ELISA detection method for biological distribution of AAV through AMD gene therapy

Abstract

The invention relates to the technical field of gene therapy, and discloses an AMD gene therapy AAV biological distribution ELISA detection method, which comprises the following steps: pre-coating an ELISA plate with a capture antibody (18B2G9) to capture the antibody, adding a sample, incubating to specifically bind a substance to be detected with the capture antibody, washing off uncombined substances, adding excessive VEGF to bind with the substance to be detected, washing off uncombined VEGF, and detecting the AAV biological distribution by using the ELISA plate. According to the method, Biotin-labeled Anti-VEGF is combined with VEGF to form a bridging compound, SA-HRP is added to be combined with biotin on the bridging compound, TMB is added for color development, a reaction is terminated through sulfuric acid, and a light absorption value of a reaction hole is read through a microplate reader. The detection method is high in sensitivity, good in specificity, accurate and reliable, and is used for preclinical biological sample detection of gene therapy products.

Description

Technical Field

[0001] The present invention relates to the technical field of gene therapy, and in particular to an ELISA detection method for AAV biodistribution in AMD gene therapy. Background Art

[0002] The prevalence of AMD in people over 70 years old in my country is 20.2%. As the aging of the Chinese population intensifies, the number of patients with AMD is also rising. AMD is the main cause of vision loss. Neovascular AMD, also known as exudative or wet AMD, accounts for 10%-20% of AMD. Wet age-related maculopathy is characterized by the growth of abnormal blood vessels, whose fluid infiltrates into the central part of the retina called the macula, causing vision loss.

[0003] At present, in addition to laser photocoagulation and PDT, the mainstream drug treatment is various anti-VEGF drugs. At present, the drugs approved for clinical use in my country include: ranibizumab, conbercept and aflibercept, all of which are drugs against vascular endothelial growth factor (VEGF) or its fragments. During treatment, the drug is directly injected into the fundus to bind and resist VEGF to reduce fluid in the macula and improve vision.

[0004] However, the chemical nature of this type of drug is a protein molecule, which is unstable in the human body and easily broken down by various enzymes. It degrades in the eye within one month. Therefore, wAMD patients usually need to be given intraocular medication once every four to eight weeks to avoid vision loss. Such frequent intraocular injections are a great challenge to medication compliance. In addition, the frequency of use of such drugs results in high treatment costs. Many patients give up or cannot tolerate treatment, resulting in blindness in the later stage. Therefore, in this treatment field, the development of safer, more effective, durable and highly accessible drugs remains an urgent clinical need.

[0005] In vivo gene therapy generally uses appropriate vectors to introduce exogenous genes into the human body by transfection or uses gene editing tools to modify disabled target genes, thereby achieving the purpose of treating the disease by replacing, compensating, blocking, and correcting specific genes.

[0006] The biodistribution of gene therapy is a key part of nonclinical studies. It refers to the distribution, persistence, and clearance of the test substance in the body, including the administration site, target tissues, and non-target tissues (such as blood, cerebrospinal fluid, and vitreous humor) in relevant animal species. It helps to explain the findings of pharmacological and toxicological studies and also provides support information for clinical trial design. Summary of the invention

[0007] 1. Technical problems solved:

[0008] In view of the deficiencies of the prior art, the present invention provides a detection method with high sensitivity, good specificity, accuracy and reliability for preclinical biological sample detection of gene therapy products.

[0009] (II) Technical solution:

[0010] (1) Add 18B2G9 antibody at a concentration of 1837 μg / mL into DPBS to prepare a coating working solution of 1.8-2.2 μg / mL. Take 90-110 μL / well of the coating working solution and add it to the ELISA plate, cover it with film, and let it stand at 2-8°C for 12-16 hours. After standing, discard the coating solution and wash the plate. Add 180-220 μL / well of protein-free rapid blocking solution to the washed ELISA plate, and place it on a shaker at 500-600 rpm / min at room temperature for blocking for 1-3 hours to obtain an ELISA plate with captured antibodies.

[0011] (2) Dilute the standard curve sample, quality control sample and the sample to be tested by 1:(19-21) times with 5% BSA-1×PBST-diluent in a dilution plate, mix them evenly, and obtain the diluted sample. Wash the diluted sample, and then add the treated sample to the capture antibody ELISA plate at 90-110 μL / well for incubation. After the sample incubation, wash the plate to obtain the ELISA plate of the sample to be tested with specific binding of the sample and the capture antibody.

[0012] (3) 90-110 μL / well of VEGF working solution is added to the ELISA plate of the object to be tested, and the plate is incubated. After incubation, the plate is washed to obtain an ELISA plate in which VEGF and the object to be tested are bound.

[0013] (4) Add 90-110 μL / well of Biotin-labeled Anti-VEGF to the ELISA plate where VEGF and the substance to be tested are bound, and incubate. After incubation, wash the plate to obtain a bridging complex.

[0014] (5) Add 90-110 μL / well of the diluted SA-HRP working solution to the bridging complex, cover the plate, incubate the plate at 10-30°C in a shaker at 500-600 rpm / min for 55-65 min, wash the plate after incubation, add 90-110 μL / well of TMB colorimetric solution, cover the plate, incubate the plate at 10-30°C in the dark for 5-15 min, and terminate the reaction by adding 100 μL / well of 0.4-0.6 mol / L H2SO4 stop solution.

[0015] Preferably, the plate washing process in steps (1)-(5) is: washing 3-4 times with 0.05% PBST washing solution, 280-320 μL / well, and patting dry.

[0016] Preferably, the incubation process in steps (2)-(4) is: incubating for 55-65 minutes at room temperature in a shaking incubator at 500-600 rpm / min.

[0017] Preferably, in step (3), the preparation process of the VEGF working solution is: adding human / crab VEGF, VEGFA, VEGF165 proteins with a concentration of 0.25 mg / mL to 5% BSA-1×PBST-diluent to prepare a VEGF working solution with a concentration of 240-260 ng / mL.

[0018] Preferably, in step (4), the process of biotin-labeled anti-VEGF is: adding anti-hVEGF with a concentration of 0.20 mg / mL into 5% BSA-1×PBST-diluent to prepare 60-65 ng / mL biotin-labeled anti-VEGF.

[0019] Preferably, in step (5), the dilution process of the SA-HRP working solution is: SA-HRP is added to 5% BSA-1×PBST-diluent to prepare a 1:(38000-42000) times diluted SA-HRP working solution.

[0020] (III) Beneficial technical effects: The ELISA plate of the present invention is pre-coated with a capture antibody (18B2G9), a sample is added for incubation to allow the analyte to specifically bind to the capture antibody, unbound substances are washed away, excess VEGF is added to bind to the analyte, unbound VEGF is washed away, and Anti-VEGF labeled with Biotin binds to VEGF to form a bridging complex, SA-HRP is added to bind to the biotin on the bridging complex, and finally TMB is added for color development. After the reaction is terminated with sulfuric acid, the absorbance of the reaction well is read using an ELISA reader.

[0021] The present invention uses a series of optimization steps, such as precise allocation of the concentration of each working solution and specific incubation, washing and other operating procedures, so that the detection method can detect a lower lower limit, can more accurately detect the protein concentration in the sample, and can effectively detect it even when the protein concentration is low; using the specific binding principle of enzyme-linked immunosorbent assay, first the capture antibody (18B2G9) is specifically bound to the analyte, and then a series of specific bindings such as VEGF and Biotin-labeled Anti-VEGF are used to form a bridging complex, etc., effectively avoiding the interference of non-specific binding, and accurately detecting the target protein, the blank background in the optimized cynomolgus monkey serum is reduced (the blank OD is about 0.07), the standard curve is linear, the fitting result is good, and the random quality control is passed. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a measurement table of target protein in organ tissues. DETAILED DESCRIPTION

[0023] Embodiment 1:

[0024] (1) Add 18B2G9 antibody at a concentration of 1837 μg / mL into DPBS to prepare a coating working solution of 2 μg / mL. Take 100 μL / well of the coating working solution and add it to the ELISA plate, cover it, and let it stand at 8°C for 16 h. After standing, discard the coating solution and wash it three times with 0.05% PBST washing solution, 300 μL / well, pat dry, add 200 μL / well of protein-free rapid blocking solution to the washed ELISA plate, and block it on a shaker at 600 rpm / min for 3 h at room temperature to obtain an ELISA plate with captured antibodies.

[0025] (2) The standard curve samples, quality control samples and samples to be tested were diluted 1:20 with 5% BSA-1×PBST-diluent in a dilution plate, mixed evenly, and diluted samples were obtained. The diluted samples were washed three times with 0.05% PBST washing solution, 300 μL / well, and patted dry. The treated samples were then added to the ELISA plate of the capture antibody at 100 μL / well, and incubated at room temperature on a shaker at 600 rpm / min for 60 minutes. After the sample incubation, the plate was washed to obtain an ELISA plate of the sample to be tested with specific binding of the sample and the capture antibody.

[0026] (3) Human / crab VEGF, VEGFA, and VEGF165 proteins at a concentration of 0.25 mg / mL were added to 5% BSA-1×PBST-diluent to prepare a 250 ng / mL VEGF working solution, and 100 μL / well of the VEGF working solution was added to the ELISA plate of the object to be tested. The plate was placed in a shaker at 600 rpm / min at room temperature and incubated for 60 min. After incubation, the plate was washed three times with 0.05% PBST washing solution, 300 μL / well, and patted dry to obtain an ELISA plate with VEGF and the object to be tested.

[0027] (4) Add anti-hVEGF at a concentration of 0.20 mg / mL to 5% BSA-1×PBST-diluent to prepare 62.50 ng / mL Biotin-labeled Anti-VEGF, add 100 μL / well of Biotin-labeled Anti-VEGF to the ELISA plate where VEGF and the test substance are bound, place it on a shaker at 600 rpm / min at room temperature, incubate for 60 minutes, wash it three times with 0.05% PBST washing solution, 300 μL / well, and pat dry to obtain a bridging complex.

[0028] (5) SA-HRP was added to 5% BSA-1×PBST-diluent to prepare a 1:40000-fold diluted SA-HRP working solution. 100 μL / well of the diluted SA-HRP working solution was added to the bridging complex, the membrane was covered, and the plate was incubated at 30°C in a shaker at 500 rpm / min for 60 min. After incubation, the plate was washed three times with 0.05% PBST washing solution at 300 μL / well. The plate was patted dry and 100 μL / well of TMB colorimetric solution was added. The plate was covered and incubated at 30°C in the dark for 15 min. 100 μL / well of 0.5 mol / L H2SO4 stop solution was added to terminate the reaction. The absorbance of the plate was read at 650 nm using an ELISA reader.

[0029] Embodiment 2:

[0030] The standard curve samples were prepared using blank mixed serum of cynomolgus monkeys. The standard curve was fitted with parameters to determine the quantitative range of the target protein. The concentration points of the standard curve samples SDT1-STD8 were: 160 ng / mL, 80.0 ng / mL, 40.0 ng / mL, 20.0 ng / mL, 10.0 ng / mL, 5.00 ng / mL, 2.50 ng / mL, and 1.25 ng / mL, of which 1.25 ng / mL was the anchor point, and the quantitative range was 2.50-160 ng / mL.

[0031] Embodiment 3:

[0032] On D6 after retinal administration of New Zealand rabbits, the following organs were collected: eyelids, conjunctiva, cornea, lens, optic nerve, optic chiasm, optic tract, iris, ciliary body, retina, choroid, sclera, aqueous humor, vitreous body, spinal cord (cervical, thoracic, lumbar, sacral), bone marrow, trigeminal nerve, cervical lymph nodes, popliteal lymph nodes, mesenteric lymph nodes, thyroid gland, testis, epididymis, uterus, ovary, heart, liver, spleen, lung, kidney, brain (including medulla oblongata, cerebrum, cerebellum), and the target protein in the tissue was determined.

[0033] Comparative Example 1:

[0034] (1) Add 18B2G9 antibody at a concentration of 1837 μg / mL into DPBS to prepare a coating working solution of 2 μg / mL. Take 100 μL / well of the coating working solution and add it to the ELISA plate, cover it, and let it stand at 8°C for 16 h. After standing, discard the coating solution and wash it three times with 0.05% PBST washing solution, 300 μL / well, pat dry, add 200 μL / well of blocking solution to the washed ELISA plate, and place it on a shaker at 600 rpm / min for 3 h at room temperature to obtain an ELISA plate with captured antibodies.

[0035] (2) The standard curve samples, quality control samples and samples to be tested were diluted 1:20 with 10% BSA-1×PBST-diluent in a dilution plate, mixed evenly, and diluted samples were obtained. The diluted samples were washed three times with 0.05% PBST washing solution, 300 μL / well, and patted dry. The treated samples were then added to the capture antibody ELISA plate at 100 μL / well, and incubated at room temperature on a shaker at 600 rpm / min for 60 minutes. After the sample incubation, the plate was washed to obtain the ELISA plate of the sample to be tested with specific binding of the sample and the capture antibody.

[0036] (3) Human / crab VEGF, VEGFA, and VEGF165 proteins at a concentration of 0.25 mg / mL were added to 10% BSA-1×PBST-diluent to prepare a 250 ng / mL VEGF working solution, and 100 μL / well of the VEGF working solution was added to the ELISA plate of the object to be tested. The plate was placed in a shaker at 600 rpm / min at room temperature and incubated for 60 min. After incubation, the plate was washed three times with 0.05% PBST washing solution, 300 μL / well, and patted dry to obtain an ELISA plate with VEGF bound to the object to be tested.

[0037] (4) Add anti-hVEGF at a concentration of 0.20 mg / mL to 10% BSA-1×PBST-diluent to prepare 62.50 ng / mL Biotin-labeled Anti-VEGF, add 100 μL / well of Biotin-labeled Anti-VEGF to the ELISA plate where VEGF and the test substance are bound, place it on a shaker at 600 rpm / min at room temperature, incubate for 60 minutes, wash it three times with 0.05% PBST washing solution, 300 μL / well, and pat dry to obtain a bridging complex.

[0038] (5) SA-HRP was added to 10% BSA-1×PBST-diluent to prepare a 1:40000-fold diluted SA-HRP working solution. 100 μL / well of the diluted SA-HRP working solution was added to the bridging complex, and the membrane was covered. The membrane was placed on a shaker at 500 rpm / min at 30°C for 55-65 min. After incubation, the membrane was washed three times with 0.05% PBST washing solution at 300 μL / well. The membrane was patted dry and 100 μL / well of TMB colorimetric solution was added. The membrane was covered and incubated at 30°C in the dark for 15 min. 100 μL / well of 0.5 mol / L H2SO4 stop solution was added to terminate the reaction.

[0039] The reaction wells of Example 1 and Comparative Example 1 were tested by reading the absorbance of the ELISA plate at 650 nm using an ELISA reader.

[0040] According to the determination method of Example 1, two replicate wells were set for each concentration point of the standard curve of Example 2, and at least two analysts performed verification of at least 6 independent analysis batches for at least 3 days. The relative error percentage between each actual concentration and the theoretical concentration in each analysis batch and the average and coefficient of variation percentage of each concentration among all analysis batches were calculated.

[0041] The target protein in the tissue of Example 3 was measured by the measurement method of Example 1.

[0042] Table 1 is the reaction hole detection of Example 1

[0043]

[0044] Table 2 is the reaction hole detection of Example 1

[0045]

[0046] In the above Tables 1 and 2, * indicates that the results do not meet the acceptance criteria, NSB indicates nonspecific binding, and RE indicates the relative error percentage between the actual concentration and the theoretical concentration in each analytical batch.

[0047] Table 3 is the reaction well detection of Example 2

[0048]

[0049] In the above Table 3, CV is the coefficient of variation percentage, RE is the relative error percentage between the actual concentration and the theoretical concentration, and RE2 is the average relative error percentage of each concentration among all analytical batches.

[0050] From the comparison of Table 1 and Table 2, it can be seen that the OD value of the standard curve is 0.46, the blank background in the optimized cynomolgus monkey serum is reduced, the blank OD is about 0.07, the standard curve is linear, the fitting results are good, and the random quality control is passed. From Table 3, it can be seen that the quantitative range of the determination method of Example 1 is 2.50-160ng / mL, and the lower limit of quantification can reach 2.5ng / mL. Figure 1 It can be seen that only the choroid, retina, sclera and vitreous body of the eye subtissues can measure effective values, and the order from high to low is choroid, retina and sclera. The target protein was not detected in other eye subtissues and systemic organs.

Claims

1. An ELISA method for detecting the biodistribution of AAV in AMD gene therapy, characterized in that: The method comprises: (1) Add 90-110 μL / well of the coating working solution to the ELISA plate, coat it, and let it stand at 2-8°C for 12-16 hours. After standing, discard the coating solution, wash the plate, add 180-220 μL / well of protein-free rapid blocking solution to the washed ELISA plate, and place it on a shaker at 500-600 rpm / min at room temperature for 1-3 hours to obtain an ELISA plate with captured antibodies; (2) washing the diluted sample, adding the treated sample to the capture antibody ELISA plate at 90-110 μL / well, incubating, washing the plate after the sample incubation, and obtaining an ELISA plate in which the sample and the capture antibody are specifically bound; (3) adding 90-110 μL / well of VEGF working solution to the ELISA plate for incubation, and then washing the plate to obtain an ELISA plate in which VEGF and the analyte are bound; (4) adding 90-110 μL / well of Biotin-labeled Anti-VEGF to the ELISA plate where VEGF and the substance to be tested are bound, incubating, and washing the plate after incubation to obtain a bridging complex; (5) Add 90-110 μL / well of the diluted SA-HRP working solution to the bridging complex, cover the plate, incubate the plate at 10-30°C in a shaker at 500-600 rpm / min for 55-65 min, wash the plate after incubation, add 90-110 μL / well of TMB colorimetric solution, cover the plate, incubate the plate at 10-30°C in the dark for 5-15 min, and terminate the reaction by adding 100 μL / well of 0.4-0.6 mol / L H2SO4 stop solution.

2. The AMD gene therapy AAV biodistribution ELISA detection method according to claim 1, characterized in that: The preparation process of the coating working solution in step (1) is as follows: adding 18B2G9 antibody with a concentration of 1837 μg / mL into DPBS to prepare a coating working solution with a concentration of 1.8-2.2 μg / mL.

3. The AMD gene therapy AAV biodistribution ELISA detection method according to claim 1, characterized in that: The plate washing process in steps (1)-(5) is as follows: washing 3-4 times with 0.05% PBST washing solution, 280-320 μL / well, and patting dry.

4. The AMD gene therapy AAV biodistribution ELISA detection method according to claim 1, characterized in that: The incubation process in steps (2)-(4) is as follows: incubate at room temperature in a shaker at 500-600 rpm / min for 55-65 minutes.

5. The AMD gene therapy AAV biodistribution ELISA detection method according to claim 1, characterized in that: In the step (2), the preparation process of the diluted sample is as follows: the standard curve sample, the quality control sample and the sample to be tested are diluted 1:(19-21) times with 5% BSA-1×PBST-diluent in a dilution plate and mixed evenly.

6. The AMD gene therapy AAV biodistribution ELISA detection method according to claim 1, characterized in that: In the step (3), the preparation process of the VEGF working solution is as follows: adding human / crab VEGF, VEGFA, and VEGF165 proteins with a concentration of 0.25 mg / mL into 5% BSA-1×PBST-diluent to prepare a VEGF working solution with a concentration of 240-260 ng / mL.

7. The AMD gene therapy AAV biodistribution ELISA detection method according to claim 1, characterized in that: In the step (4), the process of biotin-labeled anti-VEGF is as follows: adding anti-hVEGF at a concentration of 0.20 mg / mL into 5% BSA-1×PBST-diluent to prepare 60-65 ng / mL biotin-labeled anti-VEGF.

8. The AMD gene therapy AAV biodistribution ELISA detection method according to claim 1, characterized in that: In the step (5), the dilution process of the SA-HRP working solution is as follows: SA-HRP is added to 5% BSA-1×PBST-diluent to prepare a 1:(38000-42000) times diluted SA-HRP working solution.

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