A universal anti-AAV8 antibody ELISA kit and a method for detecting anti-AAV8 antibodies thereof

Abstract

The present invention discloses a universal anti-AAV8 antibody ELISA kit and a method for detecting anti-AAV8 antibodies thereof. The kit includes a capture plate and a detection solution. The capture plate is prepared by coating AAV8 and then blocking with a blocking solution; the detection solution is prepared by labeling AAV8 with biotin to obtain biotin-labeled AAV8, and then using a sample diluent. The kit of the present invention establishes a determination method suitable for rapid detection of anti-AAV8 antibody concentration in serum samples, which is not limited by the species of biological samples, and provides a simple and rapid detection tool for anti-AAV8 antibody detection. It has been verified that the kit of the present invention has good linearity, specificity, durability, precision, accuracy, detection limit, and is easy to operate and has a short detection cycle (single detection time is about 3 hours).

Description

Technical Field

[0001] The present invention belongs to the technical field of biological analysis and detection, and in particular relates to a universal anti-AAV8 antibody ELISA kit and a detection method of the anti-AAV8 antibody thereof, which are suitable for quantitatively detecting the concentration of the anti-AAV8 antibody in a sample. Background Art

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

[0003] Adeno-associated virus 8 (AAV8) was isolated from the heart of rhesus monkeys in 2002. Among various AAV vectors, AAV8 has attracted great attention because of its efficient and stable gene transfection in tissues such as the liver. Currently, recombinant AAV8 (rAAV8) has been widely used in gene therapy research for genetic diseases, tumors, autoimmune diseases and antiviral diseases.

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

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

[0006] The anti-AAV8 antibody ELISA kit is suitable for rapid detection of anti-AAV8 antibody concentration in serum samples. It is not restricted by the species of biological samples and provides a simple and rapid detection tool for the evaluation of anti-drug antibodies of gene therapy drugs based on AAV8 vectors. Summary of the invention

[0007] The purpose of the present invention is to provide a universal anti-AAV8 antibody ELISA kit and a method for detecting anti-AAV8 antibodies thereof, which is suitable for rapid detection of anti-AAV8 antibody concentration in serum samples, is not restricted by the species of biological samples, has good linearity, specificity, durability, precision, accuracy, detection limit, and is easy to operate and has a short detection cycle (single detection time is about 3 hours).

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

[0009] In a first aspect, the present invention provides a universal anti-AAV8 antibody ELISA kit, the kit comprising a capture plate and a detection solution,

[0010] The capture plate is prepared by coating with AAV8 and blocking with a blocking solution;

[0011] The detection solution is obtained by labeling AAV8 with biotin to obtain biotin-labeled AAV8, and then preparing the solution using a sample diluent.

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

[0013] S11, use 2# coating solution to dilute AAV8 to a concentration of 0.5×10 10 ~2×10 10 vg / mL solution was evenly added to the ELISA plate, sealed with sealing film and incubated at 2-8°C for 16-20 hours;

[0014] S12, discarding the 2# coating solution in the ELISA plate and washing the plate;

[0015] S13, add 3# blocking solution and block at 25-37°C for 1-4 hours;

[0016] S14, discarding the 3# blocking solution in the ELISA plate, and placing the ELISA plate at 25-37° C. to dry;

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

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

[0019] Biotin-LC-LC-NHS can react with primary amino groups to form stable, irreversible amide bonds, thereby achieving the effect of labeling AAV8 containing primary amino groups;

[0020] According to 1×10 13vgAAV8 was added with 6 μL of 10 mM Biotin-LC-LC-NHS solution, labeled in an ultrafiltration tube with a molecular weight cutoff of 100 kDa, free biotin was removed, the concentration of biotin marker protein was determined, and an equal volume of marker protection solution was added to obtain the biotin-labeled AAV8;

[0021] The biotin-labeled AAV8 is diluted to a working solution of 1.0-4.0 μg / mL using the sample diluent to obtain the detection solution.

[0022] In the above technical solution, the kit further includes a calibrator, a 10× washing solution, the sample diluent, an HRP labeling solution, a TMB substrate color developing solution and a stop solution.

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

[0024] In the above technical scheme, the preparation method of the 2# coating solution is: accurately weigh 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, and 0.20g KCl into a preparation container, add an 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;

[0025] The preparation method of the 3# blocking solution is as follows: weigh 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, 0.20g KCl, and 10.00g gelatin into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20 and 0.50mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter using a filter with a membrane pore size of 0.22μm.

[0026] In the above technical scheme, the preparation method of the 10× washing solution is: accurately weigh 11.36g Na2HPO4, 2.72g KH2PO4, 80.06g NaCl, and 2.01g KCl into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, add 5.00mL Tween-20 and 5.00mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm to obtain;

[0027] The preparation method of the calibrator is as follows: thawing the AAV8 monoclonal antibody at 2-8°C, returning to room temperature after thawing, oscillating and mixing, centrifuging instantaneously, and then using a sample diluent for gradient dilution to prepare Standard 1 of 16 ng / mL, using Standard 1 to prepare Standard 2 of 8 ng / mL, and so on, 2-fold gradient dilution to prepare Standard 7 of 0.25 ng / mL, and the sample diluent is Standard 8 of 0 ng / mL;

[0028] The HRP labeled solution is prepared by accurately measuring 300 mL of the sample diluent into a preparation container, adding 15 μL of HRP labeled streptavidin to prepare a working solution with a concentration of 50 ng / mL;

[0029] The preparation method of the stop solution is: accurately measure 270 mL of ultrapure water into a preparation container, add 30 mL of 10 mol / L hydrochloric acid solution to prepare a working solution with a concentration of 1 mol / L, and filter it using a filter with a membrane pore size of 0.22 μm.

[0030] In a second aspect, the present invention provides a method for detecting anti-AAV8 antibodies, and the detection method is performed using the above-mentioned kit.

[0031] In the above technical solution, the detection method comprises the following steps:

[0032] S21, return all reagents to room temperature and dilute 10× wash solution to 1× wash solution;

[0033] S22, return all samples to room temperature;

[0034] S23, experimental steps:

[0035] Add 100 μL of the standard and the sample to be tested to the corresponding wells of the kit, seal the plate with a sealing film, and incubate at room temperature for 30 to 90 minutes;

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

[0037] Add 100 μL of detection solution to each well, seal the plate with a new sealing film, and incubate at room temperature for 30 to 90 minutes;

[0038] Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper;

[0039] Add 100 μL of HRP labeling solution to each well, seal the plate with a new sealing film, and incubate at room temperature for 30 to 90 minutes;

[0040] Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper;

[0041] Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 5-25 minutes;

[0042] Add 100 μL of stop solution to each well to terminate the reaction;

[0043] Complete OD in 15 minutes 450nm reading;

[0044] S24, absorbance calculation:

[0045] Calculate the average absorbance of the calibrator and sample duplicate wells;

[0046] With the concentration of the calibrator as the horizontal axis and the OD value as the vertical axis, the calibration curve was generated by regression fitting using relevant software;

[0047] The mean absorbance value corresponding to the sample was inserted into the calibration curve, and the anti-AAV8 antibody concentration was calculated.

[0048] The beneficial effects of the present invention are:

[0049] 1. The present invention prepares a capture plate coated with AAV8 antigen, so that the capture plate can specifically recognize anti-AAV8 antibodies without interference from foreign proteins.

[0050] 2. The kit of the present invention can specifically identify anti-AAV8 antibodies by using a capture plate coated with AAV8 antigen and biotin-labeled AAV8 in combination.

[0051] 3. The kit of the present invention is not limited by the species of biological samples and can be used to evaluate the concentration of anti-AAV8 antibodies in various serum samples (such as humans, monkeys, dogs, and mice), providing a simple and rapid detection tool for the evaluation of anti-drug antibodies of gene therapy drugs based on AAV8 vectors.

[0052] 4. The kit of the present invention establishes a detection method suitable for rapid detection of anti-AAV8 antibody concentration in serum samples, which is not limited by the species of biological samples and provides a simple and rapid detection tool for the evaluation of anti-drug antibodies of gene therapy drugs based on AAV8 vectors. It has been verified that the kit of the present invention has good linearity, specificity, durability, precision, accuracy, detection limit, simple operation, short detection cycle (single detection time is about 3 hours), etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is the working principle diagram of the kit of the present invention;

[0054] Figure 2 The results of the positive control antibody optimization experiment in Example 2;

[0055] Figure 3 The results of the AAV8 coating concentration optimization experiment in Example 2;

[0056] Figure 4 The results of the concentration optimization experiment for biotin-labeled AAV8 in Example 2. DETAILED DESCRIPTION

[0057] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0058] 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.

[0059] Unless otherwise specified, the reagents used in the following examples can be obtained from commercial sources.

[0060] Ultra-micro-volume UV-visible spectrophotometer, Hangzhou Miou Instrument Co., Ltd., item number ND-100F.

[0061] The following solutions were prepared as follows:

[0062] 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.

[0063] 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.

[0064] Preparation of 1# blocking solution: weigh 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, 0.20g KCl, 50.00g skim milk powder into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20, 0.50mL ProClin 300 after dissolution, stir to mix, dilute to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stably stored at 2-8℃ for 2 months.

[0065] 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 stably stored at 2-8℃ for 1 year.

[0066] 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.

[0067] Preparation of sample diluent: weigh 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, 0.20g KCl, 20.00g sucrose, 10.00g BSA into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20, 0.50mL ProClin 300 after dissolution, stir to mix, 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.

[0068] 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 stably stored at 2-8℃ for 2 years.

[0069] Preparation of detection solution: Accurately measure 300 mL of sample dilution into the preparation container, add a certain volume of biotin-labeled AAV8 (Hengyu Biotechnology, homemade), and prepare a working solution with a concentration of 0.1-4 μg / mL. The solution can be stably stored at 2-8°C for 1 year.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] AAV8: AAV2 / 8-pAAV-CMV-MCS-3xFLAG-WPRE, Heyuan Biotech (Shanghai) Co., Ltd., catalog number AOV021;

[0074] Ab1: AAV8 monoclonal antibody (mouse, IgG), Beijing Subenyuanhe Biotechnology Co., Ltd., catalog number M100037;

[0075] Ab2: Mouse anti-AAV8 antibody, mAb 13C2, Beijing Tiancheng Biotechnology Co., Ltd., catalog number 05-0234-S;

[0076] Ab3: Mouse anti-AAV8 antibody, mAb 13D1, Beijing Tiancheng Biotechnology Co., Ltd., catalog number 05-0235-S;

[0077] Ab4: Anti-AAV8 (intact particle) monoclonal antibody, clone BEL9, Creative Diagnostics, product number CABT-B9065.

[0078] The preparation method of the capture plate in the following examples is:

[0079] S1, coating

[0080] The AAV8 antigen was diluted to a concentration of 0.5×10 10 ~4.0×10 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;

[0081] S2, Washing plate

[0082] 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);

[0083] S3, closed

[0084] Add blocking solution evenly at 200 μL / well and block at 25-37°C for 1-4 hours;

[0085] S4. Drying

[0086] 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;

[0087] S5. Packaging

[0088] 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.

[0089] Example 1 AAV8 biotin labeling

[0090] Biotin-LC-LC-NHS can react with primary amino groups (NH2-) to form stable, irreversible amide bonds, thereby achieving the effect of labeling AAV8 antigens containing primary amino groups (NH2-).

[0091] 1. Preparation of biotin labeling solution

[0092] 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 6 months (store in aliquots and use a new one each time).

[0093] 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 stably stored at 2-8℃ for 1 year.

[0094] 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.

[0095] 2. Calculation of biotin dosage

[0096] According to 1×10 13 vgAAV8 was added with 6 μL of 10 mM Biotin-LC-LC-NHS solution, and the amount of AAV8 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.

[0097] 3. Biotin labeling

[0098] Transfer the substance to be labeled to an ultrafiltration tube with a molecular weight cutoff of 100 kDa, add labeling buffer (10 mM PBS, pH 7.2 ± 0.2) not exceeding the maximum volume of the ultrafiltration tube, and centrifuge at 12000 × g for 10 minutes (this step can be repeated 2 to 3 times). After the last ultrafiltration, add an appropriate amount of labeling buffer to adjust the concentration of the substance to 2 × 10 13 About vg / mL.

[0099] Add an appropriate volume of Biotin-LC-LC-NHS solution to the above ultrafiltration tube and gently pipette to mix. Incubate in a 37°C incubator in the dark for 30 minutes or incubate in the dark at room temperature for 1 hour.

[0100] 4. Removal of free biotin

[0101] The labeled biotin marker was placed in a centrifuge and centrifuged at 12000×g for 10 minutes at room temperature.

[0102] Add an appropriate amount of labeling buffer to the above ultrafiltration tube, gently pipette to mix, centrifuge at 12000×g for 10 minutes at room temperature, and repeat this step 3 times.

[0103] Add an appropriate amount of labeling buffer to the ultrafiltration tube again, mix gently by pipetting, and centrifuge at 4000×g for 5 minutes.

[0104] The ultrafiltration tube membrane was placed upside down in the collection tube and centrifuged at 12000×g for 2 minutes at room temperature.

[0105] 5. Determination of biotin-labeled protein concentration

[0106] 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).

[0107] 6. Storage of biotin markers

[0108] 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.

[0109] Example 2 Establishment of a bridge ELISA detection method based on the biotin-streptavidin system

[0110] 1. Operation steps of the bridge ELISA detection method based on the biotin-streptavidin system

[0111] 1) Sample addition: Add 100 μL of calibrators (Standard 1 to Standard 8) and samples to be tested (the minimum dilution of the sample is 10 times) to the corresponding wells, seal the plate with a sealing film, and incubate at room temperature 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).

[0112] 2) Add detection solution (biotin-labeled AAV8 solution): Add 100 μL of detection solution of appropriate concentration to each well, seal the plate with a new sealing film, and incubate at room temperature or 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).

[0113] 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 room temperature or 37°C for 15 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).

[0114] 4) Color development: Add 100 μL TMB substrate color development solution to each well, protect from light, and incubate at room temperature for 15 minutes.

[0115] 5) Termination of reaction: Add 100 μL of stop solution to each well to terminate the reaction.

[0116] 6) Reading: Complete OD within 15 minutes 450nm reading.

[0117] 2. Optimization of various conditions in the establishment of the bridge ELISA detection method based on the biotin-streptavidin system

[0118] 1) Optimization of ELISA plate, coating solution and blocking solution

[0119] According to Table 1, an orthogonal test (12 different conditions) was set up to preliminarily screen suitable ELISA plates (Jie Te Bio, Catalog No.: FEP101896; Corning, Catalog No.: 42592), coating solutions (1# coating solution; 2# coating solution) and blocking solutions (1# blocking solution; 2# blocking solution; 3# blocking solution). The coating concentration was set to 2×10 10 vg / mL, the positive control was set at 8 concentrations (729ng / mL, 243ng / mL, 81ng / mL, 27ng / mL, 9ng / mL, 3ng / mL, 1ng / mL, 0ng / mL), the detection solution (biotin-labeled AAV8) concentration was set to 0.5μg / mL, and the HRP labeling solution concentration was set to 50ng / mL.

[0120] The signal-to-noise ratio (S / N) results at each concentration level showed that there was no significant difference in the reaction results between ELISA plates from different manufacturers and different coating solutions; the order of blocking solution use effect was 3# blocking solution > 2# blocking solution > 1# blocking solution, and 3# blocking solution was selected for subsequent optimization experiments.

[0121] Table 1 Results of orthogonal test for optimization of ELISA plate, coating solution and blocking solution

[0122]

[0123] 2) Positive control antibody optimization

[0124] AAV8 (AAV2 / 8-pAAV-CMV-MCS-3xFLAG-WPRE, Heyuan Biotechnology (Shanghai) Co., Ltd., Catalog No. AOV021) was prepared with 2# coating solution to a concentration of 2×10 10 vg / mL solution, blocked with 3# blocking solution, and prepared into capture plate. Four positive control antibodies [Ab1: AAV8 monoclonal antibody (mouse, IgG), Beijing Subenyuanhe Biotechnology Co., Ltd., catalog number M100037; Ab2: Mouse anti-AAV8 antibody, mAb 13C2, Beijing Tiancheng Biotechnology (Beijing) Co., Ltd., catalog number 05-0234-S; Ab3: Mouse anti-AAV8 antibody, mAb 13D1, Beijing Tiancheng Biotechnology (Beijing) Co., Ltd., catalog number 05-0235-S; Ab4: Anti-AAV8 (intact particle) monoclonal antibody, clone BEL9, Creative Biotechnology (Beijing) Co., Ltd., catalog number 05-0236-S; Ab5: Anti-AAV8 (intact particle) monoclonal antibody, clone BEL9, Creative Biotechnology (Beijing) Co., Ltd., catalog number 05-0237-S; Ab6: Anti-AAV8 (intact particle) monoclonal antibody, clone BEL9, Creative Biotechnology (Beijing) Co., Ltd., catalog number 05-0236-S; Ab7: Anti-AAV8 (intact particle) monoclonal antibody, clone BEL9, Creative Biotechnology (Beijing) Co., Ltd., catalog number 05-0236-S; Ab8: Anti-AAV8 (intact particle) monoclonal antibody, clone BEL9, Creative Biotechnology (Beijing) Co., Ltd., catalog number 05-0236-S; Ab9: Anti-AAV8 (intact particle) monoclonal antibody, clone BEL9, Creative Biotechnology (Beijing) Co., Ltd., catalog number 05-0236-S; Ab10: Anti-AAV8 (mouse, IgG), Beijing Subenyuanhe Biotechnology Co., Ltd., catalog number M100037; Ab20: Mouse anti-AAV8 antibody Diagnostics, catalog number CABT-B9065] were prepared into 8 concentrations (729 ng / mL, 243 ng / mL, 81 ng / mL, 27 ng / mL, 9 ng / mL, 3 ng / mL, 1 ng / mL, 0 ng / mL), the detection solution (biotin-labeled AAV8) concentration was set to 0.5 μg / mL, and the HRP labeling solution concentration was set to 50 ng / mL.

[0125] Test results such as Figure 2 As shown, better reaction signals can be obtained using Ab1 and Ab4, among which Ab1 is more sensitive. However, under the current conditions, there is a hook effect in high-concentration samples.

[0126] 3) Optimization of AAV8 coating concentration

[0127] AAV8 (AAV2 / 8-pAAV-CMV-MCS-3xFLAG-WPRE, Heyuan Biotechnology (Shanghai) Co., Ltd., Catalog No. AOV021) was prepared with 2# coating solution to a concentration of 8×10 10 vg / mL, 2×10 10 vg / mL, 0.5×10 10 vg / mL solution, blocked with 3# blocking solution, and prepared into capture plate. The concentration of detection solution (biotin-labeled AAV8) was set to 0.5 μg / mL, and the concentration of HRP labeling solution was set to 50 ng / mL.

[0128] Test results such as Figure 3 As shown in the figure, different coating concentrations have little effect on the reaction signal, and under different coating concentration conditions, high concentration samples all have a hook effect.

[0129] 4) Optimization of the concentration of biotin-labeled AAV8

[0130] AAV8 (AAV2 / 8-pAAV-CMV-MCS-3xFLAG-WPRE, Heyuan Biotechnology (Shanghai) Co., Ltd., Catalog No. AOV021) was prepared with 2# coating solution to a concentration of 2×10 10 vg / mL solution, blocked with 3# blocking solution, and prepared into capture plate. The concentration of detection solution (biotin-labeled AAV8) was set to 0.5μg / mL, 1.0μg / mL, 2.0μg / mL, 4.0μg / mL, and the concentration of HRP labeling solution was set to 50ng / mL.

[0131] Test results such as Figure 4 As shown, as the concentration of the detection solution (biotin-labeled AAV8) increases, the hook effect of high-concentration samples is gradually eliminated.

[0132] Example 3 Universal anti-AAV8 antibody ELISA kit

[0133] like Figure 1 The figure shows the working principle of the kit of the present invention. The kit of the present invention is suitable for rapid detection of anti-AAV8 antibody concentration 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-AAV8 antibody ELISA kit

[0135]

[0136] The method for detecting anti-AAV8 antibodies in a sample using the universal anti-AAV8 antibody ELISA kit of the present invention comprises the following specific steps:

[0137] S1. Preparation of experimental reagents

[0138] Return all reagents to room temperature before testing;

[0139] If the concentrated reagent crystallizes, it needs to be warmed at 37°C until all the crystals are dissolved;

[0140] 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);

[0141] S2. Preparation of experimental samples

[0142] Return all samples to room temperature before testing;

[0143] It is recommended to use fresh samples;

[0144] Dilute the serum sample to be tested at a dilution factor of no less than 1:10 (no more than 1:100) (e.g. 25 μL serum + 225 μL sample diluent);

[0145] S3. Experimental steps

[0146] Prepare the reagents within 30 minutes before the experiment;

[0147] 15 minutes before the experiment, equilibrate all materials and prepared reagents to room temperature;

[0148] It is recommended that all calibrators and samples to be tested (including but not limited to serially diluted samples and quality control products) be replicated;

[0149] Remove the unneeded slats, put them back into the foil bag containing the desiccant, and seal it tightly;

[0150] Add 100 μL of standard (Standard 1 to Standard 8) and the sample to be tested to the corresponding wells, seal the plate with a sealing film, and incubate at room temperature for 1 hour;

[0151] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 4 times (pat dry on absorbent paper each time the plate is washed);

[0152] Add 100 μL of detection solution to each well, seal the plate with a new sealing film, and incubate at room temperature for 1 hour;

[0153] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 4 times (pat dry on absorbent paper each time the plate is washed);

[0154] Add 100 μL of HRP labeling solution to each well, seal the plate with a new sealing film, and incubate at room temperature for 30 minutes;

[0155] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 4 times (pat dry on absorbent paper each time the plate is washed);

[0156] Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 15 minutes;

[0157] Add 100 μL of stop solution to each well to terminate the reaction;

[0158] Complete OD in 15 minutes 450nm reading;

[0159] S4. Absorbance calculation

[0160] Calculate the average absorbance of the calibrator and sample duplicate wells;

[0161] With the concentration of the calibrator as the horizontal axis and the OD value as the vertical axis, use relevant software for regression fitting to generate a calibration curve (it is recommended to use a four-parameter logistic equation);

[0162] Substitute the mean absorbance value corresponding to the sample into the calibration curve and back-calculate the corresponding concentration.

[0163] S5. Result determination

[0164] Experimental Validity: Calibration Curve: R 2 >0.99; Standard 1 absorbance (Mean OD 450nm ):>1.000;Standard 8 absorbance (Mean OD 450nm ): <0.100; the precision (coefficient of variation, CV) of the calibration standard back-calculated concentration is <20% (LLOQ <25%), and the accuracy (relative error, RE) is <±20% (LLOQ <±25%);

[0165] Calculation of sample results: Substitute the mean absorbance of the sample into the calibration curve, and the result is the anti-AAV8 antibody concentration of the diluted sample (if the mean absorbance of the sample is higher than the mean absorbance of Standard 1, the sample needs to be diluted and retested). The result multiplied by the dilution factor is the anti-AAV8 antibody concentration in the original sample.

[0166] Example 4 Kit Performance Verification

[0167] 1. Linearity & Range

[0168] The kit and detection method of Example 3 were used to measure 6 independent analysis batches on different days, and the coefficient of variation (CV) and relative error (RE) of the back-calculated concentration of the calibration standard were calculated. The results were reported in a tabular form to determine the overall robustness of the calibration curve regression model, as shown in Table 3.

[0169] Table 3 Kit linearity & range

[0170]

[0171]

[0172] The test results show that the linearity is: within the quantitative range, the calibration curve R 2 >0.99; Range: The concentration interval between the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) is the quantitative range of the calibration curve. Within this interval, the precision of the back-calculated concentration of the calibration standard is <20%, and the accuracy is <±20%. The linearity & range verification of the kit has been passed.

[0173] 2. Exclusivity

[0174] The kit and detection method of Example 3 were used to detect BSA, AAV9mAb, AAV6mAb, AAV5 mAb, Human IgG, Mouse IgG, Rat IgG, Hamster IgG, and Rabbit IgG at a concentration of 1 μg / mL, respectively. Each sample was tested twice, and the back-calculated concentration of each sample was calculated according to the calibration curve, as shown in Table 4.

[0175] Table 4 Kit specificity

[0176]

[0177]

[0178] The test results showed that the detection results of 1μg / mL BSA, AAV9 mAb, AAV6 mAb, AAV5 mAb, Human IgG, Mouse IgG, Rat IgG, Hamster IgG, and Rabbit IgG were all below the quantitative limit, and the specificity of the kit was verified.

[0179] 3. Durability

[0180] The kit and the detection method of Example 3 were used, and the reaction time (+20% and -20%), reaction temperature (+20% and -20%), and reaction volume (+20% and -20%) were changed under standard experimental parameters, and 8 calibration standards (Standard 1→Standard 8) were detected respectively, and each sample was tested twice. See Table 5 for details.

[0181] Table 5 Kit durability

[0182]

[0183]

[0184] The results showed that when there were small changes in the experimental parameters, the experiment was still valid, meeting the experimental validity standards, and the durability of the kit was verified.

[0185] 4. Precision

[0186] Using the kit and detection method of Example 3, at least 3 independent analysis batches of quality control samples (HQC, MQC, LQC) were determined within a few days. Each quality control sample in each batch was tested no less than 10 times. The mean (Mean), standard deviation (SD), and coefficient of variation (CV) of the concentration of quality control samples in each batch and between batches were calculated, as shown in Table 6.

[0187] Table 6 Kit precision

[0188]

[0189]

[0190] 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.

[0191] 5. Accuracy

[0192] Using the kit and the detection method of Example 3, at least 3 independent analysis batches of quality control samples (HQC, MQC, LQC) were determined within a few days. Each quality control sample in each batch was tested no less than 10 times, and the mean (Mean), standard deviation (SD), and relative error (RE) of the concentration of quality control samples in each batch and between batches were calculated, as shown in Table 7.

[0193] Table 7 Kit Accuracy

[0194]

[0195]

[0196] The results showed that the intra-batch accuracy and inter-batch accuracy of the kit were both less than 20%, and the accuracy of the kit passed the verification.

[0197] 6. Detection limit

[0198] Detect the blank calibration standard (Standard 8, repeated no less than 20 times), calculate the absorbance mean (Mean) and standard deviation (SD), substitute the absorbance corresponding to Mean+2SD into the calibration curve (or substitute the blank calibration standard and the lower limit of quantitation for a linear equation obtained by two-point regression fitting), and convert the corresponding concentration to the detection limit (limit of detection, LOD) of the kit; at least 3 independent analytical batches should be tested for the detection limit investigation, see Table 8 for details.

[0199] Table 8 Detection limit of the kit

[0200]

[0201] The results showed that the detection limit of the kit was <0.125ng / mL.

[0202] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A universal anti-AAV8 antibody ELISA kit, comprising a capture plate and a detection solution, characterized in that: The capture plate is prepared by coating with AAV8 and blocking with a blocking solution; The detection solution is obtained by labeling AAV8 with biotin to obtain biotin-labeled AAV8, and then preparing the solution using a sample diluent.

2. The kit according to claim 1, characterized in that: The method for preparing the capture plate comprises the following steps: S11, use 2# coating solution to dilute AAV8 to a concentration of 0.5×10 10 ~2×10 10 vg / mL solution was evenly added to the ELISA plate, sealed with sealing film and incubated at 2-8°C for 16-20 hours; S12, discarding the 2# coating solution in the ELISA plate and washing the plate; S13, add 3# blocking solution and block at 25-37°C for 1-4 hours; S14, discarding the 3# blocking solution in the ELISA plate, and placing the ELISA plate at 25-37° C. to dry; S15, after drying is completed, the coated ELISA plate is vacuum packed to obtain the capture plate.

3. The kit according to claim 1, characterized in that: The preparation method of the detection solution is: Biotin-LC-LC-NHS can react with primary amino groups to form stable, irreversible amide bonds, thereby achieving the effect of labeling AAV8 containing primary amino groups; According to 1×10 13 vgAAV8 was added with 6 μL of 10 mM Biotin-LC-LC-NHS solution, labeled in an ultrafiltration tube with a molecular weight cutoff of 100 kDa, free biotin was removed, the concentration of biotin marker protein was determined, and an equal volume of marker protection solution was added to obtain the biotin-labeled AAV8; The biotin-labeled AAV8 is diluted to a working solution of 1.0 to 4.0 μg / mL using the sample diluent to obtain the detection solution.

4. The kit according to claim 1, characterized in that: The kit also includes a calibrator, a 10× washing solution, the sample diluent, an HRP labeling solution, a TMB substrate color developing solution and a stop solution.

5. The kit according to claim 1, 3 or 4, characterized in that: The preparation method of the sample diluent is as follows: accurately weigh 2.27g Na2HPO4, 0.54g KH2PO4, 16.01g NaCl, 0.40g KCl, 40.00g sucrose, and 20.00g BSA into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, add 1.00mL Tween-20 and 1.00mL ProClin 300 after dissolution, stir to mix, make up to 2.00L with ultrapure water, and filter using a filter with a membrane pore size of 0.22μm.

6. The kit according to claim 2, characterized in that: The preparation method of the 2# coating solution is as follows: accurately weigh 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, and 0.20g KCl into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, dilute to 1.00L, and filter using a filter with a membrane pore size of 0.22μm to obtain; The preparation method of the 3# blocking solution is as follows: weigh 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, 0.20g KCl, and 10.00g gelatin into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20 and 0.50mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter using a filter with a membrane pore size of 0.22μm.

7. The kit according to claim 4, characterized in that: The preparation method of the 10× washing solution is as follows: accurately weigh 11.36 g Na2HPO4, 2.72 g KH2PO4, 80.06 g NaCl, and 2.01 g KCl into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, add 5.00 mL Tween-20 and 5.00 mL ProClin 300 after dissolution, stir to mix, make up to 1.00 L with ultrapure water, and filter through a filter with a membrane pore size of 0.22 μm to obtain the solution; The preparation method of the calibrator is as follows: thawing the AAV8 monoclonal antibody at 2-8°C, returning to room temperature after thawing, oscillating and mixing, centrifuging instantaneously, and then using a sample diluent for gradient dilution to prepare Standard 1 of 16 ng / mL, using Standard 1 to prepare Standard 2 of 8 ng / mL, and so on, 2-fold gradient dilution to prepare Standard 7 of 0.25 ng / mL, and the sample diluent is Standard 8 of 0 ng / mL; The preparation method of the HRP labeled solution is as follows: accurately measure 300 mL of the sample diluent into a preparation container, add 15 μL of HRP labeled streptavidin, and prepare a working solution with a concentration of 50 ng / mL; The preparation method of the stop solution is: accurately measure 270 mL of ultrapure water into a preparation container, add 30 mL of 10 mol / L hydrochloric acid solution to prepare a working solution with a concentration of 1 mol / L, and filter it using a filter with a membrane pore size of 0.22 μm.

8. A method for detecting anti-AAV8 antibodies, characterized in that: The detection method is carried out using the kit according to any one of claims 1 to 7.

9. The detection method according to claim 8, characterized in that: The detection method comprises the following steps: S21, return all reagents to room temperature and dilute 10× wash solution to 1× wash solution; S22, return all samples to room temperature; S23, experimental steps: Add 100 μL of the standard and the sample to be tested to the corresponding wells of the kit, seal the plate with a sealing film, and incubate at room temperature for 30 to 90 minutes; Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper; Add 100 μL of detection solution to each well, seal the plate with a new sealing film, and incubate at room temperature for 30 to 90 minutes; Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper; Add 100 μL of HRP labeling solution to each well, seal the plate with a new sealing film, and incubate at room temperature for 30 to 90 minutes; Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper; Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 5-25 minutes; Add 100 μL of stop solution to each well to terminate the reaction; Complete OD in 15 minutes 450nm reading; S24, absorbance calculation: Calculate the average absorbance of the calibrator and sample duplicate wells; With the concentration of the calibrator as the horizontal axis and the OD value as the vertical axis, the calibration curve was generated by regression fitting using relevant software; The mean absorbance value corresponding to the sample was inserted into the calibration curve, and the anti-AAV8 antibody concentration was calculated.

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