Method for detecting number of virus particles

Abstract

The invention provides application of a microplate reader in detection of the number of virus particles. The invention also provides a method for detecting the number of virus particles, especially the number of adenovirus particles, by using the microplate reader, so that the detection which is small in virus dosage, low in cost, flexible and controllable, large in flux and good in repeatability is realized, and the method is stable and reliable.

Description

Technical Field

[0001] The present disclosure belongs to the field of biological detection, and particularly relates to a method for detecting the number of virus particles using an enzyme-linked immunosorbent assay (ELISA) reader, especially for detecting the number of adenovirus particles. Background Art

[0002] Virus titer refers to the number of biologically active virus particles in a unit volume of liquid, and is an important indicator for measuring the quantity and virulence of viruses. It is an important parameter for evaluating the quality and potency of virus products such as vaccines, gene therapy vectors, or diagnostic reagents. According to different principles of virus titer determination, it can be divided into two major categories: physical titer determination and biological titer determination. Physical titers include virus particle titer (or capsid titer) and genomic titer; biological titers include infectious titer and transduction titer, etc.

[0003] The detection of the number of adenovirus particles usually uses methods such as photometry and qPCR. The photometry method for detecting physical particles mainly uses an ultraviolet spectrophotometer. This method is simple to operate, but the adenovirus yield is low, the production cycle is long, the dosage is too large, 2 - 3 mL is required at a time, and the throughput of a single detection process is low and the cost is high.

[0004] Therefore, it is necessary to develop a new method for detecting the number of adenovirus particles. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the purpose of the present disclosure is to provide a method for detecting the number of adenovirus particles using an ELISA reader, which realizes detection with less virus usage, low cost, flexibility, high throughput, and good repeatability.

[0006] According to the first aspect of the present disclosure, there is provided the use of an ELISA reader in detecting the number of virus particles.

[0007] In some embodiments, the virus includes adenovirus.

[0008] In some embodiments, the ELISA plate used for detection is selected from a 16-well ELISA plate, a 48-well ELISA plate, or a 96-well ELISA plate, preferably a 96-well plate.

[0009] The present disclosure provides a new detection form for physically quantifying the number of adenovirus particles. Detection is performed using an ELISA reader, which has an equivalent detection effect to that of an ultraviolet spectrophotometer, high repeatability, good data stability, and realizes high-throughput detection, is convenient and fast, and also reduces the detection cost.

[0010] According to the second aspect of the present disclosure, there is provided a method for detecting the number of virus particles, which includes detecting the number of virus particles using an ELISA reader.

[0011] In some embodiments, the method includes the following steps:

[0012] (1) Mix the test sample containing the virus with an SDS solution;

[0013] (2) Centrifuge the mixed sample and add the supernatant to an ELISA plate;

[0014] (3) Detect the ELISA plate in an ELISA reader.

[0015] In some embodiments, in step (1), the temperature of the mixing is 50 - 60 °C, such as 50 °C, 52 °C, 54 °C, 55 °C, 56 °C, 58 °C, 60 °C or any value therebetween. In some preferred embodiments, the temperature of the mixing is 54 - 56 °C.

[0016] In some embodiments, in step (1), the time of the mixing is 5 - 15 min, such as 5 min, 6 min, 8 min, 10 min, 12 min, 14 min, 15 min or any value therebetween. In some preferred embodiments, the time of the mixing is 8 - 12 min.

[0017] In some embodiments, in step (1), in the SDS solution, the final concentration of SDS is 0.05 - 0.5%, such as 0.05%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or any value therebetween. In some preferred embodiments, in step (1), in the SDS solution, the final concentration of SDS is 0.1 - 0.2%. In step (1), the inventor selects an SDS solution to lyse the viral capsid protein to release nucleic acid, and a specific concentration of the SDS solution is beneficial to improving the detection performance of the number of virus particles.

[0018] In some embodiments, in step (1), the test sample is a diluted sample.

[0019] In some embodiments, the dilution factor is 2 - 10 times, such as 2 times, 4 times, 6 times, 8 times, 10 times or any value therebetween.

[0020] In some embodiments, the ELISA plate is selected from a 16-well plate, a 48-well plate or a 96-well plate.

[0021] In some specific embodiments, the ELISA plate is a 96-well plate.

[0022] In some embodiments, in step (2), the centrifugation treatment includes centrifuging at 12000 - 14000 for 4 - 8 min, preferably includes centrifuging at 13000 rpm for 4 - 6 min. In some embodiments, in step (2), the centrifugation treatment includes centrifuging at 13000 for 5 min.

[0023] In some embodiments, step (1) further includes adding a blank control solution and / or a positive control solution to the microplate.

[0024] In some embodiments, the blank control solution includes an SDS solution.

[0025] In some embodiments, the blank control solution includes an SDS solution with a concentration of 0.05% - 0.5%, such as an SDS solution with a concentration of 0.05%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or any concentration value therebetween.

[0026] In some specific embodiments, the blank control solution is an SDS solution with a concentration of 0.1% - 0.2%.

[0027] In some embodiments, the sample volume of the sample to be tested is 80 - 120 μL, such as 80 μL, 90 μL, 100 μL, 110 μL, 120 μL or any value therebetween.

[0028] In some specific embodiments, the sample volume of the blank control solution and / or the sample to be tested is 100 μL.

[0029] In some embodiments, the absorbance wavelength for the detection is 250 - 270 nm, such as 250 nm, 255 nm, 260 nm, 260 nm, 265 nm, 270 nm or any value therebetween.

[0030] In some embodiments, the absorbance wavelength for the detection is 260 nm.

[0031] In some embodiments, the calculation formula for the number of virus particles is: number of virus particles (VP / mL) = (A260 待测样品 - A260 空白对照溶液 ) × dilution factor × 1.1 × 10 12 .

[0032] In some embodiments, the virus includes adenovirus.

[0033] The present disclosure uses a microplate reader for detection, operates with a 96 - well plate, has a small adenovirus dosage of about 500 μL for single - time detection, a large throughput, and is convenient and fast.

[0034] According to the third aspect of the present disclosure, there is provided the use of the method described in the second aspect in preparing a vaccine, a gene therapy vector or a diagnostic reagent containing adenovirus.

[0035] The present disclosure physically quantifies adenovirus by means of an enzyme - labeled instrument and an enzyme - labeled plate, especially a 96 - well plate. The operation is simple, rapid, with a large throughput, stable data, meeting the requirements of high - throughput detection, and the method is stable and reliable. Detailed Embodiments

[0036] To make the objectives, technical solutions and advantages of the present disclosure more clear, the following further details the present disclosure in conjunction with embodiments. The specific embodiments described herein are only used to explain the present disclosure and do not constitute any limitation to the present disclosure. In addition, in the following description, the descriptions of well - known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0037] The reagents and / or reagent kits used in the following embodiments are all commercially obtained or can be synthesized by known methods.

[0038] The test samples used in the following embodiments are purchased from the ADV finished products of Heyuan Biotechnology (Shanghai) Co., Ltd. (hereinafter referred to as No. 1), and the ADV finished products self - produced by Nanjing Yuanmai (hereinafter referred to as No. 2). Among them, the ADV finished products self - produced by Nanjing Yuanmai are mainly obtained by infecting 293A cells with adenovirus seed, harvesting cells and supernatants, lysing cells to release the virus, and then purifying through chromatography.

[0039] Comparative Example 1

[0040] Method for detecting the physical particle number of adenovirus using an ultraviolet spectrophotometer:

[0041] (1) Solution preparation

[0042] A) 10% SDS aqueous solution: Weigh 1.0 g of SDS, add 10 mL of purified water, vortex and mix well, store at room temperature, and it is valid on the day of preparation.

[0043] B) 1% SDS solution: Take 1 mL of 10% SDS solution, add 9 mL of blank preparation (PBS solution), vortex and mix well, store at room temperature, and prepare freshly before use.

[0044] C) 0.1% SDS solution: Take 1 mL of 1% SDS solution, add 9 mL of blank preparation (PBS solution), vortex and mix well, store at room temperature, and prepare freshly before use.

[0045] (2) Sample treatment

[0046] A) Preparation of blank control solution: Directly take 0.1% SDS solution

[0047] B) Preparation of the test sample: Take the test sample, restore it to room temperature, dilute it 2-fold and 4-fold for testing. Add a certain volume of 1% SDS to make the final concentration of SDS 0.1%. Taking the total preparation volume of 5 mL as an example:

[0048] Dilution 2-fold: After vortex mixing, take 2500 μL of the test sample respectively, add 2000 μL of the blank preparation solution, and add 500 μL of 1% SDS, then vortex mix.

[0049] Dilution 4-fold: After vortex mixing, take 1250 μL of the test sample respectively, add 3250 μL of the blank preparation solution, and add 500 μL of 1% SDS, then vortex mix.

[0050] C) Heat the blank control solution and the test sample in a constant-temperature metal bath at 55 °C for 10 min, centrifuge at 13000 rpm for 5 min, take the supernatant, place it in a clean EP tube, and make marks.

[0051] (3) Operation on the ultraviolet spectrophotometer:

[0052] A) First, rinse the cuvette with 400 μL of the blank control solution of 0.1% SDS solution, pat it dry on the absorbent paper, add 3 mL of the blank control solution of 0.01% SDS solution, put the cuvette into the detection chamber of the ultraviolet spectrophotometer (Mepada P4), close the lid, and perform blank correction.

[0053] B) Pour out the blank control solution of 0.1% SDS solution in the cuvette, pat it dry on the absorbent paper, add 500 μL of the test sample to rinse and then pat it dry on the absorbent paper. Put the patted-dry cuvette into the cuvette slot of the spectrophotometer, close the lid, and read the absorbance value of the test sample 3 times at 260 nm.

[0054] C) Calculation formula: Number of virus particles (VP / mL) = (A260 待测样品 - A260 空白对照溶液 ) × dilution factor × 1.1 × 10 12

[0055] The test results are shown in Table 1 below.

[0056] Table 1. Detection results of the ultraviolet spectrophotometer

[0057]

[0058] Example 1

[0059] Method for detecting the number of adenovirus physical particles using an enzyme-labeled instrument:

[0060] (1) Solution preparation

[0061] A) 10% SDS solution: Weigh 1.0 g of SDS, add 10 mL of purified water, vortex to mix evenly, store at room temperature, and it is valid on the same day.

[0062] B) 1% SDS solution: Take 1 mL of 10% SDS solution, add 9 mL of blank preparation (PBS solution), vortex to mix evenly, store at room temperature, and prepare freshly before use.

[0063] C) 0.1% SDS solution: Take 1 mL of 1% SDS solution, add 9 mL of blank preparation (PBS solution), vortex to mix evenly, store at room temperature, and prepare freshly before use.

[0064] (2) Sample treatment

[0065] A) Preparation of blank control solution: Directly take 0.1% SDS solution

[0066] B) Preparation of sample to be tested: Take the sample to be tested, restore it to room temperature, dilute it 2 times and 4 times for detection. Taking the total volume of 500 μL as an example:

[0067] Dilution by 2 times: After vortexing to mix evenly, take 250 μL of the sample to be tested respectively, add 200 μL of blank preparation solution, add 50 μL of 1% SDS, and vortex to mix evenly.

[0068] Dilution by 4 times: After vortexing to mix evenly, take 125 μL of the sample to be tested respectively, add 325 μL of blank preparation solution, add 50 μL of 1% SDS, and vortex to mix evenly.

[0069] C) Place the blank control solution and the sample to be tested in a constant temperature metal bath at 55 °C for heating for 10 min, centrifuge at 13000 rpm for 5 min, take the supernatant, place it in a clean EP tube, and make good marks.

[0070] (3) Loading onto the microplate reader

[0071] Add the 0.1% SDS solution of the blank control and the sample to be tested into the 96-well plate, 100 μL per well, with 2 replicates or 3 replicates. Avoid air bubbles, then open the software, select the absorbance wavelength of 260 nm, select Reference, select the target wells, and perform the detection.

[0072] Calculation formula: Number of virus particles (VP / mL) = (A260 待测样品 - A260 空白对照溶液 ) × dilution factor × 1.1 × 10 12 The detection results are shown in Table 2 below, and are compared with the results detected by the ultraviolet spectrophotometer method in Comparative Example 1.

[0073] Table 2

[0074]

[0075] It can be seen from the comparison of the detection data by the ultraviolet spectrophotometer and the microplate reader in Table 2 that the relative standard deviations (RSD) of the detection results of both are within 10%. The detection of the number of adenovirus physical particles by the microplate reader can replace the detection by the ultraviolet spectrophotometer.

[0076] Example 2

[0077] The number of adenovirus physical particles was detected using the same method as in Example 1. The test samples remained unchanged, and the second detection was carried out 7 days after the first detection. The detection results are shown in Table 3 below.

[0078] Table 3

[0079]

[0080]

[0081] It can be seen from the detection results in Table 3 that the repeatability RSDs are all within 10%, and the intermediate precision RSDs are all within 10%. It can be seen that the repeatability of detecting the number of adenovirus physical particles by the microplate reader is high.

[0082] The technical solution of the present disclosure is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present disclosure falls within the protection scope of the present disclosure.

Claims

1. Use of an enzyme-linked immunosorbent assay (ELISA) instrument in detecting the number of virus particles.

2. The use according to claim 1, characterized in that, The virus includes adenovirus.

3. A method for detecting the number of virus particles, which includes using an ELISA instrument to detect the number of virus particles.

4. The method according to claim 3, wherein The method includes the following steps: (1) Mix a test sample containing the virus with an SDS solution; (2) Centrifuge the mixed sample, and take the supernatant and add it to an ELISA plate; (3) Detect the ELISA plate in an ELISA instrument.

5. The method according to claim 4, wherein In step (1), the mixing time and temperature are 50 - 60 °C, preferably 54 - 56 °C; and / or the mixing time is 5 - 15 min; preferably 8 - 12 min; and / or in the SDS solution, the final concentration of SDS is 0.05 - 0.5%, preferably 0.1 - 0.2%; and / or the test sample is a diluted sample, preferably diluted 2 - 10 times.

6. The method according to claim 4, wherein In step (2), the centrifugation treatment includes centrifuging at 12000 - 14000 rpm for 4 - 8 min, preferably including centrifuging at 13000 rpm for 4 - 6 min; and / or step (2) further includes adding a blank control solution and / or a positive control solution to the ELISA plate, preferably, the blank control solution includes an SDS solution, more preferably, the concentration of the SDS solution is 0.05 - 0.5%, and further preferably 0.1 - 0.2%.

7. The method according to claim 4, characterized in that The ELISA plate is selected from a 16-well plate, a 48-well plate or a 96-well plate, preferably a 96-well plate; and / or the sample loading amount of the test sample is 80 - 120 μL; and / or the absorbance wavelength for the detection is 250 - 270 nm, preferably 260 nm.

8. The method according to any one of claims 3-7, characterized in that The calculation formula for the number of virus particles is: Number of virus particles (VP / mL) = (A260 待测样品 - A260 空白对照溶液 ) × dilution factor × 1.1 × 10 12 .

9. The method according to any one of claims 3 - 8, characterized in that, The virus includes adenovirus.

10. Use of the method according to any one of claims 3 - 8 in preparing a vaccine, a gene therapy vector or a diagnostic reagent containing adenovirus.

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