

[0053] When the sample test result is <0.9U, it is negative and no anti-AAV9 antibodies are detected in the sample.

[0054] The kit of the present invention is suitable for in vitro qualitative detection of whether anti-AAV9 antibodies are contained in serum samples. Compared with the prior art, the present invention has the following characteristics and beneficial effects:

[0055] 1. The present invention prepares a capture plate coated with AAV9 antigen, so that the capture plate can specifically recognize anti-AAV9 antibodies without interference from foreign proteins; through the combined use of the capture plate coated with AAV9 antigen and biotin-labeled AAV9, the kit of the present invention can specifically recognize anti-AAV9 antibodies;

[0056] 2. The kit of the present invention is not restricted by species and can be used to evaluate whether various serum samples (such as human, monkey, dog, mouse) contain anti-AAV9 antibodies (negative and positive samples) and their anti-AAV9 antibody titer levels.

[0057] 3. The kit of the present invention establishes a negative / positive result judgment standard, making the test results easy to interpret; it has been verified that the kit of the present invention has good specificity, durability, precision, negative / positive control compliance rate, stability, as well as simple operation and short detection cycle (single detection time is within 4 hours). BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The working principle diagram of the kit is shown below;

[0059] Figure 2 The results of the coating optimization experiment in Example 2 are as follows;

[0060] Figure 3 These are the experimental results for optimizing the concentration of the detection antigen solution (biotin-labeled AAV9) in Example 2. DETAILED DESCRIPTION

[0061] The technical solution of the present invention is further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any form of modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0062] 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.

[0063] Unless otherwise specified, the reagents used in the following examples can be obtained from commercial sources.

[0064] Ultra-micro-volume UV-visible spectrophotometer, Hangzhou Miou Instrument Co., Ltd., ND-100F;

[0065] The solutions in the following examples were prepared as follows:

[0066] Preparation of 1# coating solution: Accurately weigh 1.59g Na2CO3 and 2.94g NaHCO3 into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, 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 1 year.

[0067] Preparation of 2# coating solution: Accurately weigh 1.14g Na2HPO4, 0.27g KH2PO4, 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 stably stored at 2-8℃ for 1 year.

[0068] Preparation of 1# blocking solution: Accurately weigh 475g of 2# 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.

[0069] Preparation of 2# blocking solution: weigh 1.14g Na2HPO4, 0.27g KH2PO4, 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, dilute to 1.00L with ultrapure water, 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.

[0070] Preparation of 3# blocking solution: weigh 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, 0.20g KCl, 10.00g gelatin 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 stored stably at 2-8℃ for 1 year.

[0071] Preparation of 10× washing solution: Accurately weigh 11.36g Na2HPO4, 2.72g KH2PO4, 80.06g NaCl, 2.01g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 5.00mL Tween-20, 5.00mL ProClin300 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 stored stably at 2-8℃ for 2 years.

[0072] Preparation of sample diluent: accurately weigh 2.27g Na2HPO4, 0.54g KH2PO4, 16.01g NaCl, 0.40g KCl, 40.00g sucrose, 20.00g BSA into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 1.00mL Tween-20, 1.00mL ProClin 300 after dissolution, stir to mix, make up to 2.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stored stably at 2-8℃ for 2 years.

[0073] Preparation of detection antigen solution: Accurately measure 300 mL of sample diluent into the preparation container, add a certain volume of biotin-labeled AAV9, and prepare a working solution with a concentration of 0.1-1.2 μg / mL. The solution can be stored stably at 2-8°C for 1 year.

[0074] Preparation of HRP labeled solution: Accurately measure 300 mL of sample dilution solution into the preparation container, add a certain volume of HRP labeled streptavidin (Thermo Fisher Scientific, 21130), and prepare a working solution with a concentration of 25-100 ng / mL. The solution can be stored stably at 2-8°C for 1 year.

[0075] Preparation of TMB substrate colorimetric solution: TMB Super Sensitive One Component HRP Microwell Substrate (Surmodics, Inc., TMBS-1000-01) is directly available for aliquoting. The solution can be stored stably at 2-8°C for a period of time that is consistent with the original manufacturer's expiration date.

[0076] 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.

[0077] Preparation of negative quality control: Use sample diluent to dilute 5 kinds of animal AAV9 NAb negative mixed serum (crab-eating monkey AAV9NAb negative mixed serum, beagle dog AAV9 NAb negative mixed serum, SD rat AAV9 NAb negative mixed serum, BALB / c mouse AAV9 NAb negative mixed serum, hamster AAV9 NAb negative mixed serum) 25 times, and filter it through a filter with a membrane pore size of 0.22μm.

[0078] AAV2 / 9-pAAV-CMV-EGFP-3xFLAG-WPRE (abbreviated as AAV9), Heyuan Biotechnology (Shanghai) Co., Ltd., catalog number AOV022;

[0079] AAV9 monoclonal antibody (mouse, IgG1a), Beijing Subenyuanhe Biotechnology Co., Ltd., catalog number M100038.

[0080] The cynomolgus monkey AAV9 NAb negative mixed serum, beagle dog AAV9 NAb negative mixed serum, SD rat AAV9 NAb negative mixed serum, BALB / c mouse AAV9 NAb negative mixed serum, and hamster AAV9 NAb negative mixed serum were screened and obtained by Zhejiang Hengyu Biotechnology Co., Ltd. For specific methods, see CN117686709A "A method for detecting AAV9 neutralizing antibodies".

[0081] The preparation method of the capture plate in the following examples is:

[0082] S1, coating

[0083] AAV9 was diluted to a concentration of 0.5×10 10 ~2×10 10 vg / mL coating solution was evenly added to the ELISA plate at 100 μL / well, sealed with sealing film, and incubated at 2-8°C for 16-20 hours;

[0084] S2, Washing plate

[0085] Discard the coating solution in the ELISA plate and wash the plate twice (260 μL / well of washing solution, 30 to 60 seconds for each wash);

[0086] S3, closed

[0087] Add blocking solution evenly at 200 μL / well and block at 25-37°C for 1-4 hours;

[0088] S4. Drying

[0089] Discard the blocking solution in the ELISA plate, pat it dry on absorbent paper, and place the ELISA plate at 25-37℃ to dry for 1-8 hours;

[0090] S5. Packaging

[0091] 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.

[0092] Example 1 AAV9 biotin labeling

[0093] Biotin-LC-LC-NHS can react with primary amino groups (NH2-) to form stable, irreversible amide bonds, thereby achieving the effect of labeling AAV9 containing primary amino groups (NH2-).

[0094] 1. Preparation of biotin labeling solution

[0095] Preparation of 10mM Biotin-LC-LC-NHS: Accurately weigh 10.0mg Biotin-LC-LC-NHS into a brown sample tube, add 1.761mL DMSO, tighten the tube cap, gently mix and continue to dissolve at room temperature for 30 minutes. The solution can be stored stably at -80℃ for up to 6 months (store in aliquots and use a new one each time).

[0096] Preparation of labeling buffer: Accurately weigh 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, 0.20g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, and then dilute to 1.00L with ultrapure water, 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.

[0097] Preparation of marker protection solution: accurately weigh 0.2g BSA, 0.8g ultrapure water, and 9g glycerol into a 15mL reagent bottle (or 15mL centrifuge tube), tighten the bottle (or tube) cap, place on a rotary mixer and mix thoroughly, and filter with a filter with a membrane pore size of 0.22μm after complete dissolution. The solution can be stably stored at -20℃ for 2 years.

[0098] 2. Calculation of biotin dosage

[0099] According to 1×10 13 vgAAV9 was added with 6 μL of 10 mM Biotin-LC-LC-NHS solution, and the amount of AAV9 to be labeled (the minimum content was not less than 1 × 10 12 vg) Calculate the required volume of 10 mM Biotin-LC-LC-NHS solution.

[0100] 3. Biotin labeling

[0101] Transfer the substance to be labeled to an ultrafiltration tube, add labeling buffer (10 mM PBS, pH 7.2 ± 0.2) that does not exceed the maximum volume of the ultrafiltration tube, and centrifuge at 12000 × g for 10 min (this step can be repeated 2 to 3 times). After the last ultrafiltration is completed, add an appropriate amount of labeling buffer to adjust the concentration of the substance to 2 × 10 13 About vg / mL.

[0102] Add an appropriate volume of Biotin-LC-LC-NHS test solution to the above ultrafiltration tube and gently pipette to mix. Place in a 37°C incubator and incubate in the dark for 30 minutes or incubate in the dark for 1 hour at room temperature.

[0103] 4. Removal of free biotin

[0104] The labeled biotin marker was placed in a centrifuge and centrifuged at 12000×g for 10 min at room temperature.

[0105] Add an appropriate amount of labeling buffer to the above ultrafiltration tube, gently pipette to mix, centrifuge at 12000×g for 10 min at room temperature, and repeat this step 3 times.

[0106] Add an appropriate amount of labeling buffer to the ultrafiltration tube again, mix gently by pipetting, and centrifuge at 4000×g for 5 minutes.

[0107] The ultrafiltration tube membrane was placed upside down in the collection tube and centrifuged at 12000×g for 2 min at room temperature.

[0108] 5. Determination of biotin-labeled protein concentration

[0109] The protein concentration of the biotinylated protein was measured at 280 nm using an ultra-micro-volume UV-visible spectrophotometer (repeated twice, average value calculated).

[0110] 6. Storage of biotin markers

[0111] Collect the solution in the ultrafiltration tube into a brown sample tube, add an equal volume of marker protection solution, and store at -20°C for later use.

[0112] Example 2 Establishment of a double antigen sandwich ELISA detection method based on a biotin-streptavidin system 1. Operation steps of a double antigen sandwich ELISA detection method based on a biotin-streptavidin system

[0113] 1) Sample addition: Add 100 μL of sample (e.g., anti-AAV9 antibody of appropriate concentration, quality control product) to the prepared capture plate. Seal the plate with a sealing film and incubate at 37°C for 1 hour. Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate, and wash 5 times (pat dry on absorbent paper each time).

[0114] 2) Add detection antigen solution (biotin-labeled AAV9 solution): Add 100 μL of detection antigen solution of appropriate concentration to each well, seal the plate with a new sealing film, and incubate at 37°C for 1 hour. Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate, and wash 5 times (pat dry on absorbent paper each time).

[0115] 3) Add HRP labeling solution (HRP-labeled streptavidin solution): Add 100 μL of HRP labeling solution of appropriate concentration 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× washing solution to each well, wash the plate, and wash 5 times (pat dry on absorbent paper each time).

[0116] 4) Color development: Add 100 μL of TMB substrate color development solution to each well, protect from light, and incubate at room temperature for 15 minutes.

[0117] 5) Stop reaction: Add 100 μL of stop solution to each well to stop the reaction.

[0118] 6) Reading: Complete OD within 15 minutes 450nm reading.

[0119] 2. Optimization of various conditions in the double antigen sandwich ELISA test based on the biotin-streptavidin system

[0120] 1) Optimization of coating solution, antigen coating concentration, and blocking solution

[0121] An orthogonal test (12 different conditions) was set up according to Table 1 for preliminary screening of a suitable coating solution, a suitable antigen coating concentration, and a suitable blocking solution. The anti-AAV9 antibody concentration was set at three gradient levels (500 ng / mL, 100 ng / mL, and 20 ng / mL).

[0122] According to the results of the reaction absorbance and the reaction signal-to-noise ratio (S / N) at each concentration level described in Table 1, when the same concentration of antigen coating concentration and blocking solution were fixed, 1# coating solution could obtain a higher signal-to-noise ratio than 2# coating solution; when the coating solution and blocking solution were fixed, 2.0×10 10 Compared with the antigen coating concentration of 1.0×10 10 Antigen coating concentration can get a higher signal-to-noise ratio; when the coating solution and the same concentration of antigen coating concentration are fixed, the reaction background signal of 1# blocking solution is the lowest compared with 2# blocking solution and 3# blocking solution. 10 The antigen coating concentration and 1# blocking solution were subsequently optimized.

[0123] Table 1 Results of orthogonal test for optimization of coating solution, antigen coating concentration and blocking solution

[0124]

[0125]

[0126] 2) Optimization of coating

[0127] AAV2 / 9-pAAV-CMV-EGFP-3xFLAG-WPRE (Heyuan Biotechnology, Cat. No.: AOV022), AAV2 / 9-pAAV-CMV-Luc2 (Heyuan Biotechnology, Cat. No.: H5824), AAV2 / 9-CMV_bGI-empty-pA (Taiertu Biotechnology, Cat. No.: S0654-9, Lot No.: PDG24), AAV2 / 9-CMV_bGI-Cre-EGFP-pA (Taiertu Biotechnology, Cat. No.: S0231-9, Lot: TDP16) were prepared with 1# coating solution to a concentration of 2.0×10 10 vg / mL of coating solution, blocked with 1# blocking solution, and the prepared 4 capture plates were used to study the effects of different coating agents on the reaction system.

[0128] Test results such as Figure 2 As shown, the reaction performance of AAV9 with different structures from different companies was slightly different, and the method had good versatility. AAV2 / 9-pAAV-CMV-EGFP-3xFLAG-WPRE (Heyuan Bio, Cat. No.: AOV022) was selected for subsequent optimization.

[0129] 3) Optimization of the concentration of the detection antigen solution (biotin-labeled AAV9)

[0130] AAV2 / 9-pAAV-CMV-EGFP-3xFLAG-WPRE (Heyuan Biotechnology, Cat. No.: AOV022) was prepared with 1# coating solution to a concentration of 2.0×10 10 vg / mL of coating solution was blocked with 1# blocking solution to prepare a capture plate. During the experiment, 0.4μg / mL, 0.2μg / mL, and 0.1μg / mL of detection antigen solution (biotin-labeled AAV9 solution) were added respectively.

[0131] Test results such as Figure 3 As shown, as the concentration of the detection antigen solution (biotin-labeled AAV9) increases, the reaction signal gradually increases.

[0132] Example 3 Universal anti-AAV9 antibody ELISA kit

[0133] The kit of the present invention is used to detect anti-AAV9 antibodies in serum samples. The kit components listed in Table 2 are assembled into a kit, and the kit is stored at 2-8°C after assembly.

[0134] Table 2 Components, quantities and main ingredients of universal anti-AAV9 antibody ELISA kit

[0135]

[0136]

[0137] In Table 2, the preparation method of the capture plate is:

[0138] S1, coating

[0139] AAV2 / 9-pAAV-CMV-EGFP-3xFLAG-WPRE (Heyuan Biotechnology, Cat. No.: AOV022) was diluted to a concentration of 2×10 10 vg / mL coating solution was evenly added to the ELISA plate at 100 μL / well, sealed with sealing film, and incubated at 2-8°C for 16-20 hours;

[0140] S2, Washing plate

[0141] Discard the coating solution in the ELISA plate and wash the plate twice (260 μL / well of washing solution, 30 to 60 seconds for each wash);

[0142] S3, closed

[0143] Add blocking solution evenly at 200 μL / well and block at 25-37°C for 1-4 hours;

[0144] S4. Drying

[0145] Discard the blocking solution in the ELISA plate, pat it dry on absorbent paper, and place the ELISA plate at 25-37℃ to dry for 1-8 hours;

[0146] S5. Packaging

[0147] 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.

[0148] The universal anti-AAV9 antibody ELISA kit of the present invention is used to detect anti-AAV9 antibodies in serum samples. The working principle of the kit is as follows: Figure 1 As shown, the specific steps are:

[0149] S1 Preparation of experimental reagents

[0150] Return all reagents to room temperature before testing;

[0151] If the concentrated reagent crystallizes, it needs to be warmed at 37°C until all the crystals are dissolved;

[0152] Dilute 10× washing solution to 1× washing solution with purified water (or distilled water, deionized water) (e.g., add 60 mL of 10× washing solution to 540 mL of purified water). Gently mix the 1× washing solution and set aside (1× washing solution can be stored in a refrigerator at 2-8°C for 2 weeks);

[0153] S2 Experimental Sample Preparation

[0154] Return all samples to room temperature before testing;

[0155] It is recommended to use fresh samples;

[0156] Dilute the serum sample to be tested at 1:25 (e.g. 12 μL serum + 288 μL sample diluent);

[0157] S3 Experimental Procedure

[0158] Prepare the reagents within 30 minutes before the experiment;

[0159] 15 minutes before the experiment, equilibrate all materials and prepared reagents to room temperature;

[0160] It is recommended that all quality controls and samples be repeated;

[0161] Remove the unneeded slats, put them back into the foil bag containing the desiccant, and seal it tightly;

[0162] Add 100 μL of quality control (positive quality control, critical value quality control, negative quality control) and sample to the corresponding wells. Seal the plate with a sealing film and incubate at 37°C for 1 hour;

[0163] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time);

[0164] Add 100 μL of the detection antigen solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 1 hour;

[0165] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time);

[0166] Add 100 μL of HRP labeling solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes;

[0167] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time);

[0168] Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 15 min;

[0169] Add 100 μL of stop solution to each well to terminate the reaction;

[0170] Complete OD in 15 minutes 450nm reading;

[0171] S4 absorbance calculation

[0172] Calculate the average absorbance of the quality control and sample duplicate wells;

[0173] Calculate the absorbance ratio of each sample to the cut-off control (CC).

[0174] S5 Result Determination

[0175] Experimental validity: positive control absorbance (OD 450nm ):>1.000,Cut-offControl absorbance (MeanOD 450nm ): 0.150~0.500, Negative Control absorbance (Mean OD 450nm ):<0.150;

[0176] Sample result determination: Sample results are determined in relative units (Units, U), sample OD 450nm / CC OD 450nm =U, the judgment basis is shown in Table 3.

[0177] Table 3 Sample test results

[0178] Critical value 1U Response levels that define samples as positive or negative results Positive ＞1.1U Anti-AAV9 antibodies were detected in the samples Suspicious results 0.9U~1.1U Retesting required Negative ＜0.9U No anti-AAV9 antibodies were detected in the samples

[0179] Note: To ensure the reliability of the test results, it is recommended to retest samples with suspicious results or use other methods (such as WB, etc.) for further verification.

[0180] Example 4 Kit Performance Verification

[0181] 1. Exclusivity

[0182] The kit described in Example 3 and the detection method described therein were used to detect PC, Assay Buffer, NC, 100 μg / mL Mouse IgG, 100 μg / mL Rat IgG, 100 μg / mL Hamster IgG, 100 μg / mL Rabbit IgG, 100 μg / mL BSA, 1 μg / mL AAV8 mAb, 1 μg / mL AAV6 mAb, 1 μg / mL AAV5 mAb, and CC, respectively. Each sample was tested 3 times, and the U value was calculated. See Table 4 for details.

[0183] Table 4 Kit specificity

[0184]

[0185] The test results showed that the PC test result was positive, and the test results of Assay Buffer, NC, 100μg / mLMouse IgG, 100μg / mL Rat IgG, 100μg / mL Hamster IgG, 100μg / mL Rabbit IgG, 100μg / mL BSA, 1μg / mL AAV8mAb, 1μg / mLAAV6 mAb, and 1μg / mLAAV5 mAb were all negative, and the specificity of the kit was verified.

[0186] 2. Durability

[0187] The kit described in Example 3 and the detection method described therein were used. The reaction time (+20% and -20%), reaction temperature (+20% and -20%), and reaction volume (+20% and -20%) were changed under standard experimental parameters. Assay Buffer, NC, CC, PC, and MPC (1 / 5PC) were tested respectively. Each sample was tested 4 times (MPC was tested 10 times). See Table 5 for details.

[0188] Table 5 Kit durability

[0189]

[0190]

[0191] The results showed that the Assay Buffer, OD 450nm <0.150; detect NC, OD 450nm <0.150; detection CC, 0.150<OD 450nm <0.500; detection PC, OD 450nm >1.000; detect MPC, OD 450nm The coefficient of variation (%CV) was less than 10%; the durability of the kit was verified.

[0192] 3. Precision

[0193] At least 3 independent analytical batches of MPC were determined within a few days using the kit and the detection method described in Example 3. Each batch of MPC was tested no less than 10 times, and the mean value, standard deviation (SD) and coefficient of variation (CV) of the absorbance of MPC for each batch and between batches were calculated, as shown in Table 6.

[0194] Table 6 Kit precision

[0195]

[0196] The test results showed that the intra-batch precision and inter-batch precision of the kit were both less than 10%, and the precision verification of the kit passed.

[0197] 4. Negative control compliance rate & positive control compliance rate

[0198] Using the kit described in Example 3 and the detection method described therein, 10 negative controls and 10 positive controls were taken and tested using the kit respectively, and the U value of each sample was calculated, as shown in Table 7.

[0199] Table 7 Kit Negative Control & Positive Control Compliance Rate

[0200]

[0201] The results showed that all 10 negative control test results were negative (negative control compliance rate was 100%), and all 10 positive control test results were positive (positive control compliance rate was 100%). The negative control compliance rate & positive control compliance rate of the kit were verified.

[0202] 5. Stability

[0203] The kit and the detection method described in Example 3 were used, the kit was stored in a 37°C environment, and quality inspections were performed at regular intervals to examine its stability under accelerated conditions, as shown in Table 8.

[0204] Table 8 Kit stability

[0205]

[0206]

[0207] The results showed that the accelerated stability of the kit at 37°C was no less than 7 days.

[0208] The above is a detailed introduction to a universal anti-AAV9 antibody ELISA kit and a method for detecting anti-AAV9 antibodies provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A universal anti-AAV9 antibody ELISA kit, comprising a capture plate, a positive quality control, a critical value quality control and a negative quality control, characterized in that: The capture plate is prepared by coating AAV9 and blocking with a blocking solution. The preparation method of the capture plate is: AAV9 was diluted to a concentration of 2×10 using 50 mM CBS solution with a pH of 9.6±0.2 as the coating solution. 10 vg / mL, add to the ELISA plate, seal with sealing film and incubate at 2-8℃ for 16-20 hours; Discard the coating solution in the ELISA plate and wash the plate; Add a blocking solution containing 5% to 6% by weight of skimmed milk powder, and block at 25 to 37° C. for 1 to 4 hours; Discard the blocking solution in the ELISA plate and place the plate at 25-37°C to dry for 1-4 hours; After drying, the coated ELISA plate is vacuum packed to become the capture plate of the test kit.

2. A universal anti-AAV9 antibody ELISA kit according to claim 1, characterized in that: Preparation of coating solution: Take 1.59g Na2CO3 and 2.94g NaHCO3 into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, make up to 1.00L with ultrapure water, and filter through a filter with a membrane pore size of 0.22μm; Preparation of blocking solution: Take 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, and 0.20g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, make up to 1.00L, and filter with a filter with a membrane pore size of 0.22μm to obtain a mixed solution; take 475mL of the mixed solution into a preparation container, add 25.00g skimmed milk powder to obtain a blocking solution.

3. A universal anti-AAV9 antibody ELISA kit according to claim 1, characterized in that: The universal anti-AAV9 antibody ELISA kit also includes 10× washing solution, sample diluent, detection antigen solution, HRP labeling solution, TMB substrate color development solution and stop solution.

4. A universal anti-AAV9 antibody ELISA kit according to claim 1, characterized in that: The negative quality control is a dilution of cynomolgus monkey AAV9 NAb negative mixed serum, beagle dog AAV9 NAb negative mixed serum, SD rat AAV9 NAb negative mixed serum, BALB / c mouse AAV9 NAb negative mixed serum, and hamster AAV9 NAb negative mixed serum; The critical value quality control is 20ng / mL AAV9 antibody prepared using negative quality control; The positive quality control is 500 ng / mL AAV9 antibody prepared using the negative quality control.

5. A universal anti-AAV9 antibody ELISA kit according to claim 1, characterized in that The components of this universal anti-AAV9 antibody ELISA kit are: Capture plate coated with AAV9 antigen, negative quality control containing 1 / 25 negative mixed serum, critical value quality control containing 20ng / mAAV9 antibody, positive quality control containing 500ng / mAAV9 antibody, 10× washing solution, sample diluent, detection antigen solution, HRP labeling solution, TMB substrate color development solution and stop solution.

6. A universal anti-AAV9 antibody ELISA kit according to claim 1, characterized in that The configuration method of each reagent in the kit is as follows: Preparation of 10× washing solution: weigh 11.36g Na2HPO4, 2.72g KH2PO4, 80.06g NaCl, 2.01g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 5.00mL Tween-20, 5.00mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter through a filter with a membrane pore size of 0.22μm; Preparation of sample diluent: weigh 2.27g Na2HPO4, 0.54g KH2PO4, 16.01g NaCl, 0.40g KCl, 40.00g sucrose, 20.00g BSA into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 1.00mL Tween-20, 1.00mL ProClin 300 after dissolution, stir to mix, make up to 2.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm; Preparation of detection antigen solution: Measure 300 mL of sample diluent into the preparation container, add 30 μL of biotin-labeled AAV9, and prepare a working solution with a concentration of 1.2 μg / mL; Preparation of HRP labeled solution: Measure 300 mL of sample dilution solution into the preparation container, add 30 μL of HRP labeled streptavidin to prepare a working solution with a concentration of 100 ng / mL; Preparation of stop solution: 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.

7. A universal anti-AAV9 antibody ELISA kit according to claim 1, characterized in that The preparation method of the biotin-labeled AAV9: Biotin-LC-LC-NHS can react with primary amino groups (NH2-) to form stable and irreversible amide bonds, thereby achieving the effect of labeling AAV9 containing primary amino groups (NH2-). 13 vgAAV9 was added with 6 μL of 10 mM Biotin-LC-LC-NHS test solution and labeled in an ultrafiltration tube to remove free biotin. The concentration of biotin-labeled protein was determined and an equal volume of label protection solution was added to obtain the result.

8. A method for detecting anti-AAV9 antibodies of a universal anti-AAV9 antibody ELISA kit according to claim 1, characterized in that The method comprises the following steps: S1 Preparation of experimental reagents Return all reagents to room temperature before testing; If the concentrated reagent crystallizes, it needs to be warmed at 37°C until all the crystals are dissolved; Dilute the 10× wash solution to 1× wash solution with water, gently mix the 1× wash solution and set aside; S2 Experimental Sample Preparation Before testing, all samples were returned to room temperature and the serum samples to be tested were diluted 1:25; S3 Experimental Procedure Add 100 μL of quality control (positive quality control, critical value quality control, negative quality control) and sample to the corresponding wells; seal the plate with a sealing film and incubate at 37°C for 1 hour; Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time); Add 100 μL of the detection antigen solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 1 hour; Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time); Add 100 μL of HRP labeling 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× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time); Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 15 min; Add 100 μL of stop solution to each well to terminate the reaction; Complete OD in 15 minutes 450nm reading; S4 absorbance calculation Calculate the average absorbance of the quality control and sample duplicate wells; Calculate the absorbance ratio of each sample to the critical value quality control (Cut-off Control, CC); S5 Result Determination Experimental validity: positive control absorbance (OD 450nm ):>1.000,Cut-offControl absorbance (Mean OD 450nm ): 0.150~0.500, Negative Control absorbance (Mean OD 450nm ):<0.150; Sample result determination: Sample results are determined in relative units (Units, U), sample OD 450nm / CC OD 450nm =U; When the sample test result is >1.1U, it is positive and anti-AAV9 antibodies are detected in the sample; When the sample test result is between 0.9U and 1.1U, it is a suspicious result and needs to be retested; When the sample test result is <0.9U, it is negative and no anti-AAV9 antibodies are detected in the sample.

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