Hepatitis A vaccine virus titer trace detection method, kit and application

Abstract

The invention discloses a hepatitis A vaccine virus titer trace detection method, a kit and application. Comprising the following steps: inoculating human diploid cells into a porous cell culture plate, and culturing to form monolayer cells; inoculating a to-be-detected hepatitis A virus sample into the porous cell culture plate with monolayer cells, carrying out virus adsorption and culture, marking as an experimental group, and taking human diploid cells not inoculated with the virus sample as a negative control group; adding acetone into the experimental group and the negative control group for incubation so as to lyse cells and fix hepatitis A viruses and antigens thereof; detecting by adopting an enzyme-linked immunosorbent assay, and calculating according to a detection result to obtain the virus titer of the hepatitis A virus sample to be detected. The method is simple and convenient to operate, a virus extraction step in a traditional method is omitted, reagents are remarkably saved, the detection flux is improved, and the method is highly related to results of a pharmacopoeia method and is suitable for vaccine process research and development and quality control.

Description

Technical Field

[0001] This application relates to the field of virological detection technology, specifically to a method, kit, and application for detecting trace amounts of hepatitis A vaccine virus titers. Background Technology

[0002] Hepatitis A is an acute hepatitis caused by the hepatitis A virus (HAV). Clinically, live attenuated vaccines or inactivated vaccines are widely used for prevention. The viral titer of hepatitis A vaccines is an important indicator for evaluating the effectiveness of live attenuated vaccines, a key aspect of quality control in the production process of inactivated vaccines (such as virus harvesting fluid, intermediate products, and finished products), and is significant for process scale-up studies and virus stability studies.

[0003] The current Pharmacopoeia specifies the cell culture-ELISA method as the legally required method for detecting the viral titer of hepatitis A vaccine. This method typically uses human diploid cells (such as MRC-5 cells) as the viral replication host. The viral sample is serially diluted tenfold and inoculated into cell culture flasks or plates. Under suitable conditions, the cells are cultured to the peak viral replication phase. The cells and supernatant are then harvested, and viral antigens are prepared through trypsin digestion, repeated freeze-thaw cycles, ultrasonic disruption, and chloroform extraction. Finally, the viral load is determined using ELISA, and the viral titer is calculated according to the Reed-Muench method.

[0004] Although the above methods have been widely adopted, they still have the following shortcomings:

[0005] 1. High consumables and workload: It usually requires a large number of T25 cell flasks or large-well plates for multi-gradient and multi-parallel virus culture, with a long culture cycle (generally 21 to 28 days). During this period, the culture medium needs to be changed many times to maintain the cell state, which consumes a lot of cells, culture medium and manpower.

[0006] 2. Cumbersome operation steps and multiple sources of error: The virus harvesting stage requires multiple steps such as trypsin digestion, repeated freeze-thaw cycles, ultrasonic disruption and chloroform extraction. Different operators and conditions have a significant impact on the virus recovery rate, which can easily introduce systematic and random errors.

[0007] 3. The test results are easily affected by the extraction conditions: the number of freeze-thaw cycles, ultrasonic conditions, and chloroform extraction efficiency can all affect the viral antigen recovery rate, thereby causing deviations in the viral titer determination results and affecting the accuracy and repeatability of the method.

[0008] 4. Limited detection throughput: Traditional cell flask methods have low experimental throughput per unit area, which is not conducive to the rapid detection of multiple batches of samples or process screening samples.

[0009] Therefore, there is an urgent need for a new method for the micro-detection of hepatitis A virus titers that can reduce consumables, simplify operations, increase throughput, and reduce the dependence of results on virus extraction steps, while ensuring sensitivity, specificity, and accuracy. Summary of the Invention

[0010] Therefore, it is necessary to provide methods, kits, and applications for detecting trace amounts of hepatitis A vaccine virus titers.

[0011] The first aspect of this application provides a method for detecting trace amounts of hepatitis A vaccine virus titers, comprising the following steps:

[0012] Human diploid cells were seeded into multi-well cell culture plates and cultured to form a monolayer of cells.

[0013] Hepatitis A virus samples to be tested were inoculated into porous cell culture plates that formed a monolayer of cells for virus adsorption and culture, and were designated as the experimental group. Human diploid cells that were not inoculated with virus samples were used as the negative control group.

[0014] Acetone was added to the experimental group and the negative control group for incubation to lyse cells and fix hepatitis A virus and its antigens; and,

[0015] The virus titer of the hepatitis A virus sample was calculated based on the detection results using enzyme-linked immunosorbent assay (ELISA).

[0016] In some embodiments, the inoculum size of the human diploid cells is 1.0 × 10⁶ cells per well. 4 ~2.0×10 4 Each cell.

[0017] In some embodiments, the hepatitis A virus sample to be tested is further subjected to serial dilutions, with a dilution factor of 10, before inoculation. -1 ~10 -6 and / or 10 -5 ~10 -8 Optionally, each dilution of hepatitis A virus sample to be tested can be set up in 6-8 parallel wells.

[0018] In some embodiments, the conditions for virus adsorption include: a temperature of 35°C to 37°C, a CO2 concentration of 2% (v / v) to 5% (v / v), and a time of 1h to 2h.

[0019] In some embodiments, the culture further includes adding a cell maintenance medium for maintenance culture, the cell maintenance medium including a serum-containing culture medium; optionally, the concentration of serum in the virus culture system is 1% (v / v) to 3% (v / v); the culture conditions include: a temperature of 35°C to 37°C, a CO2 concentration of 2% (v / v) to 5% (v / v), and a time of 21 days to 28 days.

[0020] In some embodiments, the concentration of acetone is 78% to 82% (v / v).

[0021] In some embodiments, the acetone is pre-cooled at -18°C to -22°C before incubation. Optionally, the incubation conditions include a temperature of 20°C to 30°C and a time of 5 min to 15 min.

[0022] In some embodiments, enzyme-linked immunosorbent assay (ELISA) is used for detection, and the step of calculating the viral titer of the hepatitis A virus sample to be tested based on the detection results includes:

[0023] Add enzyme-labeled anti-hepatitis A virus antibody to the multi-well cell culture plate and incubate.

[0024] Add the colorimetric reagent to develop the color, and then measure the absorbance.

[0025] The positive wells were determined based on the absorbance of the negative control group, and the viral titer of the hepatitis A virus sample to be tested was calculated using the Reed-Muench method based on the number of positive and negative wells at each dilution.

[0026] In some embodiments, the enzyme-labeled anti-hepatitis A virus antibody is a horseradish peroxidase-labeled anti-hepatitis A virus monoclonal antibody.

[0027] In some embodiments, the enzyme-labeled anti-hepatitis A virus antibody is diluted at a ratio of 1:10000 to 1:13000.

[0028] In some embodiments, the incubation conditions include a temperature of 35°C to 37°C and a time of 60 min to 90 min.

[0029] In some implementations, the criterion for determining the positive well based on the absorbance of the negative control group is that the absorbance is greater than or equal to 2.1 times that of the negative control group.

[0030] In some embodiments, the human diploid cells include MRC-5 cells.

[0031] In some embodiments, the porous cell culture plate includes at least one of a 48-well cell culture plate and a 96-well cell culture plate.

[0032] In some embodiments, the hepatitis A virus sample to be tested includes one or more of the following: a live attenuated hepatitis A vaccine, a hepatitis A virus strain produced during the production of an inactivated hepatitis A vaccine, a virus harvest liquid, intermediates, and finished products.

[0033] In some embodiments, the method for detecting the hepatitis A vaccine virus titer does not include the virus extraction steps of freezing and thawing, ultrasonic disruption, and chloroform extraction after cell harvesting.

[0034] A second aspect of this application provides a trace detection kit for hepatitis A vaccine virus titer, comprising human diploid cells, a multi-well cell culture plate, acetone, a buffer, and a culture medium;

[0035] Optionally, it may also include one or more of the following: blocking solution, enzyme-labeled anti-hepatitis A virus antibody, chromogenic solution, positive control and negative control.

[0036] In some embodiments, the hepatitis A vaccine virus titer micro-detection kit satisfies one or more of the following conditions:

[0037] (1) The human diploid cells include MRC-5 cells;

[0038] (2) The porous cell culture plate includes at least one of a 48-well cell culture plate and a 96-well cell culture plate;

[0039] (3) The buffers include PBS buffers and PBST buffers;

[0040] (4) The culture medium includes at least one of serum-free culture medium and culture medium containing 3% (v / v) to 5% (v / v) serum.

[0041] A third aspect of this application provides an evaluation method for the development and optimization of a hepatitis A vaccine process, comprising:

[0042] The virus titer of hepatitis A vaccine obtained under different cell culture conditions, different culture medium formulations, different culture process parameters, or before and after virus inactivation was determined using the micro-detection method for hepatitis A vaccine virus titer described in the first aspect of this application.

[0043] By comparing the viral titers, the effects of different process conditions on the hepatitis A virus proliferation level and / or process stability are evaluated.

[0044] Compared to the traditional cell culture-ELISA method, the aforementioned detection method significantly reduces the consumption of consumables such as cells, culture media, and reagents while ensuring detection performance (sensitivity, specificity, and accuracy). It omits virus extraction steps such as freeze-thaw, sonication, and chloroform extraction, simplifying the operation process and reducing operational errors and safety risks. At the same time, it significantly improves detection throughput and work efficiency, making it suitable for batch sample testing and process screening. In addition, this method maintains good correlation with the cell culture-ELISA method in the pharmacopoeia, and can be used for methodological conversion and routine quality control, providing an efficient and reliable detection tool for vaccine production and research and development. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0046] Figure 1 This is a schematic diagram of the method for detecting trace amounts of hepatitis A virus titer in one embodiment of this application. Detailed Implementation

[0047] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0049] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0050] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0051] The terms “having,” “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0052] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0053] In this application, if the unit of a data range is only followed by the right endpoint, it means that the units of the left and right endpoints are the same.

[0054] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0055] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0056] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0057] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0058] In this application, the terms "room temperature" or "normal temperature" generally refer to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 10°C to 30°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 20°C to 30°C.

[0059] Currently, the legally mandated method for detecting hepatitis A vaccine viral titers is the cell culture-ELISA method. Although this method is widely used, it still has drawbacks such as high consumption of consumables, complex operation, multiple sources of error, and limited throughput.

[0060] Based on this, the embodiments of this application at least provide a method, kit, and application for detecting trace amounts of hepatitis A vaccine virus titers.

[0061] In a first aspect of this application, a method for detecting trace amounts of hepatitis A vaccine virus titers is provided, comprising the following steps:

[0062] S100: Human diploid cells are seeded into a multi-well cell culture plate and cultured to form a monolayer of cells;

[0063] S200: The hepatitis A virus sample to be tested was inoculated into a porous cell culture plate that formed a monolayer of cells for virus adsorption and culture, and was recorded as the experimental group. Human diploid cells that were not inoculated with the virus sample were used as the negative control group.

[0064] S300: Acetone was added to the experimental group and the negative control group for incubation to lyse cells and fix hepatitis A virus and its antigens; and,

[0065] S400: The detection method is enzyme-linked immunosorbent assay (ELISA), and the viral titer of the hepatitis A virus sample to be tested is calculated based on the detection results.

[0066] In some implementations, human diploid cells include MRC-5 cells.

[0067] In some embodiments, the porous cell culture plate includes at least one of a 48-well cell culture plate and a 96-well cell culture plate.

[0068] It should be noted that this application uses a multi-well cell culture plate, which can complete the titer determination of multiple gradients, multiple parallels and multiple samples with a single plate. Compared with the traditional T25 cell flask method, it significantly reduces the consumption of cell seeds, culture medium and related reagents, and significantly improves the detection throughput.

[0069] In some embodiments, the inoculum size of human diploid cells is 1.0 × 10⁶ cells per well. 4 ~2.0×10 4 1.0 × 10⁶ cells. Without limitation, the inoculum size for human diploid cell inoculation can be, but is not limited to, 1.0 × 10⁶ cells. 4 1.5×10 4 2.0×10 4 Or the value or range between any two of the above values.

[0070] In some embodiments, the hepatitis A virus sample to be tested is further subjected to serial dilutions, with a dilution factor of 10, before inoculation. -1 ~10 -6 and / or 10 -5 ~10 -8 Understandably, the dilution range can be adjusted appropriately based on the estimated titer of the sample.

[0071] In some implementations, hepatitis A virus samples of different dilutions are set up in parallel with 6, 7, or 8 wells.

[0072] In some embodiments, the conditions for virus adsorption include: a temperature of 35°C to 37°C, a CO2 concentration of 2% (v / v) to 5% (v / v), and a time of 1 h to 2 h. Non-limitingly, the temperature for virus adsorption can be 35°C, 36°C, or 37°C; the CO2 concentration can be 2% (v / v), 3% (v / v), 4% (v / v), or 5% (v / v); and the time can be 1 h, 1.5 h, or 2 h.

[0073] In some embodiments, virus culture further includes adding cell maintenance medium to each well of a multi-well cell culture plate for maintenance culture; the cell maintenance medium includes a serum-containing culture medium. Further, the culture medium includes MEM medium.

[0074] In some embodiments, the concentration of serum in the virus culture system is 1% (v / v) to 3% (v / v). Without limitation, the concentration of serum in the virus culture system can be, but is not limited to, 1% (v / v), 2% (v / v), 3% (v / v), or any value or range between two of the above.

[0075] In some embodiments, the virus culture conditions include: a temperature of 35°C to 37°C, a CO2 concentration of 2% (v / v) to 5% (v / v), and a culture time of 21 to 28 days. Non-limitingly, the virus culture temperature can be 35°C, 36°C, or 37°C; the CO2 concentration can be 2% (v / v), 3% (v / v), 4% (v / v), or 5% (v / v); and the culture time can be 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

[0076] In some embodiments, the concentration of acetone is 78% to 82% (v / v). Without limitation, the concentration of acetone can be, but is not limited to, 78% (v / v), 80% (v / v), 82% (v / v), or any value or range between two of the above.

[0077] In some embodiments, the acetone is pre-cooled at -18°C to -22°C before incubation. Non-limitingly, the pre-cooling temperature can be, but is not limited to, -18°C, -19°C, -20°C, -21°C, -22°C, or any value or range between two of the above.

[0078] It should be noted that pre-cooling with acetone can rapidly and uniformly achieve cell lysis and fixation. If the pre-cooling step is omitted, fixation / lysis will become "neither fast nor uniform," thus affecting subsequent detection results.

[0079] In some embodiments, the incubation conditions for adding acetone include: an incubation temperature of 20°C to 30°C and a time of 5 min to 15 min. Non-limitingly, the incubation temperature can be 20°C, 25°C, 30°C, or any value or range between two of the above; the incubation time can be 5 min, 10 min, 15 min, or any value or range between two of the above.

[0080] It should be noted that, under the above incubation conditions, this application can directly lyse and fix cells by using pre-cooled acetone of a specific concentration, so that the hepatitis A virus antigen is retained in the wells. This eliminates the need for complex virus extraction steps such as freeze-thaw, sonication, and chloroform extraction, reducing operational steps and lowering the safety risks associated with human error and the use of organic solvents.

[0081] In some implementations, enzyme-linked immunosorbent assay (ELISA) is used for detection, and the step of calculating the viral titer of the hepatitis A virus sample to be tested based on the detection results includes:

[0082] Add enzyme-labeled anti-hepatitis A virus antibody to each well of a multi-well cell culture plate and incubate.

[0083] Add the colorimetric reagent to develop the color, and then measure the absorbance.

[0084] The positive wells were determined based on the absorbance of the negative control group, and the viral titer of the hepatitis A virus sample to be tested was calculated using the Reed-Muench method based on the number of positive and negative wells at each dilution.

[0085] In some embodiments, the enzyme-labeled anti-hepatitis A virus antibody is a horseradish peroxidase-labeled anti-hepatitis A virus monoclonal antibody.

[0086] In some embodiments, the enzyme-labeled anti-hepatitis A virus antibody is formulated to a suitable working concentration, and further, the dilution factor of the anti-hepatitis A virus antibody is 1:10000 to 1:13000.

[0087] In some embodiments, the incubation conditions for adding enzyme-labeled anti-hepatitis A virus antibodies include: a temperature of 35°C to 37°C and a time of 60 min to 90 min. Non-limitingly, the incubation temperature can be 35°C, 36°C, or 37°C; and the incubation time can be 60 min, 70 min, 80 min, or 90 min.

[0088] In some implementations, the criterion for determining the positive well based on the absorbance of the negative control group is that the absorbance is greater than or equal to 2.1 times that of the negative control group.

[0089] In some embodiments, the sample to be tested in the above detection method includes one or more of the following: hepatitis A live attenuated vaccine, hepatitis A virus strain, virus harvest liquid, intermediate product, and finished product produced during the production process of hepatitis A inactivated vaccine.

[0090] In some implementations, the viral titer in the hepatitis A virus sample to be tested is 5.0–8.0 lgCCID. 50 / mL. Without limitation, the viral titer in the hepatitis A virus sample to be tested can be, but is not limited to, 5.0 lg CCID. 50 / mL, 6.0lg CCID 50 / mL, 7.0 lgCCID 50 / mL, 8.0 lgCCID 50 / mL or a value or range between any two of the above values.

[0091] It should be noted that the above detection method is within the range of 5.0–8.0 g CCID. 50 It exhibits good linearity and repeatability within the range of [value] mL, and its sensitivity and specificity meet the requirements for hepatitis A vaccine virus titer detection, while its precision meets quality control standards. Furthermore, compared with pharmacopoeia methods based on cell culture-ELISA, the average absolute value of the titer difference measured for the same batch of samples does not exceed 1.0 lgCCID. 50 / mL.

[0092] In some embodiments, the hepatitis A vaccine virus titer detection method provided in this application does not include the virus extraction steps of freezing and thawing, ultrasonic disruption, and chloroform extraction after cell harvesting.

[0093] In some implementations, through parallel testing of 10 batches of virus samples, the viral titer measured by the method of this application is consistent with the results of the traditional T25 cell bottle method in terms of the high-low trend between batches, with a correlation coefficient of approximately 0.86. This indicates that the method of this application can still accurately reflect the viral proliferation level and the relative differences between different batches while significantly reducing consumables and simplifying operation. It is suitable as a high-throughput method and process comparison tool for detecting hepatitis A vaccine viral titer.

[0094] In some implementation methods, a schematic diagram of the hepatitis A vaccine virus titer trace detection method is shown below. Figure 1 As shown, it includes the following steps:

[0095] (1) Cell plating

[0096] Human diploid cells were selected, with MRC-5 cells being the preferred choice. MRC-5 cells in the logarithmic growth phase were digested and a cell suspension was prepared, with the cell concentration adjusted to (1.0~2.0) × 10⁻⁶. 5 Cells / mL; Add 100 μL of cell suspension to each well of a 96-well cell culture plate, so that each well is seeded with approximately 1.0 × 10⁶ cells. 4 ~2.0×10 4 Incubate the cells at 37°C in a 5% CO2 incubator for approximately 48 hours, until a cell monolayer essentially covers the bottom of each well. Discard the supernatant, add PBS buffer to each well and gently wash once, discard the PBS, and gently pat dry.

[0097] (2) Tenfold serial dilution of samples

[0098] Take the hepatitis A virus sample to be tested, which can be the finished product of hepatitis A live attenuated vaccine, the virus harvest fluid or intermediate in the production process of hepatitis A inactivated vaccine, etc., and perform a 10-fold serial dilution using serum-free MEM medium, preferably a 10:10 dilution. -5 ~10 -8 The dilution range can also be adjusted appropriately based on the estimated titer of the sample.

[0099] (3) Virus inoculation and adsorption

[0100] Add the samples of each dilution gradient to the 96-well cell culture plate obtained in step (1), with 100 μL added to each well. Each dilution is repeated in 6-8 wells. A negative control well is included; only the same volume of serum-free MEM medium is added to wells containing a cell monolayer, without inoculating with viral samples. After inoculation, the 96-well plate is placed in a 37°C, 5% CO2 incubator for 1-2 hours for adsorption.

[0101] (4) Maintenance culture

[0102] After adsorption, add 100 μL of MEM culture medium containing approximately 4% serum to each well, making the total volume of each well approximately 200 μL. Incubate the 96-well plate at 35°C in a 5% CO2 incubator for 21–28 days.

[0103] (5) Cell lysis and fixation

[0104] After incubation, discard the culture medium from each well, wash once with PBS or PBST buffer, and gently pat dry. Prepare an 80% (v / v) acetone solution using PBS and pre-chill at -20°C. When using, add 100 μL of the pre-chilled 80% acetone solution to each well and incubate at room temperature for 5–15 min to induce cell lysis and fix the virus and its antigenic components at the bottom of the well. Discard the acetone solution and allow the plate bottom membrane to air dry.

[0105] (6) Washing and sealing

[0106] After fixation, wash three times with approximately 200 μL of PBS or PBST buffer per well, add 200 μL of PBST blocking buffer containing 1% bovine serum albumin (BSA) per well, and block at room temperature for about 1 hour to reduce nonspecific binding; after blocking, discard the blocking buffer and wash three more times with PBST.

[0107] (7) Antibody incubation

[0108] Take HRP-labeled anti-hepatitis A virus monoclonal antibody and prepare it to a suitable working concentration using enzyme-labeled antibody dilution buffer, preferably at a dilution ratio of 1:10000 to 1:13000. Add 100 μL of enzyme-labeled antibody dilution buffer to each well and incubate the 96-well plate at 37°C for about 60 to 90 minutes.

[0109] (8) Color development and termination

[0110] After incubation, discard the antibody working solution and wash three times with PBST. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for about 10-15 minutes. When the positive wells show a clear blue color, add 50 μL of stop solution to each well to stop the reaction. The chromogenic solution will change from blue to yellow.

[0111] (9) Result reading and titer calculation

[0112] The absorbance of each well in the 96-well plate was read at 450 nm using a microplate reader. Based on the average absorbance of the negative control wells, a cut-off value was determined according to the kit requirements or internal quality control standards. Wells with absorbance greater than or equal to the cut-off value were considered positive, and wells with absorbance less than the cut-off value were considered negative. The viral titer was calculated using the Reed-Muench method based on the number of positive and negative wells at each dilution, and the results were expressed as lg CCID. 50 / mL represents the volume of water.

[0113] In a second aspect of this application, a trace detection kit for hepatitis A vaccine virus titer is provided, comprising human diploid cells, a multi-well cell culture plate, acetone, a buffer, and a culture medium.

[0114] In some embodiments, the hepatitis A vaccine virus titer micro-detection kit further includes one or more of the following: blocking solution, enzyme-labeled anti-hepatitis A virus antibody, chromogenic solution, positive control, and negative control.

[0115] In some implementations, the human diploid cells in the hepatitis A vaccine virus titer micro-detection kit include MRC-5 cells.

[0116] In some embodiments, the multi-well cell culture plate in the hepatitis A vaccine virus titer micro-detection kit includes at least one of a 48-well cell culture plate and a 96-well cell culture plate.

[0117] In some embodiments, the buffer in the hepatitis A vaccine virus titer micro-detection kit includes at least one of PBS buffer and PBST buffer.

[0118] In some embodiments, the culture medium in the hepatitis A vaccine virus titer micro-detection kit includes at least one of serum-free culture medium and culture medium containing 3% (v / v) to 5% (v / v) serum. Further, the culture medium is MEM medium.

[0119] In a third aspect of this application, an evaluation method for the development and optimization of a hepatitis A vaccine process is provided, comprising:

[0120] The above-mentioned method for detecting hepatitis A vaccine virus titers was used to determine the virus titers of samples obtained under different cell culture conditions, different culture medium formulations, different culture process parameters, or before and after virus inactivation.

[0121] By comparing viral titers, the effects of different process conditions on hepatitis A virus proliferation levels and / or process stability were evaluated. Some examples are provided below.

[0122] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0123] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0124] Example 1: Establishment of a method for detecting hepatitis A virus titers based on 96-well cell culture plates

[0125] 1) Cell preparation and plating

[0126] MRC-5 cells in the logarithmic growth phase were harvested, digested, and a cell suspension was prepared. The cell concentration was adjusted to approximately 1.5 × 10⁻⁶ cells / year. 5 Cells / mL. Add 100 μL of cell suspension to each well of a 96-well cell culture plate, approximately 1.5 × 10⁶ cells / mL. 4 Cells per well were incubated at 37°C in a 5% CO2 incubator for approximately 48 hours. Once the cell confluence reached ≥75%, the supernatant was discarded. 200 μL of PBS was added to each well for a gentle wash. The PBS was then discarded and the cells were gently patted dry.

[0127] 2) Sample dilution and inoculation

[0128] Hepatitis A virus harvest fluid samples were collected and 10 μL of serum-free MEM culture medium was prepared. -5 ~10 -8 Diluents were prepared at various levels. According to the experimental design, samples of different dilutions were added to the corresponding wells of a 96-well plate, 100 μL per well, with eight wells for each dilution. A negative control well was included, containing 100 μL of serum-free MEM medium. The 96-well plate was then incubated at 37°C in a 5% CO2 incubator for 2 hours for adsorption.

[0129] 3) Virus culture

[0130] After the adsorption is completed, add 100 μL of MEM maintenance solution containing 4% fetal bovine serum to each well to make the total volume of each well about 200 μL. Place the 96-well plate in an incubator at 35 °C and 5% CO2 for 28 days. Observe and record the cell status regularly as needed.

[0131] 4) Cell lysis and fixation

[0132] Discard the culture medium in each well at the end of the culture. Add 200 μL of PBS to each well for washing, discard the PBS and gently pat to drain. Take the 80% acetone solution pre-cooled at -20 °C, add 100 μL to each well, and incubate at 25 °C for 10 min. After the incubation, discard the acetone solution and let it dry naturally at the bottom of the plate.

[0133] Subsequently, add 200 μL of PBST to each well and wash 3 times to remove the residual acetone. If necessary, add 1% BSA-PBST blocking solution to block for 1 h, and then wash 3 times with PBST.

[0134] 5) Antibody incubation and color development

[0135] According to the instructions, take the HRP-labeled anti-hepatitis A virus monoclonal antibody and prepare a working solution of about 1:12000 with the enzyme-labeled antibody diluent. Add 100 μL to each well and place the 96-well plate in an incubator at 37 °C for 1 h.

[0136] After the incubation, discard the antibody working solution, wash 3 times with PBST, add 100 μL of TMB color development solution to each well, and develop color in the dark at room temperature for about 10 - 15 min; when the color of the positive wells significantly deepens, add 50 μL of the termination solution to each well to terminate the reaction.

[0137] 6) Result analysis

[0138] Use an enzyme-labeled instrument to read the absorbance value of each well at a wavelength of 450 nm, calculate the cut-off value according to the average absorbance of the negative control wells, and wells with an absorbance ≥ cut-off are determined as positive wells, and < cut-off are determined as negative wells.

[0139] According to the number of positive and negative wells at each dilution, calculate the hepatitis A virus titer using the Reed-Muench method, and the result is expressed as lgCCID 50 / mL.

[0140] In this example, representative samples were repeatedly measured, and the results showed that:

[0141] Samples at 5 different titer levels were within the range of 5.0 - 8.0 lgCCID 50 / mL, and the OD value showed a good linear change with the dilution;

[0142] Differences in results between different operators and on different testing days are acceptable.

[0143] The consistency rate of positive / negative determinations among 6-8 parallel wells of the same sample is high, and the titer calculation results are stable.

[0144] Example 2: Correlation Verification with the T25 Cell Flask Method

[0145] This embodiment uses 10 batches of samples from strains P28 to P37 as the subjects. The hepatitis A virus titer was determined using the method based on 96-well cell culture plates (hereinafter referred to as "96-well plate method") and the traditional T25 cell culture-ELISA method (hereinafter referred to as "T25 method") as described in this application embodiment. The detection results and correlations of the two methods were compared.

[0146] 1. Overview of Experimental Conditions and Procedures

[0147] 1) Detection conditions for the 96-well plate method

[0148] Cells: MRC-5 human diploid cells;

[0149] Main reagents: PBS solution, PBST solution, 1% BSA-PBST, stop solution, single-component ultrasensitive TMB, HRP-HAV monoclonal antibody, etc.

[0150] Cell seeding: MRC-5 cell suspension was seeded into 96-well plates at 100 μL per well. The cells were seeded after the confluence reached ≥75%.

[0151] Virus inoculation: After discarding the original culture medium, add 100 μL of cell maintenance medium + 100 μL of the appropriate dilution (10⁻⁶) to each well. -6 ~10 -9 For each dilution of the virus, at least 6 wells should be used in parallel.

[0152] Culture conditions: 35℃, CO2 volume fraction approximately 2%, culture for 21-28 days;

[0153] Fixation and color development: After washing with PBS, add 80% acetone (pre-cooled at -20℃, 100μL / well) for 5-15 min to fix. After washing 3 times with PBS, block with 1% BSA-PBST for 1 h. Then add HRP-HAV monoclonal antibody (1:10000, 100μL / well, incubate at 37℃ for 1 h), and develop color with TMB for 15 min. Stop the reaction with stop solution. Read the value at 450 nm using a microplate reader.

[0154] Judgment and calculation: A positive result is defined as "the OD of the positive well is ≥2.1 times that of the negative control". The titer is calculated using the Reed-Muench method based on the number of positive / negative wells at each dilution.

[0155] 2) Detection conditions for the T25 cell flask method

[0156] Cell culture: MRC-5 cells were cultured into a monolayer in T25 cell flasks;

[0157] ELISA plate and coating system: Rabbit polyclonal antibody against hepatitis A virus was used as the coating antibody (prepared at 2000-fold dilution, 100 μL / well, incubated overnight at 2-8°C in the dark), blocked with 1% BSA-PBST (200 μL / well, incubated at 37°C for 1.5 h), and then the sample after freeze-thaw, sonication and cell harvesting was added.

[0158] Antibody system: 3 μL of HRP-HAV monoclonal antibody was added to 39 mL of enzyme-labeled antibody dilution buffer to prepare a working solution diluted approximately 13,000 times. 100 μL / well was incubated at 37 °C for 1.5 h. The incubation was terminated after 15 min of TMB color development, and the absorbance was measured at 450 nm.

[0159] Viral titers were calculated using the Reed-Muench method based on the number of positive / negative wells at each dilution.

[0160] 2. Titer determination results for each batch of samples

[0161] The viral titers of samples P28-P37 were detected using the methods (96-well plate method) and T25 method of this application. The results are shown in Table 1.

[0162] Table 1. Comparison of viral titers (lgCCID) of samples P28-P37 using the T25 method and the 96-well plate method. 50 / mL)

[0163]

[0164] Note: The titer results of the T25 method in Table 1 were calculated according to the cell harvest-ELISA method; the 96-well plate method was obtained by directly detecting viral antigens after in-situ lysis and fixation of cells in a 96-well plate according to the method of this application.

[0165] As can be seen from Table 1:

[0166] The titers of samples P28 to P37, determined by the T25 method, were generally between 7.6 and 8.5 lgCCID. 50 / mL range;

[0167] The titers of the same batch of samples measured by the 96-well plate method ranged from 6.6 to 8.0 lgCCID. 50 The titer was slightly lower than that of the T25 method in the / mL range, but the trend and relative differences between batches remained consistent. High-titer samples showed high titers in both methods.

[0168] 3. Statistical correlation analysis

[0169] Based on the titer results of 10 batches of samples in Table 1, a linear correlation analysis was performed on the titer values ​​of the T25 method and the 96-well plate method:

[0170] The mean titer of the 10 samples using the T25 method was approximately 8.15 lgCCID. 50 / mL, the average titer using the 96-well plate method is approximately 7.22lgCCID. 50 / mL, the average difference between the two is about 0.9 log;

[0171] Pearson correlation analysis showed that the correlation coefficient r between the titer values ​​of the T25 method and the 96-well plate method for 10 batches of samples was approximately 0.86, indicating that the two methods have a good positive correlation. This suggests that the ranking and relative differences among different batches are basically consistent, which can meet the requirements for consistency of methods and trends in process research and quality control (correlation analysis was calculated based on the batch data in Table 1).

[0172] Overall, the results of this embodiment show that:

[0173] The titer measured by the method in this application embodiment is slightly lower than that of the traditional T25 method, but there is a good linear correlation and consistent high and low trends between the two.

[0174] Since the method in this application omits the multiple freeze-thaw cycles, ultrasonication, and chloroform extraction steps in the T25 method, it reduces the error caused by fluctuations in viral antigen extraction efficiency and is more suitable as a process comparison, trend monitoring, and high-throughput screening method within an enterprise.

[0175] The above-mentioned correlation verification can provide data support for the application of the methods in the embodiments of this application as alternative or supplementary methods to the pharmacopoeia method in process validation, process change research and daily quality control.

[0176] Example 3: Methodological Performance Verification and Application Evaluation

[0177] Based on Examples 1 and 2, the linear range, repeatability, precision, specificity, and robustness of the methods in the embodiments of this application are validated methodologically, and the practical application is evaluated in conjunction with the data on pages 28 to 37.

[0178] 1. Linearity and Applicable Scope

[0179] Representative viral titers of 5.0–8.0 lgCCID were selected. 50 Multiple batches of samples within the range of / mL were tested for titer using the method described in this application. The results showed that:

[0180] For titers of approximately 6.6–8.0 lgCCID 50For the virus samples P28~P37 per mL, the methods in the embodiments of this application can all give stable and reproducible titer results;

[0181] Validation, using samples of other intermediate products from the production process (such as harvested liquid and concentrated liquid), showed that this method is effective within the range of 5.0–8.0 lgCCID. 50 It exhibits a good practical linear range within / mL, which can cover the routine requirements for viral titer in the registration standards for live attenuated hepatitis A vaccines and inactivated vaccines.

[0182] 2. Precision and repeatability

[0183] Taking samples P28~P37 as an example, multi-well parallel detection was performed under the same batch of reagents and the same operator:

[0184] Each dilution was run in parallel with 6-8 wells. The Reed-Muench titer calculation showed a smooth positive rate curve, and the proportion of positive wells decreased as expected with increasing dilution, such as samples P30, P31, P33, and P35. -5 ~10 -8 The proportion of positive wells decreased stepwise at different dilutions;

[0185] When the same batch of samples is repeatedly tested on different plates or on different testing days, the titer difference is controlled within the preset acceptance standard (e.g., |Δtiter|≤0.5 log), which meets the requirements of intra-batch and inter-batch precision for virus titer methods.

[0186] 3. Specificity and background signal

[0187] In 96-well plate assays, blank control wells and negative control wells are set up: negative control wells contain normal cells that have not been inoculated with the virus, while blank control wells contain no cells; the OD value of the blank control wells should not be higher than that of the negative control wells; the blank background between plates should remain stable, and background fluctuations should be controlled within an acceptable range (CV≤10%).

[0188] The OD values ​​of positive wells were significantly higher than those of negative wells. The positive determination criteria (positive control was 2.1 times that of negative control) could effectively distinguish between wells with and without viral infection, indicating that this method has good specificity for the detection of hepatitis A virus antigen.

[0189] 4. Durability and Key Reagent Change Assessment

[0190] By replacing key reagents such as PBS, PBST, BSA-PBST, TMB, and HRP-HAV monoclonal antibody from different batches, titer results comparable to the original reagent batches can be obtained while maintaining standard operating conditions, indicating that this method has a certain tolerance to reagent batch variations.

[0191] Based on the correlation results of the T25 method (Example 2), it can be concluded that the method of this application has good methodological stability and practicality in seed stock management, production process validation, process scale-up studies and batch-to-batch consistency monitoring.

[0192] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0193] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for detecting trace amounts of hepatitis A vaccine virus titer, characterized in that, Includes the following steps: Human diploid cells were seeded into multi-well cell culture plates and cultured to form a monolayer of cells. Hepatitis A virus samples to be tested were inoculated into porous cell culture plates that formed a monolayer of cells for virus adsorption and culture, and were designated as the experimental group. Human diploid cells that were not inoculated with virus samples were used as the negative control group. Acetone was added to the experimental group and the negative control group for incubation to lyse cells and fix hepatitis A virus and its antigens; Furthermore, the virus titer of the hepatitis A virus sample to be tested was calculated based on the detection results using enzyme-linked immunosorbent assay (ELISA).

2. The method for detecting trace amounts of hepatitis A vaccine virus titer as described in claim 1, characterized in that, The inoculation amount of the human diploid cells was 1.0 × 10⁶ cells per well. 4 ~2.0×10 4 Each cell.

3. The method for detecting trace amounts of hepatitis A vaccine virus titer as described in claim 2, characterized in that, Before inoculating the hepatitis A virus sample to be tested, the procedure also includes serially diluting the sample to be tested, with a dilution of 10-1. -1 ~10 -6 and / or 10 -5 ~10 -8 Optionally, each dilution of hepatitis A virus sample to be tested can be set up in 6-8 parallel wells.

4. The method for detecting trace amounts of hepatitis A vaccine virus titer as described in claim 1, characterized in that, One or more of the following conditions must be met: (1) The conditions for virus adsorption include: a temperature of 35℃~37℃, a CO2 concentration of 2% (v / v)~5% (v / v), and a time of 1h~2h; (2) The virus culture also includes adding cell maintenance medium for maintenance culture, wherein the cell maintenance medium includes a culture medium containing serum; optionally, the concentration of serum in the virus culture system is 1% (v / v) to 3% (v / v); the virus culture conditions include: temperature of 35℃ to 37℃, CO2 concentration of 2% (v / v) to 5% (v / v), and time of 21 days to 28 days; (3) The concentration of the acetone is 78%~82% (v / v); (4) Before adding acetone for incubation, the acetone is pre-cooled at -18℃ to -22℃. Optionally, the incubation conditions for adding acetone include: temperature of 20℃ to 30℃ and time of 5min to 15min.

5. The method for detecting trace amounts of hepatitis A vaccine virus titer as described in claim 1, characterized in that, The steps for detecting hepatitis A virus in the sample using enzyme-linked immunosorbent assay (ELISA) and calculating the viral titer based on the detection results include: Add enzyme-labeled anti-hepatitis A virus antibody to the multi-well cell culture plate and incubate. Add the colorimetric reagent to develop the color, and then measure the absorbance. The positive wells were determined based on the absorbance of the negative control group, and the viral titer of the hepatitis A virus sample to be tested was calculated using the Reed-Muench method based on the number of positive and negative wells at each dilution. Optionally, one or more of the following conditions must be met: (1) The enzyme-labeled anti-hepatitis A virus antibody is a horseradish peroxidase-labeled anti-hepatitis A virus monoclonal antibody; (2) The enzyme-labeled anti-hepatitis A virus antibody is diluted at a ratio of 1:10000 to 1:13000; (3) The incubation conditions include: a temperature of 35℃~37℃ and a time of 60min~90min; (4) The standard for determining the positive well based on the absorbance of the negative control group is that the absorbance is greater than or equal to 2.1 times that of the negative control group.

6. The method for detecting trace amounts of hepatitis A vaccine virus titer as described in any one of claims 1 to 5, characterized in that, It also meets one or more of the following conditions: (1) The human diploid cells include MRC-5 cells; (2) The porous cell culture plate includes at least one of a 48-well cell culture plate and a 96-well cell culture plate; and, (3) The hepatitis A virus sample to be tested includes one or more of the following: hepatitis A live attenuated vaccine, hepatitis A virus strain, virus harvest liquid, intermediate product and finished product produced during the production process of hepatitis A inactivated vaccine.

7. The method for detecting trace amounts of hepatitis A vaccine virus titer as described in any one of claims 1 to 5, characterized in that, The method for detecting the viral titer of hepatitis A vaccine does not include the viral extraction steps of freezing and thawing, ultrasonic disruption, and chloroform extraction after cell harvesting.

8. A trace detection kit for hepatitis A vaccine virus titer, characterized in that, Includes human diploid cells, porous cell culture plates, acetone, buffers, and culture medium; Optionally, it may also include one or more of the following: blocking solution, enzyme-labeled anti-hepatitis A virus antibody, chromogenic solution, positive control and negative control.

9. The hepatitis A vaccine virus titer trace detection kit as described in claim 8, characterized in that, The hepatitis A vaccine virus titer micro-detection kit meets one or more of the following conditions: (1) The human diploid cells include MRC-5 cells; (2) The porous cell culture plate includes at least one of a 48-well cell culture plate and a 96-well cell culture plate; (3) The buffers include PBS buffers and PBST buffers; (4) The culture medium includes at least one of serum-free culture medium and culture medium containing 3% (v / v) to 5% (v / v) serum.

10. An evaluation method for the development and optimization of hepatitis A vaccine processes, characterized in that, It includes: The viral titer of hepatitis A vaccine virus was determined using the micro-detection method for hepatitis A vaccine virus titer as described in any one of claims 1 to 7, under different cell culture conditions, different culture medium formulations, different culture process parameters, or before and after virus inactivation. By comparing the viral titers, the effects of different process conditions on the hepatitis A virus proliferation level and / or process stability are evaluated.

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